Biotin (Vitamin B8) Synthesis in Plants: Advances in Botanical Research October 2011

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Biotin (Vitamin B8) Synthesis

in Plants
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From: Claude Alban, Biotin (Vitamin B8) Synthesis in Plants.
In Fabrice Rbeill and Roland Douce, editors:
Advances in Botanical Research, Vol. 59,
Amsterdam, The Netherlands, 2011, pp. 39-66.
ISBN: 978-0-12-385853-5
Copyright 2011 Elsevier Ltd.
Academic Press.
Author's personal copy
Biotin (Vitamin B8) Synthesis in Plants
CLAUDE ALBAN*,{,{,},1
*Laboratoire de Physiologie Cellulaire Vegetale, CNRS,

UMR5168, Grenoble, France
{
CEA, DSV, iRTSV, Grenoble, France
{
INRA, UMR1200, Grenoble, France
}
Universite Joseph Fourier, Grenoble, France
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. Significance ......................................................................
B. Distribution and Nutritional Aspects .......................................
C. Biotin-Containing Proteins ...................................................
II. The Biosynthetic Pathway. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
A. The Origin of Pimeloyl-CoA..................................................
B. 7-Keto-8-Aminopelargonic Acid Synthase .................................
C. 7,8-Diaminopelargonic Acid SynthaseDethiobiotin Synthetase......
D. Biotin Synthase .................................................................
III. Protein Biotinylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IV. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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ABSTRACT
Biotin, also known as vitamin H or B8, is an essential cofactor for CO2-manipulating
enzymes found in all three domains of life. The past few years have seen decisive
progress accomplishments on the elucidation of biotin metabolism in plants, at both
1
Corresponding author: E-mail: claude.alban@cea.fr
Advances in Botanical Research, Vol. 59

Copyright 2011, Elsevier Ltd. All rights reserved.
0065-2296/11 $35.00
DOI: 10.1016/B978-0-12-385853-5.00005-2

40
C. ALBAN
the molecular and cellular levels, and several unique features are emerging. Noticeably, biotin synthesis in plants is split between cytosol and mitochondria. Biotinutilizing enzymes are also quartered between different compartments of the plant cell.
Among these compartments, mitochondria play a central role. In this review, I will
summarize the most recent discoveries about the synthesis, manipulation and compartmentalization of biotin in plant cells. These advances open challenging prospects
for plant biotechnology purposes through a better understanding of regulation,
storage and utilization of the vitamin. Understanding how the biotin biosynthetic
pathway interacts with other metabolic pathways and the emerging involvement of
mitochondria in plant growth and development, through its intimate implication in
vitamins synthesis are also particularly challenging.
ABBREVIATIONS
50 -UTR
AdoMet
AdoMTOB
ADR
ADX1
DAPA
DTB
KAPA
PLP
uORF
50 -untranslated region
S-adenosyl-L-methionine
4-(methylthioadenosyl)-2-oxobutanoate
adrenodoxin reductase
adrenodoxin 1
7,8-diaminopelargonic acid
dethiobiotin
7-keto-8-aminopelargonic acid
pyridoxal 50 -phosphate
upstream open reading frame
I. INTRODUCTION
Biotin, also known as vitamin H or B8, is a cofactor for some carboxylases,

decarboxylases and transcarboxylases dealing with crucial metabolic processes such as fatty acid and carbohydrate metabolism (Alban et al., 2000;
Knowles, 1989). In mammals, biotin is also known to regulate gene expression through different ways including histone biotinylation (Beckett, 2007;
Zempleni, 2005). Despite its essential functions, de novo synthesis of this
vitamin is restricted to bacteria, a few fungi and plants. All animals including
humans cannot synthesize biotin as part of their normal metabolism and
therefore rely on the supply of biotin from exogenous sources. Biotin is a
fusion of an imidazolinone ring with a tetrahydrothiophene ring bearing a
valeric acid side chain (Fig. 1). There are three chiral carbon atoms in biotin,
leading to eight possible stereoisomers. However, only one is biologically
active; this isomer is denoted () or (D)-biotin. The absolute stereochemistry
of D-biotin established by X-ray crystallography revealed that the

BIOTIN (VITAMIN B8) SYNTHESIS IN PLANTS
41
O
HN
HOOC
Valeric acid
Fig. 1.
NH
Ureido ring
S
H
H Tetrahydrothiophene ring
H
The structure of biotin.
imidazolinone and the tetrahydrothiophene rings are fused in a cis configuration, producing a bottle structure (Fig. 1).
A. SIGNIFICANCE
Biotin was discovered in the search for the nutritional factor that prevents
egg white injury in experimental animals, and the use of the biotin antagonist
in egg white, the biotin-binding protein avidin, was further useful in producing biotin deficiency in animal models (Kogel and Tonnis, 1936). The detrimental effect of feeding high doses of raw egg white most often involves
dermatologic lesions such as dermatitis or alopecia. This explains the name
of vitamin H (Haut, German word for skin) given to biotin at that time. In
addition to primary deficiencies of the vitamin, genetic disorders in biotin
metabolism have been identified. These are rare, affecting infants and children, but usually having serious consequences (neurologic abnormalities
such as hypotonia, altered consciousness, seizures and ataxia, and skin
damages such as rash and alopecia) (Baumgartner and Suormala, 1999).
Congenital defects fall into two major categories. The first involves the
absence of a biotin apoenzyme. In the second, multiple carboxylases have
defective activities due to absence of biotinidase, the enzyme responsible for
biotin recycling, or altered holocarboxylase synthetase (HCS), the enzyme in
charge of biotin-dependent carboxylases activation by biotinylation (Fig. 2).
These last congenital disorders usually respond to high doses of biotin.
B. DISTRIBUTION AND NUTRITIONAL ASPECTS
Biotin exists under two forms in living cells, free or covalently bound to
proteins. In bacterial and animal cells, free biotin content is low or even
undetectable. In Escherichia coli, for example, free biotin never accumulates
above a nanomolar concentration range. In contrast, plant cells contain a
large pool of free biotin. In pea leaves, for instance, free biotin accumulates in
the cytosol of mesophyll cells to a concentration of about 11 M (Baldet

42
C. ALBAN
Dietary biotin
Free
Protein-bound
150300 mg/day
Biotinidase
O
HN
Holocarboxylase
synthetase
NH
COOH
S
Biotinidase
Biotin
O
HN
NH
Apocarboxylases
(PCC, MCC, PC, ACC)
O
COOH
N
H
Biocytin
NH2
Proteolytic
Degradation
HN
NH
Holocarboxylases
Congenital disorders
Fig. 2.
Proteins
Aminoacid
catabolism
Lipids
Fatty acid
synthesis
N
H
Carbohydrates
Gluconeogenesis
The biotin cycle in mammalian cells.
et al., 1993a). The pool of protein-bound biotin associated to biotin-dependent carboxylases is mainly present within organelles (1.2 M within chloroplast stroma and 13 M within mitochondrial matrix). The free/bound-biotin
ratio in the whole cell is > 6 (Baldet et al., 1993a). In Arabidopsis cultured
cells, the free biotin pool is somewhat lower with a ratio free/bound of
around 1.5 (Claude Alban and Virginie Pautre, unpublished observation).
To date the precise fate of free biotin in plant cells is still poorly understood
but it could behave as a reserve pool for maintaining biotin-dependent
carboxylases activity and thus cell viability under stress conditions affecting
biotin synthesis or availability. This is well illustrated in the following example. After 3 days of treatment of Arabidopsis cultured cells with sublethal
concentrations of acidomycine, an inhibitor of biotin synthesis, the pool of
free biotin was found to be drastically reduced while that of bound biotin and
the activity of biotin-dependent carboxylases were poorly affected. After
6 days of treatment, the pool of bound biotin and biotin enzyme activities
were, in turn, significantly reduced with as consequences a global alteration
of respiration, photosynthetic activity and cell division. These effects
were reversed by supplementation with free biotin (Claude Alban and
Virginie Pautre, unpublished data).

