Biochemical characterization

of predicted Precambrian
RuBisCO
Prado, Arturo Jr. A
Far Eastern University Manila
Sampaloc, Manila , Metro Manila

Introduction
Precambrian (0.5424 Gyr ago)
environments is limited to inferences gained
from geological proxies. However, the
inherent property of evolution in biological
systems offers a means by which ancient
proteins can be probed to gain fresh insight
into the corresponding palaeoenvironments.
One particular enzyme that has had
profound implications for life on Earth is
RuBisCO,
the
predominant
enzyme
responsible for carbon fixation, whose
activity over billions of years has linked
biological systems to their inorganic
environment through global biogeochemical
cycles. Form 1 RuBisCO is composed of
eight large (LSUs) and eight small (SSUs)
subunits, forming a hexadecameric (L8S8)
holoenzyme.
RuBisCO
exhibits
dual
substrate specificity (CO2 versus O2).
Carboxylation of the acceptor
molecule, ribulose-1,5-bisphosphate (RuBP)
generates
two
molecules
of
3phosphoglycerate that are used in the
regeneration of RuBP and synthesis of
biomolecules. Oxygenation of RuBP
generates
one
molecule
of
3phosphoglycerate and one molecule of 2phosphoglycolate, a toxic metabolite that is
catabolized through photorespiration. Loss
of CO2 and energy through photorespiration

decreases the efficiency of carbon fixation,

yet the oxygenase activity of RuBisCO has
persisted across all known forms of
RuBisCO.
The decreasing CO2 and increasing
O2 atmospheric concentrations over Earths
history, changes in the proportions of these
two gases have been suggested to be the
major evolutionary force driving RuBisCO
activity towards higher carboxylation activity.
In this study, I reconstructed
ancestral versions of RuBisCO based on
phylogenetic analyses. The predicted
protein sequences were then resurrected
using gene synthesis to probe the
biochemical properties of these ancient
enzymes. I expressed ancestral LSUs in an
extant cyanobacterial strain to determine
whether they could be incorporated into the
carboxysome, the core component of the
cyanobacterial
carbon
concentrating
mechanism (CCM) that evolved in response
to the changing atmosphere and is essential
for survival at current CO2 levels. I findings
are presented in the context of the
Precambrian palaeoatmosphere.

Methods
Alignment and phylogeny
A total 125 RbcL and 131 RbcS
sequences spanning the Form 1 RuBisCO

were calculated for all amino-acid residues

clade

structural

across the sequence, and the residue with

information from a variety of RuBisCO

the highest probability was assigned to each

crystal structures in the Protein Data Bank

site. Predicted ancestral sequences are the

(PDB IDs: 1SVD, 1IR1, 1GK8, 1RBL) using

same length as the alignment used; this

the

Maximum-likelihood

may result in ancestral sequences that are

generated

using

longer than they should be. To address this

PhyML with 100 bootstrap replicates. The

issue, Hall describes a method used to

LG amino-acid substitution model was

estimate the positions of ancestral gaps

chosen based on ProtTest with gamma-

owing to insertions and deletions, where

distributed variation (four categories) and

gaps were coded as g and amino acids

estimation of a proportion of variable sites.

were coded as a to predict the inclusion or

The tree was rooted to the Form 1C and 1D

exclusion of certain positions. The ancestral

monophyletic subclade.

sequence reconstruction of insertions and

were

aligned

PROMALS3D.

phylogenies

were

using

deletions was implemented using BaseML

Ancestral sequence reconstruction

Maximum-likelihood
implemented

in

PAML

methods
were

used

to

resurrect ancestral sequences. Sequences

at internal nodes of interest (-MRCA, MRCA and /-MRCA) from both the RbcL
and RbcS phylogeny were inferred by
maximum likelihood using CodeML within
the PAML package. The alignment and the
maximum-likelihood phylogenies generated
by PhyML were used as input files for
CodeML. PAML utilizes a discrete-gamma
model, and sequences were reconstructed
with the LG substitution model, based on
ProtTest results. The parameter was fixed
with the default parameter of 0. Posterior
probabilities

of the PAML package, using the JC69

model

and

maximum-likelihood

phylogenetic trees used above as input

files. Positions that were predicted to be
ancestral gaps were omitted from the final
ancestrally reconstructed sequences, as
described by Hall.

