Cyanobacterial CP12 Proteins Journal research area … · Biochemical Processes and Macromolecular ... different types based on primary structure ... GAPDH can undergo a thiol-disulfide

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Cyanobacterial CP12 Proteins

Journal research area most appropriate for the paper: Bioenergetics and Photosynthesis or Biochemical Processes and Macromolecular Structures

Corresponding author: Cheryl Kerfeld

Mailing address: Department of Energy Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, CA 94598. Phone: (925) 296-5691. E-mail: [email protected]

Plant Physiology Preview. Published on November 26, 2012, as DOI:10.1104/pp.112.210542

Copyright 2012 by the American Society of Plant Biologists

www.plantphysiol.orgon September 8, 2018 - Published by Downloaded from Copyright © 2012 American Society of Plant Biologists. All rights reserved.

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Comparative Analysis of 126 Cyanobacterial Genomes Reveals Evidence of Functional Diversity Among Homologs of the Redox-Regulated CP12 Protein

Desirée N. Stanleya,b, c, Christine A. Rainesd and Cheryl A. Kerfelda,b,e,*

a DOE Joint Genome Institute

b Department of Plant and Microbial Biology – University of California, Berkeley

c Present Address: Department of Biochemistry – University of California, San Francisco

d School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, UK

e Berkeley Synthetic Biology Institute



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Footnotes:

Financial support:

CAK and DNS acknowledge the support of the NSF (MCB 0851094 and EF1105897). This work of CAR was underpinned by BBSRC grant P19403 and the University of Essex Research Promotion Fund.

Corresponding author: Cheryl Kerfeld ([email protected])



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Abstract

CP12 is found almost universally among photosynthetic organisms where it plays a key role in

regulation of the Calvin cycle by forming a ternary complex with GAPDH (glyceraldehyde 3-

phosphate dehydrogenase) and PRK (phosphoribulokinase). Newly available genomic

sequence data for the phylum Cyanobacteria reveals a heretofore unobserved diversity in

cyanobacterial CP12 proteins. Cyanobacterial CP12 proteins can be classified into eight

different types based on primary structure features. Among these are CP12-CBS (cystathionine-

β-synthase) domain fusions. CBS domains are regulatory modules for a wide range of cellular

activities; many of these bind adenine nucleotides through a conserved motif that is also present

in the CBS domains fused to CP12. In addition, a survey of expression datasets shows that the

CP12 paralogs are differentially regulated. Furthermore, modeling of the cyanobacterial CP12

protein variants based on the recently available three-dimensional structure of the canonical

cyanobacterial CP12 in complex with GAPDH suggests that some of the newly identified

cyanobacterial CP12 types are unlikely to bind to GAPDH. Collectively these data show that, as

is becoming increasingly apparent for plant CP12 proteins, the role of CP12 in cyanobacteria is

likely more complex than previously appreciated, possibly involving other signals in addition to

light. Moreover, our findings substantiate the proposal that this small protein may have multiple

roles in photosynthetic organisms.



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INTRODUCTION

CP12 is a small (~80 amino acid) protein present in most photosynthetic organisms,

including cyanobacteria, diatoms, red and green algae, and higher plants (Pohlmeyer et al.

1996; Wedel and Soll 1998; Oesterhelt et al. 2007; Erales et al. 2008a; Groben et al. 2010). In

eukaryotic organisms CP12 is located in the chloroplast, where the only function thus far

identified is in the regulation of the Calvin cycle in response to changes in light availability by

reversibly binding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and, subsequently,

phosphoribulokinase (PRK) (Wedel et al. 1997; Graciet et al. 2003a; Graciet et al. 2003b; Marri

et al. 2005a; Erales et al. 2008b; Howard et al. 2008; Marri et al. 2008; Carmo-Silva et al. 2011).

The formation of the GAPDH-PRK-CP12 complex inhibits GAPDH and PRK activity leading to

down-regulation of the Calvin cycle; this has been demonstrated in plants, Chlamydomonas

reinhardtii and cyanobacteria (Wedel et al. 1997; Graciet et al. 2003b; Marri et al. 2005b; Tamoi

et al. 2005). The GAPDH-PRK-CP12 complex dissociates under reducing conditions mediated

by thioredoxin, thereby restoring GAPDH and PRK activity (Lebreton et al. 2003; Marri et al.

2005b; Howard et al. 2008; Marri et al. 2009). Erales et al. (2008b) showed that CP12 binds

aldolase, another Calvin cycle enzyme, implying involvement of CP12 in additional Calvin cycle

regulation. Further studies in higher plants have proposed a role for CP12 outside of Calvin

cycle regulation. CP12 is expressed in non-photosynthetic A. thaliana tissues, and anti-sense

suppression of tobacco CP12 disrupts a variety of developmental processes (Singh et al. 2008;

Howard et al. 2011a; Howard et al. 2011b). Moreover, higher plant genomes encode up to three

forms of CP12, which are differentially expressed, but all have been shown to bind GAPDH and

PRK (Singh et al. 2008; Marri et al. 2010).

In CP12 disulfide bonds between two pairs of cysteine residues create peptide loops. In

vitro analysis of Arabidopsis thaliana CP12 mutants has shown that both pairs of redox-

regulated cysteine residues are required for ternary complex formation: an N-terminal pair for

PRK binding and a C-terminal pair for GAPDH binding (Wedel et al. 1997). The red alga

Galdieria sulphuraria CP12 does not have an N-terminal cysteine pair and, although the

GAPDH-PRK-CP12 complex forms, PRK is not completely inactivated (Oesterhelt et al. 2007).

In the cyanobacterium Synechococcus elongatus PCC7942 (hereafter S. elongatus), the N-

terminal cysteine pair is not required for PRK binding (Tamoi et al. 2005). In the green alga C.

reinhardtii, CP12 and GAPDH have complex interactions centered on the cysteine pairs.