43
Because of the dual nature of biotin in living cells, dietary biotin is

present in two forms: free and protein bound (Fig. 2). The protein-bound
form, in which vitamin is covalently linked to polypeptides through a
specific lysine residue, is degraded by digestive proteases to the biotinyllysine adduct biocytin. Then, biotinidase is thought to be responsible for
the cleavage of biocytin, liberating the free vitamin that can be absorbed by
the intestine and then transferred to the cytosolic space of other tissues
where it is directly assimilated (Hymes and Wolf, 1996). The existence of
biotinidase in the plant kingdom has not yet been reported. Since, in
general, foodstuffs of plant origin have a greater free biotin content
than foodstuffs of animal origin (with the exception of milk), plant biotin
is more rapidly absorbed and metabolized than animal sources of the
vitamin.
In humans, the daily requirement of biotin has been estimated between 150
and 300 g. Biotin is widely distributed in foods and feedstuffs, although the
absolute amount of biotin present in even the richest dietary sources are low.
Milk, liver, egg yolk and a few vegetables (nuts, fruits and unpolished rice)
are the most important natural sources for human nutrition (Table I).
The oilseed or alfalfa meals and dried yeasts are the most important natural
sources for the feeding of nonruminant animals. A second potential source of
biotin for higher organisms is the microbial synthesis by gut microflora. As a
consequence, simple deficiencies of biotin in animals or humans are extremely rare. Few cases of biotin deficiencies have been reported in humans. Most
of these involved nursing infants whose mothers milk contained inadequate
supplies of the vitamin, chronic ingestion of egg white, or patients receiving
incomplete parenteral nutrition (Combs, 1998a). It is also thought that
marginal biotin status plays a causative role in the aetiology of sudden infant
death syndrome.
C. BIOTIN-CONTAINING PROTEINS
Biotinylated proteins are not widespread in nature. For example, the only
biotin-dependent carboxylase in E. coli is acetyl-CoA carboxylase (EC
6.4.1.2), a multisubunit enzyme, in which one of the subunits is biotinylated
and corresponds to the biotin carboxyl carrier protein (BCCP). Other bacteria contain one to no more than three biotinylated proteins (Fall, 1979).
Eukaryotic cells appear to contain a slightly greater number of biotinylated
proteins. For example, Saccharomyces cerevisiae contains four or five biotinylated proteins depending on growth conditions (Lim et al., 1987), whereas
mammals (Jitrapakdee and Wallace, 2003) are reported to contain four

44
C. ALBAN
TABLE I
Biotin Contents of Food
Food
Dairy products
Milk
Cheeses
Meats
Beef
Liver
Calf kidney
Cereals
Barley
Sorghum
Rice
Oilseed
Rapeseed
Soybean
Vegetables
Carrots
Cauliflower
Potatoes
Soybeans
Nuts
Peanuts
Walnuts
Others
Eggs
Brewers yeast
Alfalfa meal
Biotin (g/100 g)
2
35
3
52
100
14
29
25
99
27
3
17
0.1
60
34
37
20
80
54
Compilation of values by Combs (1998b) and Bonjour (1991).
biotinylated proteins. All these enzymes play crucial cellular housekeeping

functions. More specifically, acetyl-CoA carboxylase that catalyses the
ATP-dependent carboxylation of acetyl-CoA is recognized as the regulatory
enzyme of lipogenesis; methylcrotonoyl-CoA carboxylase (EC 6.4.1.4) catalyses the conversion of methylcrotonoyl-CoA to methylglutaconyl-CoA, a
key reaction in the degradation pathway of leucine; propionyl-CoA carboxylase (EC 6.4.1.3) is a key enzyme in the catabolic pathway of odd-numbered
fatty acids and the amino acids, Ile, Thr, Met and Val; and pyruvate carboxylase (EC 6.4.1.1) has an anaplerotic role in the formation of oxaloacetate.
The common feature of these reactions is the transfer of a carboxyl group
from bicarbonate to an acceptor substrate, utilizing biotin as a carboxyl
carrier. The reactions catalysed by these enzymes take place in two steps (1)
and (2), resulting in the overall reaction (3):

45
HCO
3 Enzyme - Biotin ATP - Mg
! Enzyme - Biotin CO
2 ADP - Mg Pi
Enzyme - Biotin CO

2 Acceptor
! Acceptor CO
2 Enzyme - Biotin

HCO
3 Acceptor ATP - Mg ! Acceptor CO2
ADP - Mg Pi
The features that distinguish the reactions of each of these enzymes are the
acceptor substrates. The family of biotin enzymes also includes oxaloacetate,
methylmalonyl-CoA and glutaconyl-CoA decarboxylases that are involved
in sodium transport in anaerobic prokaryotes (Dimroth, 1985) as well as
transcarboxylase (EC 2.13.1) that participates in propionic acid fermentation
in Propionibacterium shermanii (Wood and Kumar, 1985). The latter two
classes of enzymes do not require ATP as a substrate. In all biotin enzymes
described to date, the biotin is covalently linked to the e-amino group of a
specific Lys residue located within a highly conserved (Ala/Val)-Met-Lys(Met/Leu) tetrapeptide motif.
Plant acetyl-CoA carboxylase has been documented since 1961 (Hatch and
Stumpf, 1961). Investigations of plant acetyl-CoA carboxylases increased in
the late 1980s because of its regulatory role in fatty acid biosynthesis and also
because this enzyme is the molecular target of powerful herbicides in use
since the early 1980s and effective against grasses (the Graminaceae) including grass weeds (Harwood, 1988). Since then, other biotin-containing proteins with variable structure and subcellular localizations have been
discovered in plants. These include two structurally distinct isoforms of
acetyl-CoA carboxylases in cytosol and plastids (Alban et al., 1994), a
geranyl-CoA carboxylase in plastids (Guan et al., 1999), a methylcrotonoyl-CoA carboxylase in mitochondria (Alban et al., 1993) and a cytosolic
seed storage biotin-protein (SBP) with an atypical biotinylation motif (Duval
et al., 1994). Comprehensive information on the structure, regulation and
function of plant biotin-containing proteins are available on leading reviews
(Alban et al., 2000; Nikolau et al., 2003) and will not be detailed here. In this
chapter, I have attempted to summarize the recent advances about
biotin biosynthesis and protein biotinylation processes in higher plants and
their implications for industry, for example, the rational design of new
herbicides.