example, through the formation of a C4 acid

by combination of CO2 with a C3 acid).
Protein purification
Reconstructed
synthesized

and

sequences

were

codon-optimized

for

RuBisCO activity assay

For determination of the Vmax and KM for

expressionin E.coli (Genscript).

the carboxylase (Vc and Kc, respectively)

Corresponding

RbcL

and

RbcS

and

of

theKM for

the

oxygenase

sequences were subcloned into the pET11a

activities,

vector as previously described. Modified

cyanobacterial and ancestral -MRCA and

pET11a vectors and pBAD33ES/EL were

-MRCA RuBisCO were rapidly extracted

co-transformed
cells.

into E.

previously

preparations

of

(Ko)

extant

coli BL21

(DE3)

from E.coli cultures at 0C by sonication,

described

strain

followed by debris removal by centrifugation

pET101/D-

and passage through PD-10 (Sephadex G-

of E. coli co-transformed

with

TOPO MIT9313 cbbL/S and pGroESL was

25)

used

equilibrated with 0.1M BicineNaOH pH

to

heterologously

express

columns

(GE

Healthcare)

pre-

8.0, 10mM MgCl2, 1mM EDTA, 1mM -

the Prochlorococcus RuBisCO.

aminocaproic acid, 1mM benzamidine, 1

The Synechococcus RuBisCO

was

mM KH2PO4, 2% (w/v) PEG-4000, 10mM

heterologously expressed as previously

NaHCO3 and 5mM DTT. Fractions (0.5ml)

described using the vector pAn92 in the

with the most protein were combined (1.0

expression strain BL21 (DE3). Protein

1.5ml

expression was performed as previously

immediately in liquid nitrogen, until assayed.

described. Assays for the determination of

These

the Vmax and KM for

controls to establish (a) the dependence of

the

carboxylase

in

total)

assays

were

and

frozen/stored

accompanied

by

(Vc and Kc, respectively) and of the KM for

the

the

were

presence of RuBP; (b) the abolition of this

accompanied by controls to establish (a) the

activity by pretreatment of the extract with

dependence of the measured acid-stable C

an excess of CABP; and (c) that products of

on the presence of RuBP; (b) the abolition

the carboxylase activity are not themselves

of this activity by pretreatment of the extract

the cause of acid-stable C (for example,

with an excess of CABP; and (c) that

through the formation of a C4 acid by

products of the carboxylase activity are not

combination of CO2 with a C3 acid).

oxygenase

(Ko)

activities

themselves the cause of acid-stable C (for

measured

acid-stable C

on

the

For
factor,

determination
purer,

specificity

developed with (50mM BicineNaOH pH 8,

concentrated

20mM MgCl2, 0.2mM EDTA, 2mM DTT).

of

more

preparations of extant cyanobacterial and

The

ancestral -MRCA and -MRCA RuBisCO

concentrated by ultrafiltration using 20ml

were

capacity/150kDa

necessary,

necessitating

prior

resulting

protein

peak

cut-off

was

centrifugal

purification on a larger scale than necessary

concentrators (Thermo Pierce). The quality

for the kinetic determinations.

of preparations was verified by SDS

polyacrylamide

All steps in the purification process

were conducted at 04C. The harvested

gel

electrophoresis

and

confirmed by western blot using antisera

raised against wheat RuBisCO.

cultures expressing all forms of RuBisCO

were

sonicated

and

clarified

by

Measurement

of Vc, Kc and Ko in

centrifugation, as before. PEG-4000 and

solutions equilibrated with nitrogen, oxygen,

MgCl2 were

resulting

air (79% N2, 21%O2) and 40% N2, 60% O2,

supernatants giving final concentrations of

were as described elsewhere. After careful

20% (w/v) and 20mM, respectively. After 30

thawing, RuBisCO preparations were kept

min at 0C, the PEG/MgCl 2 precipitated

at 0C for 60min to ensure full activity, after

RuBisCO was sedimented by centrifugation

which 1-min assays were conducted in

(20min

pellet

glass scintillation vials, fitted with caps and

resuspended in 25mM triethanolamine (pH

silicone rubber septa, in a final volume of 1

7.8, HCl), 5mM MgCl2, 0.5mM EDTA 1

ml at 25C. At each O2 concentration, six

1mM

different concentrations of bicarbonate were

benzamidine, 12.5% (v/v) glycerol, 2mM

used, chosen to provide CO2 (aq) between 5

DTT and 5mM NaHCO3. This material was

and

subjected

radioactivity

at

added

to

22,095g)