46
C. ALBAN
II. THE BIOSYNTHETIC PATHWAY

In all known microbes, biotin is synthesized from pimeloyl-CoA through
four enzymatic steps comprising 7-keto-8-aminopelargonic acid (KAPA)
synthase (EC 2.3.1.47), 7,8-diaminopelargonic acid (DAPA) synthase (EC
2.6.1.62), dethiobiotin synthetase (EC 6.3.3.3) and biotin synthase (EC
2.8.1.6) encoded by bioF, bioA, bioD and bioB genes, respectively (Fig. 3).
Enzymes encoded by these genes in E. coli, Bacillus sphaericus and more
recently in Bacillus subtilis or Mycobacterium tuberculosis have been totally
or partially characterized biochemically, and/or structurally, and their reaction mechanisms elucidated (Dey et al., 2010; Schneider and Lindqvist, 2001;
Streit and Entcheva, 2003). In plants, the biosynthetic pathway appears to
follow the same pattern as identified for bacteria (Fig. 3). This was deduced
from measuring pools of the different intermediates of biotin biosynthesis,
employing lavender (Lavandula vera) cells cultures treated with radiolabelled
B. sphaericus
B. subtilis
A. thaliana
Gram +
Plants
?
E. coli
bioI
Gram
Pimelic acid
Malonyl-CoA (ACP)
bioC, FabH-G-Z-I-B, bioH
bioW
Pimeloyl-CoA (ACP)
L-ala
bioF (BIO4), KAPA synthase
7-keto-8-aminopelargonic acid (KAPA)

bioA (BIO1), DAPA synthase
7,8-diaminopelargonic acid (DAPA)
bioD (BIO3), DTB synthetase
Dethiobiotin (DTB)
bioB (BIO2), biotin synthase
Biotin
Fig. 3. The biotin biosynthetic pathway in bacteria and plants. The bacterial gene
names are given in lower case letters; the respective plant homologs are shown in
bracketed uppercase letters.

47
precursors (Baldet et al., 1993b). An alternative approach used to study

biotin synthesis in plants has been the isolation and characterization of
auxotrophic mutants. The Arabidopsis bio1 mutant was the first plant auxotroph shown to result in embryo lethality (Schneider et al., 1989). Seeds
homozygous for the mutation failed to develop unless exogenous biotin,
DTB or DAPA was supplied to the plant (Shellhammer and Meinke,
1990). The E. coli bioA gene, which codes for DAPA synthase, could genetically complement the bio1 mutation, demonstrating that bio1/bio1 mutant
plants are defective in this enzyme (Patton et al., 1996). Later on, a second
biotin auxotroph mutant of Arabidopsis was identified, defective in the final
step of biotin synthesis, that is, the conversion of DTB to biotin (Patton et al.,
1998). Genetic and phenotypic characterization of this bio2 mutant also
showed embryo lethality as a consequence of the BIO2 gene knockout and
efficient phenotypic reversion on addition of exogenous biotin (Arnal et al.,
2006). Thus, biotin biosynthesis is an indispensable procedure for plant
growth and inhibition of the enzymes of the pathway is potentially an
attractive target for herbicide development (Alban et al., 2000). As a proof,
inhibition of KAPA synthase reaction by triphenyltin acetate (TPTA),
DAPA synthase reaction by KAPA analogs or biotin synthase reaction by
acidomycin is lethal for the plant (Baldet et al., 1993b; Hwang et al., 2010;
Nudelman et al., 2004). However, until the past decade, none of these
enzymes had been characterized.
A. THE ORIGIN OF PIMELOYL-COA
If the last four steps of biotin biosynthesis, from pimeloyl-CoA to biotin, are
common to most bacteria, fungi and plants, the origin of this precursor is
much less clear. Alternate pathways to pimeloyl-CoA seem to coexist in
nature (Fig. 3). The gram-positive bacteria such as B. sphaericus or B. subtilis
are capable of forming pimeloyl-CoA from pimelic acid with a single gene
encoding a pimeloyl-CoA synthetase (EC 6.2.1.14; bioW; for a review, see
Streit and Entcheva, 2003). Further, in Bacillus species, the bioI gene, which
appears to be restricted to these organisms, encodes a cytochrome P450
family member that makes the pimeloyl moiety by cleaving long-chain
acyl-ACPs precursors (Stok and De Voss, 2000). Gram-negative bacteria
like E. coli do not synthesize pimeloyl-CoA from pimelic acid. Genetic
analysis in E. coli identified two genes essential for pimeloyl-CoA synthesis,
bioC and bioH whose exact function remained unknown for more than
15 years (Ifuku et al., 1994). Recently, the group of Cronan demonstrated
that the pimeloyl moiety in E. coli is synthesized by a modified fatty acid
synthetic pathway in which !-carboxyl group of a malonyl-thioester is

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C. ALBAN
methylated by BioC, which allows recognition of this atypical substrate by

the classical fatty acid synthetic enzymes. The malonyl-thioester methyl ester
enters fatty acid synthesis as the primer and undergoes two reiterations of the
fatty acid elongation cycle to give pimeloyl-ACP methyl ester, which is finally
hydrolysed to pimeloyl-ACP and methanol by BioH (Lin et al., 2010). This
work also suggests that pimeloyl-ACP rather that pimeloyl-CoA is the
physiological substrate for biotin synthesis. In eukaryotes, our knowledge
of the origin of the pimeloyl moiety is still fragmentary. Recently, the protein
encoded by the BIO1 gene in yeasts was found to have the function of a
pimeloyl-CoA synthetase (Hall and Dietrich, 2007). In plants, labelled pimelic acid has been efficiently incorporated into biotin using lavender cell
cultures, suggesting the existence of pimeloyl-CoA synthetase activity also
in plants (Baldet et al., 1993b). However, to date, no plant gene homologous
to gram-positive bacterial bioW or yeast BIO1 genes has been identified.
B. 7-KETO-8-AMINOPELARGONIC ACID SYNTHASE
KAPA synthase, the first committed enzyme in the pathway, catalyses the
decarboxylative condensation of pimeloyl-CoA and L-Alanine to produce
KAPA, CoASH and carbon dioxide:
Pimeloyl - CoA L - alanine ! KAPA CoA - SH CO2
The structure and reaction mechanism of KAPA synthase place it in the
subfamily of -oxoamine synthases, a small group of pyridoxal 50 -phosphate
(PLP)-dependent enzymes of the -family (Alexeev et al., 1998; Ploux and
Marquet, 1996; Webster et al., 2000). Searches of the Arabidopsis genome
database detected a single gene (here named AtBIOF or BIO4) encoding a
predicted protein with 2732% identity to protein sequences of well-characterized bacterial KAPA synthases (Pinon et al., 2005). Despite the relatively
low overall amino acid identity with its bacterial counterparts, the plant
protein was able to complement an E. coli bioF-mutant and to catalyse
KAPA synthase reaction when assayed using pimeloyl-CoA and L-Ala as
substrates. Biochemical, kinetic and spectroscopic studies of purified recombinant enzyme evidenced high substrate specificities and allowed determination of the reaction mechanism. Essential steps of this mechanism are
formation of an external aldimine between PLP cofactor and the substrate
L-Ala. Abstraction of the C2-H proton of the aldimine, possibly by Lys-319,
leads to a quinonoid intermediate, which then attacks the thioester carbonyl
of pimeloyl-CoA. Release of CoASH produces a -ketoacid aldimine, which
after decarboxylation is converted into the product (Pinon et al., 2005). More
importantly, the salient fact of this study concerned the surprising cellular

49
distribution of KAPA synthase as determined by two independent methods.

Both GFP-fusions targeting experiments and subcellular fractionation studies showed that this initial step in biotin synthesis in Arabidopsis takes place
in the cell cytosol, which contrasts with the mitochondrial location of the
remaining pathway (see below).
C. 7,8-DIAMINOPELARGONIC ACID SYNTHASEDETHIOBIOTIN SYNTHETASE
The antepenultimate step in the biotin biosynthetic pathway, the conversion

of KAPA to DAPA, is catalysed by DAPA synthase, another PLP-dependent enzyme. In most bacteria, the enzyme uses S-adenosyl-L-methionine
(AdoMet) as amino group donor, an unusual feature among aminotransferases (Breen et al., 2003; Izumi et al., 1975; Mann and Ploux, 2006):
KAPA AdoMet ! DAPA AdoMTOB
Interestingly, B. subtilis uses L-Lysine, another unusual amino donor for
the reaction (Van Arsdell et al., 2005). The chemical TPTA is a potent
inhibitor of KAPA synthase reaction in Arabidopsis with strong herbicidal
activity (Hwang et al., 2010). The supplement of biotin or biotin biosynthesis
intermediate such as DTB, DAPA, KAPA AdoMet, but not KAPA alone
rescued germination and plant growth inhibited by TPTA, suggesting that
AdoMet might be an essential amino group donor for the synthesis of DAPA
in plants, as well. DTB synthetase carboxylates DAPA to form the ureido
ring of DTB in an ATP-Mg-dependent penultimate step of the pathway
(Alexeev et al., 1995; Huang et al., 1995):
DAPA ATP CO2 ! DTB ADP Pi
In Arabidopsis, genetic studies revealed that DAPA synthase (BIO1; BioA
ortholog) and DTB synthetase (BIO3; BioD ortholog) are encoded in adjacent genes defining a single genetic locus and are expressed in both single and
chimeric BIO3BIO1 transcripts, through alternative splicing events
(Muralla et al., 2008). One of the fused transcripts is monocistronic and
encodes a bifunctional protein capable of complementing the orthologous
auxotrophs of E. coli (bioD and bioA). The second one includes 10 more
nucleotides that introduce a premature stop codon. As a consequence, this
splice variant is bicistronic, with distinct but overlapping reading frames.
This bicistronic transcript is potentially capable of producing separate BIO3
and BIO1 proteins. The existence of a monocistronic BIO3BIO1 transcript
is not a unique feature of Arabidopsis. Homology searches among eukaryotic
and prokaryotic genomes revealed the presence of a bifunctional BIO3BIO1
homologue gene in others flowering plants, mosses, green and red algae and

50
C. ALBAN
in most ascomycete and basidiomycete fungi (Hall and Dietrich, 2007;

Magliano et al., 2010; Muralla et al., 2008; Fig. 4). These data suggest that
a fusion event between prokaryotic bioD and bioA ancestor genes occurred
Vitis vinifera (XM_002270515.1)

Arabidopsis thaliana (EU089963.1)
Brassica rapa subsp. pekinensis (AC189479.2)
Medicago truncatula (barrel medic) (AC174353.16)
Sorghum bicolor (sorghum)(XM_002468273.1)
Zea mays (BT065649.1)
Oryza sativa Japonica Group (NM_001067889.1)
Physcomitrella patens subsp. Patens (XM_001764409.1)
Ostreococcus lucimarinus CCE9901 (XM_00T422822.1)
Micromonas sp. RCC299 (XM_002503155.1)
Chlamydomonas reinhardtii (XM_001690622.1)
Cyanidioschyzon merolae strain 10D (CMG023C)
Aspergillus nidulans FGSC A4 (XM_659156.1)
Aspergillus oryzae RIB40 (XM_001816971.1)
Aspergillus flavus NRRL3357 (XM_002382995.1)
Aspergillus terreus NIH2624 (XM_001209788.1)
Aspergillus niger CBS 513.88 (XM_001396701.1)
Aspergillus fumigatus Af293 (XM_74618.1)
Neosartorya fischeri NRRL 181 (XM_001257570.1)
Aspergillus clavatus NRRL 1 (XM_001270182.1)
Penicillium chrysogenum Wisconsin 54-1255 (XM_002563776.1)
Uncinocarpus reesii 1704 (XM_002541682.1)
Coccidioides immitis RS (XM_001247545.1)
Penicillium marneffei ATCC 18224 (XM_002143123.1)
Talaromyces stipitatus ATCC 10500 (XM_002479411.1)
Aiellomyces dermatitidis SLH14081 (XM_002627316.1)
Aiellomyces capsulatus NAm1 (XM_001538071.1)
Phaeosphaeria nodorum SN15 (XM_001805235.1)
Pyrenophora tritici-repentis Pt-1 C-BFP (XM_001930446.1)
Sclerotinia sclerotiorum 1980 UF-70 (XM_001590649.1)
Botryotinia fuckeliana B05.10 (XM_001558049.1)
Podospora anserina DSM 980 (XM_001903481.1)
Podospora anserina (CAP61291.1)
Gibberella zeae PH-1 (anamorph: Fusarium graminearum) (XM_389224.1)
Yarrowia lipolytica CLIB122 (XM_504233.2)
Malassezia globosa CBS 7966 (XM_001729066.1)
Ustilago maydis 521 (XM_753969.1)
Crytococcus neoformans var. neoformans JEC21 (XM_569073.1)
Laccaria bicolor S238N-H82 (XM_001880692.1)
Coprinopsis cinerea okayama7#130 (XM_001836166.1)
Schizosaccharomyces iaponicus yFS275 (XM_002171908.1)
Fungi
Bacteria
Monofunctional
Hydrogenobaculum sp. Y04AAS1 (ACG57182.1)