mM -aminocaproic

the

and

the

acid,

to

anion-exchange

of

each
3.7

with

specific

10 Bqmol1 and
10

containing 375nmols RuBP and 25l of the

columns (GE Healthcare) pre-equilibrated

protein extract. Values of Ko were calculated

with the same buffer. RuBisCO was eluted

from the relationship:

chromatography

using

5ml

550M,

HiTrap

with a 0600mM NaCl gradient in the same

most

Kc (at stated O2 concentration)=Kc (in

RuBisCO activity (as judged by RuBP-

N2) (1+([O2]/Ko)). Estimates of Vo were

dependent CO2 assimilation) were further

derived from the equation, =[Vc/Kc]/[Vo/Ko],

purified and desalted by size-exclusion

the specificity factors () having been

chromatography using a 20cm 2.6cm

determined separately (see below). The

diameter column of Sephacryl S-200 HR

concentration of RuBisCO active sites was

(GE

determined using [C]CABP.

buffer.

Fractions

Healthcare)

containing

the

pre-equilibrated

and

at 30C. Wild-type cells were transformed

and selected for on BG11 plates with
antibiotics.

For

co-localization

studies,

strains expressing CFP-tagged ancestral

RbcL were subsequently transformed with
The specificity factor for -MRCA
and -MRCA RuBisCO was determined at
25C by the total consumption of RuBP in
an

oxygen

electrode,

as

described

previously. A series of assays containing

pure wheat RuBisCO were interspersed
with

those

containing

the

ancestral

RuBisCO, and the results normalized to the

average

value

obtained

from

wheat

RuBisCO (100.0 at 25C). Comparisons

between this kinetic data and that of extant
forms of RuBisCO are summarized.

BioBrick Assembly standard 21 (BglBrick

E.

coli and

subsequently cloned into neutral site vector

pAM1573PMS for genomic integration into
the Synechococcus genome at Neutral Site
2.

pAM1573PMS

was

modified

from

pAM1573 (ref.62) to be BglBrick compatible.

Ancestral RbcL sequences were fused to a
carboxy (C)-terminal cerulean fluorescent
protein (CFP) and constitutively expressed
with the rplC promoter, as described by
Savage et.al.
Wild

enhanced yellow fluorescent protein driven

with

CcmK2

promoter.

This

vector

(pAM2314PMS) is a modified version of a

BglBrick-modified

version

of

pAM2314

(ref. 62) and mediates genomic integration

into the Synechococcus genome at Neutral
Site 1 describes the various strains used in
this study.
Fluorescence microscopy
were spotted on to 1% agarose pads (w/v in

All constructs were cloned using

format in

protein (CcmN) fused to a C-terminal

Cells grown on solid BG11 media

Synechococccus strains

assembly)

another vector for expressing carboxysomal

type Synechococcus was

grown in BG11 medium with constant light

BG11) in a 16-well chamber slide (Lab-Tek,

Scotts Valley, CA) and covered with a 0.17
mm coverglass. Images were acquired on a
Zeiss

LSM

710

inverted

confocal

microscope (Carl Zeiss Inc, Thornwood,

NY) using laser lines at 405, 514 and 633
nm and a 63/1.4 NA oil-immersion
objective. Images were captured using Zen
2010 (Carl Zeiss, Inc.) and analysed using
ImageJ.

were able to purify ancestral RuBisCO

enzymes from the -MRCA and -MRCA.
Soluble /-MRCA enzyme could not be
recovered, possibly due to misfolding or
misprediction of the ancestral sequence;
this is not surprising owing to the fact that
this is the most remote of the three

Results

ancestral variants synthesized. Remarkably,

purified

Biochemical characterization of ancestral

RuBisCO
520 kiloDalton hexadecameric L8S8 -MRCA
To

investigate

the

biochemical

and -MRCA

holocomplexes

were

fully

properties of ancestral RuBisCO, the genes

assembled as determined by size-exclusion

were synthesized, expressed in Escherichia

chromatography and were enzymatically

coli (E. coli), and the resulting proteins

active.

subsequently purified (further described in

the Methods section). In addition, Form 1A

Examining

and Form 1B bacterial RuBisCOs from

ancestral RuBisCO and CCM

Prochlorococcus

marinus MIT9313

and Synechococcus sp.