Hydrogenobaculum sp. Y04AAS1 (YP_002121160.1)
Aquifex aeolicus (O66557.1)
Hydrogenivirga sp. 128-5-R1-1 (EDP73255.1)
Sulfurihydrogenibium sp. YO3AOP1 (ACD66647.1)
Acidithiobacillus ferrooxidans ATCC 53993 (ACH83818.1)
Acidithiobacillus ferrooxidans ATCC 53993 (YP_002220025.1)
Methanocaldococcus jannaschii (Q58696)
Kurthia sp. 538-KA26 (BAB39453.1)
Staphylococcus carnosus subsp. carnosus TM300 (YP_002635311.1)
Bacillus subtilis (P53555.1)
Brevibacillus brevis NBRC 100599 (BAH46441.1)
Helicobacter pylori J99 (Q9ZKM5.1)
Helicobacter pylori (025627.1)
Helicobacter pylori B38 (YP_003057676.1)
Helicobacter acinonychis str. Sheeba (CAJ99442.1)
Herminiimonas arsenicoxydans (CAL60336.1)
Azoarcus sp. BH72 (YP_933392.1)
Lysinibacillus sphaericus (P22805.1)
Rhizobium leguminosarum bv. viciae 3841 (CAK12322.1)
Rhodopirellula baltica SH 1 (NP_865422.1)
Zymomonas mobilis subsp. mobilis ZM4 (AAV90542.1)
Mycobacterium tuberculosis (P0A4X6.1)
Mycobacterium bovis (P0A4X7.1)
Mycobacterium leprae (P45488.1)
Corynebacterium glutamicum (P46395.2)
Thiomicrospira crunogena XCL-2 (ABB40873.1)
Haemophilus influenzae 86-028NP (AAX88383.1)
Haemophilus influenzae 86-028NP (YP_249043.1)
Haemophilus influenzae (P44426.1)
Neisseria meningitidis 8013 (CAX50480.1)
Neptuniibacter caesariensis (ZP_01167088.1)
Campylobacter hominis ATCC BAA-381 (ABS52358.1)
Lachancea thermotolerans (CAR23468.1)
Buchnera aphidicola (Baizongia pistaciae) (Q89AK4.1)
Buchnera aphidicola str . Bp (Baizongia pistaciae) (AAO26998.1)
Buchnera aphidicola (Schizaphis graminum) (Q8K9P0.1)
Buchnera aphidicola (Acyrthosiphon pisum) (P57379.1)
Pichia stipitis CBS 6054 (EAZ63280.2)
Debaryomyces hansenii (CAR66048.1)
Saccharomyces cerevisiae (P50277.1)
Zygosaccharomyces rouxii (CAR30704.1)
Kluyveromyces lactis (CAH01942.1)
Escherichia coli (P12995.2)
Escherichia vulneris (P53656.1)
uncultured bacterium pCosAS1 (AAG53588.1)
Salmonella thyphimurium (P12677.2)
uncultured bacterium pCosHE1 (AAG60563.1)
Erwinia pyrifoliae DSM 12163 (CAY74978.1)
Serratia marcescens (P36568.1)
Serratia odorifera 4Rx13 (ZP_06189072.1)
Providencia rustigianii DSM 4541 (EFB74154.1)
Providencia alcalifaciens DSM 30120 (ZP_03320306.1)
Providencia stuartii ATCC 25827 (EDU58416.1)
Proteus penneri ATCC 35198 (ZP_03806407.1)
Photorhabdus luminescens subsp. laumondii TTO1 (CAE13777.1)
Mesorhizobium loti MAFF303099 (BAB52209.1)
Xanthobacter autotrophicus Py2 (ABS68156.1)
Gemmatimonas aurantiaca T-27 (YP_002762353.1)
Prochlorococcus marinus MED4 (CAE19931.1)
Prochlorococcus marinus subsp. marinus str. CCMP1375 (AAQ00670.1)
Thermosynechococcus elongatus BP-1 DNA (BAC09487.1)
Flavobacterium johnsoniae UW101 (ABQ03844.1)
Capnocytophaga ochracea DSM 7271 (ACU93703.1)
Pedobacter heparinus DSM 2366 (ACU04770.1)
Chlamydophila pneumoniae LP CoLN (ACZ32943.1)
Bifunctional
Plants
Hemiascomycetes
Fig. 4. Phylogenetic analysis of eukaryotic bifunctional DTB synthetase/DAPA

synthase and monofunctional DAPA synthase prokaryotic orthologs. Protein
sequences were first aligned using CLUSTALW. The phylogenetic tree was constructed using the protpars and neighbour modules from the PHYLIP package and
the BLOSUM 62 similarity matrix.

51
early in the evolution of modern-day eukaryotes. By contrast, in the large

majority of species in the Saccharomycotina and Schizosaccharomycetes
classes, the bioA and bioD gene orthologues (named BIO3 and BIO4 in
yeasts) are separate (Hall and Dietrich, 2007; Magliano et al., 2010). Hall
and Dietrich (2007) propose that much of the biotin pathway was lost in
Saccharomycotina ancestors of Saccharomyces and Candida and that the
bioA and bioD orthologs were reacquired through a separate, horizontal
gene transfer from an unidentified prokaryotic donor. Noticeably, although
numerous bacteria have neighbouring bioD and bioA genes in the same
orientation, a bacterial gene fusion event does not seem to have occurred.
The ability to produce a bicistronic transcript through differential splicing
appears to have been a more recent event because it is limited to selected
angiosperms (Muralla et al., 2008). Biotin biosynthesis in plants thus provides an intriguing example of a bifunctional locus that catalyses two sequential reactions in the same metabolic pathway. This complex locus exhibits
several unusual features that distinguish it from biotin operons in bacteria
and from other genes known to encode bifunctional enzymes in plants
(Muralla et al., 2008). Interestingly, the BIO3BIO1 protein contains an
N-terminal sequence that is predicted to target the protein to mitochondria
by all the major prediction programs for intracellular localization of plant
proteins (Muralla et al., 2008). This localization is supported by experimental
proteomic data in the case of Chlamydomonas reinhardtii BIO3BIO1 protein
(Chlamydomonas Mitochondrial Proteome; Atteia et al., 2009). It therefore
appears that in plants, both DAPA synthase and DTB synthetase activities
take place in mitochondria. Finally, the capacity of a single plant enzyme to
convert KAPA into DAPA and then DTB may have implications for ongoing efforts to design herbicides that interfere with biotin production and with
biotechnological strategies to increase biotin levels in crop plants (for biofortification or phytofarming aims; Muralla et al., 2008).
D. BIOTIN SYNTHASE
Biochemical and molecular characterization of the biotin biosynthetic pathway in plants has dealt primarily with biotin synthase, the final enzyme of the
pathway, because this reaction is a rate-limiting step and also because its
mechanism still remains an enigma for chemists and biologists. Biotin
synthase is an AdoMet-dependent radical enzyme, undoubtedly the most
complex of the pathway generating biotin. Its activity aims to sulphur
insertion at the C6 and C9 position in DTB and the intimate chemistry of
the underlying reaction is not yet fully understood.

52
C. ALBAN
DTB AdoMet S ! Biotin 50 - deoxyadenosine L - methionine

Also, the role of specific proteins and cofactors, and the ultimate source of
sulphur are still matter of debate (for a review, see Jarrett, 2005a). In E. coli, the
conversion of dethiobiotin to biotin is catalysed by a complex involving at least
three proteins (including flavodoxin, flavodoxin reductase and MioC) in addition to biotin synthase which alone is not able to support this reaction (Sanyal
et al., 1996). Interestingly, plant biotin synthase (here named BIO2) seems to
accommodate these bacterial partners of the reaction, yielding a functional
biotin synthase complex. This capacity accounted for the cloning of the Arabidopsis BIO2 gene by functional complementation of an E. coli bioB mutant
(Baldet and Ruffet, 1996). In addition, subcellular fractionation studies and
Western blot analyses using antibodies raised against the plant recombinant
enzyme clearly demonstrated its mitochondrial location (Baldet et al., 1997).
As its bacterial counterparts (Berkovitch et al., 2004), purified BIO2
protein is a homodimer that, in its active reconstituted form, coordinates a
[2Fe2S]2 and a [4Fe4S]2 cluster per monomer (Daouda Traore, Antoine
Picciocchi and Claude Alban, unpublished data; Fig. 5). The purified enzyme
alone is not able to support biotin synthesis. Combination experiments using
purified BIO2 protein and extracts from pea leaf or potato tuber organelles
(plastids and mitochondria) showed that only mitochondrial fractions could
0.45
BIO2 [2Fe2S]/[4Fe4S]
330
Absorbance
2Fe/2S
4Fe/4S
N (Pro44)
AdoMet
DTB
0.35
410
0.25
540
0.15
0.05
0
A 280 = 0.69
300
400
500
600
700
Wavelength (nm)
Fig. 5. (A) Structure of Arabidopsis BIO2 monomer modelized by Swiss-model

program (www.expasy.ch) using E. coli biotin synthase structure as a matrix
(Berkovitch et al., 2004). AdoMet, S-adenosyl-L-methionine; DTB, dethiobiotin.
(B) UVvisible spectrum of purified recombinant BIO2 protein incubated with excess
iron and sulphide under strict anaerobiosis. The spectrum profile is consistent with
the presence on the enzyme of both a [2Fe2S] and a [4Fe4S] cluster (Daouda
Traore, Antoine Picciocchi, Claude Alban, Lilian Jacquamet, unpublished data).

53
elicit biotin formation at catalytic rates in the plant reconstituted system, in

keeping with the specific location of BIO2 protein (Picciocchi et al., 2001).
A biochemical screening of potato mitochondrial matrix (fractionation of
proteins onto chromatographic columns and in vitro reconstitution experiments of biotin synthase activity) together with a genomic-based search in
the Arabidopsis genome database resulted in the identification of an adrenodoxin-like protein (ADX1), an adrenodoxin reductase (ADR) and a cysteine
desulphurase (NFS1) as essential components for the reaction (Picciocchi
et al., 2003). ADX1 and ADR form a physiological reduction system, fuelling
the reaction with electrons from NADPH. The role of this system is to
sustain the reductive cleavage of S-adenosylmethionine (AdoMet), an obligatory cofactor of the reaction, through the [4Fe4S]2 centre of BIO2. This
two-step reaction, which generates AdoMet radical intermediates, is involved
in the activation of the CH bonds of the DTB substrate where the sulphur
atom is to be introduced, with production of 9-mercaptodethiobiotin as an
intermediate (Baldet et al., 1993b; Taylor et al., 2008; Tse Sum Bui et al.,
2004). Thus, BIO2 is part of the group of Adomet-radical enzymes to which
belongs a variety of enzymes using such radical intermediates, but for quite
different purposes (for a review, see Jarrett, 2005a). The in vitro stimulation
of biotin synthase activity by the NFS1 protein strongly supports the idea
that cysteine is the initial sulphur donor for biotin in plant mitochondria. The
desulphurase is thought to take part in the recycling of BIO2 activity by
providing a renewable source of sulphur for the reaction. The sulphur atom
may be attached to DTB via the [2Fe2S]2 cluster of BIO2, suggesting that
BIO2 is actually a real catalyst (Picciocchi et al., 2001, 2003), in contrast to
what was originally suggested from in vitro studies with bacterial biotin
synthase (Choi-Rhee and Cronan, 2005a,b; Jarrett, 2005b). In vitro formation of biotin at catalytic rates by biotin synthase has been recently confirmed
(Farrar et al., 2010). This work demonstrates that low in vitro biotin synthase
activities usually reported in the literature are mainly due to strong synergic
inhibition by both 50 -deoxyadenosine, an end-product of biotin synthase
reaction, and S-adenosyl-L-homocysteine, a major contaminant commonly
present in commercial AdoMet preparations. Different mechanisms have
been hypothesized to explain sulphur insertion into DTB. One mechanism
proposes that sulphide from the [2Fe2S]2 cluster is attached in a stepwise
manner to the C9 and C6 positions of DTB, with concomitant reduction and
loss of the residual cluster (Ugulava et al., 2001). An alternate mechanism
suggests that reduction and loss of the cluster precedes catalysis, and that
sulphur insertion is from an enzyme-bound cysteine persulphide that is
formed either during cluster degradation (Jameson et al., 2004) or via the
action of a cysteine desulphurase (Ollagnier-de-Choudens et al., 2002).

54
C. ALBAN
In addition to their implication in biotin synthase reaction, mitochondrial

ADX1/ADR redox system and NFS1 protein could be also involved in the
synthesis of the lipoate cofactor which also occurs in plant mitochondria
(Douce et al., 2001), the lipoate synthase and biotin synthase reactions being
mechanistically related. Also, ADX1, ADR and NFS1 and the yeast homologous proteins (namely Yah1p, Arh1p and Nfs1p, respectively) have been
identified as key components of mitochondrial ironsulphur cluster (ISC)
assembly machinery (Balk and Lobreaux, 2005; Lill and Muhlenhoff, 2005).
Consequently, these proteins could have a dual function, a specific function
in the biotin synthase and lipoate synthase reactions and a more general role
in biosynthesis of FeS clusters for other redox enzymes. Interestingly,
attempts to complement a bio2 mutant with a truncated version of BIO2
lacking the mitochondrial targeting sequence failed, even with provision
of the substrate DTB, suggesting that biochemical constraints, and the
apparent close connection with the mitochondrial FeS machinery,
may account for the reaction being retained within mitochondria (Arnal
et al., 2006).
III. PROTEIN BIOTINYLATION

As mentioned in Section I, biotin is a cofactor for some carboxylases dealing
with crucial metabolic processes such as fatty acid synthesis and carbohydrate metabolism. The biotinylation of these enzymes is a post-translational
modification allowing the transformation of inactive apo-proteins into their
active holo forms. Therefore, this original and very specific post-translational
modification can be considered as the ultimate step of the biotin biosynthetic
pathway, and as such, it was included in this review. The covalent attachment
of biotin is catalysed by biotin-protein ligase (BPL) also called HCS (EC
6.3.4.11-15). D-Biotin is attached to a specific Lys residue of newly synthesized apo enzyme, via an amide linkage between the biotin carboxyl group
and a unique e-amino-group of Lys residue (Samols et al., 1988). It occurs in
two steps (4) and (5) as follows:
D
- Biotin ATP ! D - biotinyl 50 -AMP PPi
- Biotinyl 50 -AMP apocarboxylase ! holocarboxylase AMP 5
In plants, four different biotin-dependent carboxylase activities have

been identified, two acetyl-CoA carboxylase activities, one in cytosol
and one in plastids; one geranoyl-CoA carboxylase in plastids; and one

55
methylcrotonoyl-CoA carboxylase in mitochondria (Alban et al., 2000).

Moreover, sequencing of the Arabidopsis genome confirmed the occurrence
of two genes encoding two distinct isoforms of acetyl-CoA carboxylase and
one gene for methylcrotonoyl-CoA carboxylase (Nikolau et al., 2003). As a
result, plants offer a unique case of triple compartmentalization of biotindependent carboxylases. HCS activity localization in plant cell parallels this
complexity. In pea leaf cells, HCS activity was mainly located in cytosol of
fractionated protoplasts, but a significant activity was also identified in both
highly purified chloroplasts and mitochondria (Tissot et al., 1997). In Arabidopsis cultured cells, HCS activity was also essentially recovered in cytosol of
fractionated protoplasts and, to a lesser extent, in chloroplasts and mitochondria (Puyaubert et al., 2008). This suggests that carboxylases are biotinylated in their compartment of residence. Two HCS genes have been
evidenced in Arabidopsis (Tissot et al., 1997). Firstly, HCS1 cDNA has
been isolated by functional complementation of an E. coli mutant (Tissot
et al., 1997). Subsequently, the systematic sequencing of Arabidopsis genome
enabled the identification of HCS1 gene. Moreover, it confirmed the existence of a second HCS gene (HCS2), localized in the pericentromeric region
of chromosome 1 (Arabidopsis Genome Initiative, 2000). HCS1 and HCS2
genes present very large similarities and probably result from the duplication
of a common ancestor gene (Denis et al., 2002). HCS1 presents a broad
specificity of substrates and is able to biotinylate efficiently in vitro all
recombinant biotin-dependent apo-carboxylases identified in Arabidopsis
and E. coli, as well as the seed-specific biotinyl protein, SBP, albeit to a
lower extent (Denis et al., 2002; Puyaubert et al., 2008; Tissot et al., 1996,
1998). Interestingly, HCS2 expression produces a highly diverse family of
alternatively spliced mRNAs (Denis et al., 2002). However, none of the
putative HCS2 proteins, produced by alternative splicing of HCS2, were
active in vitro when overproduced in E. coli, nor rescued an E. coli mutant
affected in protein biotinylation (Denis et al., 2002). Moreover, reverse
genetics studies evidenced that HCS1 gene is essential for plant viability,
whereas disruption of HCS2 gene in Arabidopsis does not lead to any
obvious phenotype when plants are grown under standard conditions.
These findings suggested that HCS1 is the only protein responsible for
HCS activity in Arabidopsis cells, including the cytosolic, mitochondrial
and plastidial compartments. A close scrutiny of HCS1 gene expression
and splicing enabled Puyaubert et al. (2008) to propose an original mechanism to account for this multiplicity of localizations. Located in HCS1
mRNA 50 -untranslated region, an upstream open reading frame (uORF)
regulates the translation initiation of HCS1 and the subsequent targeting
of HCS1 protein. Moreover, an alternative splicing of HCS1 mRNA can