PCC6301,

the

evolutionary

context

of

were

Given their biochemical properties,

and

extant cyanobacterial RuBisCOs could still

enzyme

perform photosynthesis efficiently under

comparisons. Before this study, only one

early Phanerozoic CO2 (1520 times PAL)

Form 1A RuBisCO had ever been fully

and O2 (at PAL) concentrations. Thus, it has

characterized (from Chromatium vinosum);

been hypothesized that CCMs were not

thus, the full biochemical characterization of

necessary and did not evolve until ~0.4Gyr

the Prochlorococcus RuBisCO

provides

ago. Owing to the genetic complexity and

much needed additional data on Form 1A

prerequisites needed to evolve the multiple

RuBisCOs.

components involved in CCMs (for example,

heterologously

expressed,

assayed,

provide

to

purified

extant

transporters, carboxysomes), the factors

Although most ancestral sequence
reconstruction

studies

have

focused

most directly amenable to evolution in

response

to

the

drastically

changing

primarily on monomeric proteins owing to

atmosphere would have been the catalytic

the potential challenges of reconstituting

properties of RuBisCO. Consistent with this

ancestral proteinprotein interactions, we

evolutionary scenario, extant cyanobacterial

RuBisCOswhich operate in concert with a

the

CCMdisplay high Vc and low specificity

examined the distribution of fluorescently

factor values, which would complement the

tagged

ability

carboxysomal subunit, CcmN, fused to a

of

the

CCM

to

increase

the

CO2/O2ratios within the cell.

carboxysome,

we

ancestral

LSU

simultaneously
and

of

the

spectrally distinct fluorescent tag, within the

-carboxysome

of

an

extant

cyanobacterium, Synechococcus
elongatus PCC 7942. All tagged ancestral
RbcL subunits displayed spatially discrete
To further examine the origin of
CCMs, we focused on the carboxysome, the
core component of the bacterial CCM. The
carboxysome is an organelle composed of
an array of proteins, including RuBisCO and
carbonic anhydrase, encapsulated in a
protein shell that functions to increase the
local CO2 concentration around RuBisCO.
Cyanobacteria encapsulate the majority of
the

cellular

RuBisCO

carboxysomes

or

within -

-carboxysomes,

containing Form 1A and Form 1B RuBisCO,

respectively.
Because RuBisCO is packed into
the

carboxysome

through

an

ordered

assembly pathway, we utilized the unique

reference point of our ancestral sequences
to probe the evolutionary history of the
CCM. Assembly of multi-protein complexes
are under strong evolutionary pressure, thus
we aimed to determine whether ancestral
RuBisCOs could be encapsulated within the
carboxysomes

of

extant

cyanobacteria.

Because the LSU has been shown to

determine holoenzyme encapsulation within

fluorescent

puncta

across

coincident

with

CcmN:

described

signature

of

the

cell,

previously

carboxysome

association. Our results indicate that the

ancestral

LSUs

are

able

to

be

encapsulated; selection for this property

may have enabled successful lineages to
avoid

extinction

during

Proterozoic atmosphere.

the

changing

studies are based on the uniformitarian

assumption

that

discrimination

values

displayed by extant enzymes are similar to

those in the geological past. The isotope
ratios of sediments that have long been
believed to contain the isotopic signature of
ancient forms of photosynthetic metabolism
are already know. It will be interesting to see
whether data from the ancestral RuBisCOs
will corroborate these isotope ratios, thus

Discussion
Here,

we

cross-

confirming the uniformitarian assumption.

disciplinary study addressing the nature of

However, given that the measured kinetics

the

of ancestral RuBisCOs differ from extant

geologically

enzyme,

present
and

RuBisCO.

bioinformatic
predictive

globally

characterize

relevant

Phylogenetic

analysis
basis

to

and

enzymes, it would be surprising if these

provided

the

proteins fractionated to the same degree as

synthesize

and

their modern counterparts.

the in

vitro and in

vivo biochemical properties of resurrected

enzymes, thus allowing the results to be
integrated with information gathered from
the geological and fossil record. Even with a
perfect sampling of all existing extant
sequences, the empirically derived models
used to reconstruct the ancestral sequences
are unlikely to be perfect. However, future
efforts to characterize the C/C isotope
discrimination of ancestral RuBisCOs may
allow testing against the C isotope data
from

the

geological

record.

Isotopic

compositions have long been used to infer

the

contribution

of

various

metabolic

pathways and enzymes to the geochemical

composition of ancient biospheres. These

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