56
C. ALBAN
regulate the presence and absence of this uORF, thus controlling organelle
versus cytosolic localization of HCS1 gene product (Fig. 6). This provides a
possibility for fine molecular regulation and, beyond the specific issue of
HCS1 protein, unveils the general complexity of plant metabolism compartmentalization. The physiological role of HCS2 gene is much less clear. HCS2
gene does not seem to bear any fundamental function in carboxylases biotinylation in plants. It has been proposed that HCS2 could be an inactive
pseudogene in Arabidopsis or may have a regulatory function as a noncoding RNA (Puyaubert et al., 2008). Alternatively, HCS2 proteins might
be involved in histones biotinylation. Indeed, beside its classical role in
carboxylases biotinylation, evidence is emerging that HCS in mammalian
cells nuclei participates in the epigenetic control of chromatin structure and
gene expression, through biotinylation of histones (Narang et al., 2004;
Zempleni, 2005). However, these conclusions are matter of debate and
controversy, and it is not clear whether histones are truly biotinylated
in vivo or not. Indeed, to date, no direct evidence for the existence of natural
biotinylated histones, from mass spectroscopic analyses, for example, has
been provided. All available data rely on secondary detection systems such as
streptavidinHRP, and/or on in vitro biotinylation assays using recombinant
mammalian HCS. A recent study has called into question the reliability of
streptavidin detection of biotin on histones. It concluded that binding
of streptavidin to histones occurs independently of the biotin-binding site
on streptavidin (Bailey et al., 2008). Also, Healy et al. (2009) critically
examined a number of methods used to detect biotin attachment on histones,
including [3H]-biotin uptake, Western blot analysis of histones and mass
spectrometry of affinity-purified histone fragments with the objective of
determining if the in vivo occurrence of histone biotinylation could be definitively established. Their conclusion was that biotin is not a natural histone
modification. Our initial efforts to demonstrate in vivo plant histones biotinylation have also not been successful (Claude Alban, unpublished data).
For example, treatment of Arabidopsis cultured cells with [3H]-biotin specifically labelled biotin-dependent carboxylases, but no [3H]-biotin incorporation by histones could be evidenced (Fig. 7A). On the other hand, plant
histones were poor substrates for in vitro biotinylation by Arabidopsis HCS,
compared to carboxylases. Further, since similar low levels of biotin incorporation into unrelated basic proteins (with pKa > 9, i.e. comparable to those
of histone proteins), such as lysosyme (Fig. 7B), RNAse A or cytochrome c,
were also measured, this suggested that in vitro biotinylation of histones by
plant HCS is also artefactual. Collectively, these data suggest that the wellestablished regulatory impact of biotin on gene expression in eukaryotes
must be through alternate mechanisms. For example, in mammals an

57
Chromosome 2
Nucleus
Transcription and splicing
HCS1
ATG
0 1
Transcription
HCS1 gene
2
1
AUG
0 1
TGA
6
HCS1 mRNAs 1
10
Splicing
HCS1.s
HCS1.un
HCS1.un
HCS1.s
Cytosol
Translation and targeting
HCS1.s
HCS1.un
Translation
100%
Re-initiation HCS1
HCS1 proteins
TP
HCS1
TP-HCS1
HCS1
Targeting
HCS1
HCS1
Cytosol
Plastids
HCS1
Mitochondria
= UpstreamORF
HCS1.un = Unspliced HCS1 mRNA
HCS1.s = Spliced HCS1 mRNA

TP = Transit peptide
= Ribosome
Fig. 6. A model of uORF-mediated translational control and HCS1 compartmentalization in Arabidopsis cells. HCS1 gene is represented in chromosome 2 of
Arabidopsis thaliana. Following its transcription, alternative splicing produces two
mRNA variants HCS1.un (unspliced) and HCS1.s (spliced). After their export into
the cytosol, HCS1.un and HCS1.s are translated. HCS1.un produces a short protein
starting at AUG2, which by eluding the transit peptide, leads to a cytosolic localization. HCS1.s produces a longer protein starting at AUG1 and dual-targeted into the
plastids and mitochondria. Boxes figure a schematic view of the molecular mechanisms controlling this sketch of events in the nucleus and the cytosol. When HCS1 50 UTR is unspliced, the persistence of the uORF (starting at AUG0) disengages the
ribosomes from the mRNA. They fail to reinitiate at the close AUG1: translation
starts from AUG2 and produces a cytosolic HCS1. When HCS1 50 -UTR is spliced
out, uORF inhibition on translation initiation at AUG1 is abolished. Translation
starts from AUG1 and produces a HCS1 protein headed by a transit peptide.

58
ble
one
s
Anti-biotin-HRP
Hist
Solu
prot
eins
Solu
ble
prot
eins
Hist
one
s
H is
to
Lys nes
o
BC zyme
CP
2
H is
ton
Lys es
o
BC zyme
CP
2
C. ALBAN
97
66
45
31
21
Ponceau stain
14
Coomassie blue
3H-phosphor
15 min
2h
Incubation time with HCS1
Fig. 7. Attempts in biotinylation of plant histones by Arabidopsis HCS1 (Claude

Alban, unpublished data). (A) [3H]-biotin uptake into Arabidopsis cultured cells.
Arabidopsis cells were treated for 4 days with 0.5 mM acidomycin, an inhibitor of
biotin synthesis, in order to deplete the endogenous pool of biotin, and then cultured
for 8 days in the presence of 0.1 M [3H]-biotin (25 Ci/mmol). Cells were then washed,
resuspended into fresh culture medium supplemented with acidomicine and finally
cultured for 10 more days. Total soluble proteins and histone proteins were extracted
and analysed by SDS-PAGE, electrotransfer onto PVDF membrane and exposition
of the membrane to a tritium-specific phosphor screen for 11 weeks. Detection of
radioactive bands was performed by scanning the screen on a phosphoimager. The
molecular weight of the observed bands matches that of carboxylases. No radioactive
bands for histones were detected. Molecular mass markers (M) values are given on the
left in kDa. (B) Substrate specificity of Arabidopsis HCS1. The enzyme was incubated
for 15120 min in the presence of biotin and apo-BCCP2 (the biotinyl subunit of
Arabidopsis acetyl-CoA carboxylases in its apo-form), extracted Arabidopsis histones
or lysosyme as protein substrates. Biotinylated proteins were analysed by western
blotting using anti-biotin-HRP as a probe and chemifluorescence detection.
alternative route involving a cGMP-dependent signalling cascade has been

proposed. This mechanism requires HCS, guanylate cyclase and cGMPdependent protein kinase (Solorzano-Vargas et al., 2002).
IV. CONCLUDING REMARKS

The past few years have seen dramatic advances in our understanding of the
enzymes that manipulate biotin in plants, including the characterization
of biotin-containing carboxylases, biotin synthesizing enzymes and BPLs.

59
Most of these proteins have been purified and/or their genes cloned and
characterized. With these achievements, a better understanding of the regulation and of the interconnection between these different pathways is now
possible. A new challenge will be also to discover other cell functions for
biotin, especially in regard to the identification of novel biotinylated proteins
differing from the well-characterized carboxylases.
As it was underlined in this review, enzymes involved in biotin metabolism
are scattered among cell compartments, with mitochondria playing a central
role (Fig. 8). Such complex situation involving several compartments of the
plant cell is also found in other plant vitamin pathways such as those of
folates, ascorbate, pantothenate, niacin or phylloquinones (Lunn, 2007;
Rebeille et al., 2007). This highlights the complexity and the peculiarity of
plant metabolism. The complex compartmentalization of biotin, biotinmediated reactions and biotin synthesis in the plant cell implies an intracellular trafficking of biotin and precursors (Fig. 8). Biotin synthesis requires at
Mitochondrion
Cytosol
KAPA
KAPA
Pimeloyl-CoA
BIO4
AdoMet
BIO1
DAPA
Plastid
BIO3
ADR
ADX1
Biotinyl-protein
Dethiobiotin
AdoMet
BIO2
NFS1
Biotin
HCS1
Biotin
HCS1
Biotinyl-protein
Biotinyl-protein
biotin
HCS1
HCS1 gene
Nucleus
Fig. 8. Compartmentation of biotin metabolism in the plant cell. Biotin synthesizing enzymes are KAPA synthase (BIO4); bifunctional DAPA synthase (BIO1)/
dethiobiotin synthetase (BIO3) (BIO3BIO1 protein); biotin synthase (BIO2) associated with stimulatory proteins as redox partners ADR and ADX1, and cysteine
desulphurase NFS1. Protein biotinylation in both the organelles and the cytosol is
mediated by HCS1 protein variants originating from the same gene (HCS1)

60
C. ALBAN
least one mitochondrial transporter to permit the counter-exchange of

KAPA (synthesized in the cytosol) and biotin. Indeed, once synthesized,
biotin must be exported outside mitochondria to cytosol and to chloroplasts
for protein biotinylation reactions. In plants, only one biotin transporter has
been identified so far. Arabidopsis AtSuc5 is a plasma membrane sucrose/
biotin co-transporter, possibly involved in biotin uptake and allocation
between the various plant tissues (Ludwig et al., 2000). Intracellular trafficking of biotin and intermediates is, however, largely unknown, and the
proteins responsible for these activities have never been isolated or identified
hitherto. Interestingly, searches of the Arabidopsis genome database detected
a gene (At2g01170) encoding a predicted protein with about 45% similarity
to the protein sequence of S. cerevisiae Bio5p (Claude Alban, unpublished
observation). Bio5p is a KAPA/DAPA translocator found in biotin auxotrophic yeast strains (Phalip et al., 1999). The expression of the plant protein
into S. cerevisiae Bio5 mutant partially complemented yeast growth in the
presence of KAPA but not DAPA, suggesting that this protein could be
involved in KAPA transport in plant cells (Claude Alban, unpublished
observation). Also, synthesis of biotin depends on the presence of AdoMet
in plant mitochondria. In plant cells, AdoMet is synthesized in the cytosol
and a specific carrier is thus required to ensure the import of AdoMet into the
mitochondrial matrix (Palmieri et al., 2006). Understanding how
these carriers operate is particularly challenging because they are key
elements to understand how different parts of the pathway are co-ordinately
regulated.
In conclusion, recent and forthcoming advances in plant biotin metabolism comprehension will allow for more rational and directed efforts at
manipulation of biotin pathway by genetic engineering. Indeed, some of
the processes that involve biotin generate biochemicals that serve a broad
range of nutritional and industrial purposes. For example, plant storage oils,
the biosynthesis of which requires acetyl-CoA carboxylase, are a major
resource for both human and animal nutrition, and also for a number of
non-food uses including pharmaceutical, cosmetics, detergents and even
biofuels. Biotin itself is also added to many food, feed and cosmetic products,
but it is mainly produced in a chemical process. Thus, the production of
biotin in natural and particularly plant environments as compared to synthetic chemistry might be advantageous, especially for meeting positive
public acceptance. Finally, we anticipate that the future elucidation of
the structure of the enzymes of the plant biotin synthesis pathway will
allow the design of new inhibitor families having herbicidal activities, affecting plants in a specific manner and therefore having a lower impact on the
environment.

61
ACKNOWLEDGEMENTS
Dr. Olivier Bastien is gratefully acknowledged for his invaluable expertise in
phyllogenetic tree construction. I thank all my collaborators and students
who were involved in the plant biotin metabolism project. Finally, I would
like to thank Professor Roland Douce for his indefectible enthusiasm and the
exciting discussions we have had during the past 25 years.
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Biotin (Vitamin B8) Synthesis

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Biotin (Vitamin B8) Synthesis in Plants

*Laboratoire de Physiologie Cellulaire Vegetale, CNRS,

Corresponding author: E-mail: claude.alban@cea.fr

Advances in Botanical Research, Vol. 59

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Biotin, also known as vitamin H or B8, is a cofactor for some carboxylases,

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The structure of biotin.

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The biotin cycle in mammalian cells.

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Because of the dual nature of biotin in living cells, dietary biotin is

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Compilation of values by Combs (1998b) and Bonjour (1991).

biotinylated proteins. All these enzymes play crucial cellular housekeeping

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Enzyme - Biotin CO

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II. THE BIOSYNTHETIC PATHWAY

bioF (BIO4), KAPA synthase

7-keto-8-aminopelargonic acid (KAPA)

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precursors (Baldet et al., 1993b). An alternative approach used to study

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methylated by BioC, which allows recognition of this atypical substrate by

B. 7-KETO-8-AMINOPELARGONIC ACID SYNTHASE

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distribution of KAPA synthase as determined by two independent methods.

C. 7,8-DIAMINOPELARGONIC ACID SYNTHASEDETHIOBIOTIN SYNTHETASE

The antepenultimate step in the biotin biosynthetic pathway, the conversion

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in most ascomycete and basidiomycete fungi (Hall and Dietrich, 2007;

Vitis vinifera (XM_002270515.1)

Hydrogenobaculum sp. Y04AAS1 (ACG57182.1)

Fig. 4. Phylogenetic analysis of eukaryotic bifunctional DTB synthetase/DAPA

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early in the evolution of modern-day eukaryotes. By contrast, in the large

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DTB AdoMet S ! Biotin 50 - deoxyadenosine L - methionine

Fig. 5. (A) Structure of Arabidopsis BIO2 monomer modelized by Swiss-model

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elicit biotin formation at catalytic rates in the plant reconstituted system, in

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In addition to their implication in biotin synthase reaction, mitochondrial

III. PROTEIN BIOTINYLATION

- Biotin ATP ! D - biotinyl 50 -AMP PPi

- Biotinyl 50 -AMP apocarboxylase ! holocarboxylase AMP 5

In plants, four different biotin-dependent carboxylase activities have

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methylcrotonoyl-CoA carboxylase in mitochondria (Alban et al., 2000).

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BIOTIN (VITAMIN B8) SYNTHESIS IN PLANTS

HCS1.s = Spliced HCS1 mRNA

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Incubation time with HCS1

Fig. 7. Attempts in biotinylation of plant histones by Arabidopsis HCS1 (Claude