GAPDH can undergo a thiol-disulfide exchange reaction with CP12, cleaving the C-terminal



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disulfide bond (Erales et al. 2009a). CP12 has also been shown to regulate the Calvin cycle in a

redox-independent manner, acting as a specific chaperone for GAPDH to prevent thermal

inactivation and aggregation (Erales et al. 2009b). It is oxidized, but not reduced, CP12 that is

able to complex with GAPDH and PRK (Graciet et al. 2003b; Marri et al. 2009b). Oxidized

CP12 is composed of helix and coil segments and is flexible, whereas reduced CP12 is

unstructured (Graciet et al. 2003a). Interestingly, the bulk of CP12’s primary structure fits the

criteria for intrinsically disordered proteins (IDP). IDPs are conformationally very flexible, which

allows tight regulatory control of important processes, including the cell cycle, and

transcriptional and translational regulation (Wright and Dyson 1999; Tompa 2002). Likewise,

IDPs have been shown to “moonlight”, assuming multiple conformations upon binding their

partners, leading to distinct or even opposing actions (Tompa et al. 2005).

The importance of CP12 in cyanobacteria was demonstrated by analysis of a S.

elongatus insertion mutant which showed that, under both light and dark conditions, the

formation of the GAPDH-PRK-CP12 ternary complex regulated the activities of both enzymes

and thus carbon flow from the Calvin cycle to the oxidative pentose phosphate cycle (Tamoi et

al. 2005). More recently, it was shown that cyanophages of the marine picoplanktonic

cyanobacteria genera Synechococcus and Prochlorococcus encode and express a CP12

protein similar to that of their hosts (Thompson et al. 2011). Cyanophage CP12 expression

results in decreased Calvin cycle activity in infected hosts, leading to an increased level of

NADPH; this is proposed to fuel the phage ribonucleotide reductase for production of phage

nucleotides. Together, these data underscore the key role of CP12 in the regulation of the

Calvin cycle in both redox-dependent and -independent mechanisms in cyanobacteria.

Recently, structures have been determined of both the S. elongatus and A. thaliana

CP12 in complex with GAPDH (Matsumura et al. 2011; Femarni et al. 2012). These structures

provide unprecedented detail of the intermolecular interactions between CP12 and GAPDH and

suggest how PRK may be recruited to the complex. The C-terminal domain of CP12 (residues

53-75 of the S. elongatus CP12) inserts into the active site of GAPDH, while the N-terminus of

CP12 (residues 1-52) remains disordered in the CP12-GAPDH complex and, presumably,

interacts with PRK. In S. elongatus, CP12 binding to GAPDH creates a patch of negative charge

on the binary complex that is proposed to electrostatically attract positive charges on PRK

(Matsumura et al. 2011). However, this may be a feature specific to cyanobacterial CP12

because this negatively charged patch is not present in the A. thaliana structure (Fermani et al.

2012).



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All cyanobacterial genomes sequenced to-date (75 publically available) contain at least

one gene encoding a CP12 or CP12-like protein (except Cyanobacterium sp. UCYN-A and

Moorea producta 3L). Recently, the amount of sequence data available for cyanobacteria more

than doubled as a result of the CyanoGEBA project (Submitted data Shih PM, Wu D, Latifi A,

Axen SD, Fewer DP, Talla E, Calteau A, Cai F, de Marsac T, Rippka R, Herdman M, Sivonen K,

Coursin T, Laurent T, Goodwin L, Nolan M, Davenport KW, Han CS, Rubin EM, Eisen JA,

Woyke T, Gugger M, Kerfeld CA). We report here on the bioinformatic analysis of a large

number of newly available cyanobacterial genome sequences, including members of

Subsections II and V that previously had no sequenced representatives. This analysis has

identified a previously unknown diversity of CP12 homologs among cyanobacteria indicating a

diversity of roles for these proteins, including possibly some outside the Calvin cycle.

RESULTS

Classification of Cyanobacterial CP12 Types and Comparison of Primary Structure

A bioinformatic analysis of 126 cyanobacterial genomes representing the five

morphological subsections revealed that all but UCYN-A and Moorea producta 3L contained

between one and five CP12 paralogs. A total of 274 CP12 genes were identified (Table S1).

They were subdivided into eight groups based on key primary structural features: the N-terminal

cysteine pair, the C-terminal cysteine pair, a core “AWD_VEEL” sequence, and an N-terminal

CBS domain fusion (Fig. 1). Of the 274 cyanobacterial sequences analyzed, 120 resemble the

canonical higher plant-like CP-12 (CP12-N/C; Fig. 2A): CP12-N/C proteins are approximately 80

amino acids long and contain both the N-terminal cysteine pair associated with PRK binding and

the C-terminal cysteine pair required for GAPDH binding. Other types of cyanobacterial CP12s

include a variant lacking both cysteine pairs (CP12-0; eight sequences), CP12 containing only

the N-terminal cysteine pair (CP12-N; two sequences) and CP12 with only the C-terminal

cysteine pair (CP12-C; 53 sequences). Furthermore, the CP12-C group contains a subset of 39

sequences, found only in marine picoplanktonic cyanobacteria, that lack the otherwise

conserved core CP12 “AWD_VEEL” sequence (CP12-C-M; residues 66-73 in CP12-N/C logo).

This core sequence is implicated in GAPDH binding by trypsin-protection experiments (Lebreton

et al. 2006); however, this region of CP12 is not visible in the electron density of either of the

CP12-GAPDH structures (Matsumura et al. 2011; Fermani et al. 2012). Additionally, the N-

terminal Gly24, Ser27, and His46, and C-terminal Pro65 residues are strongly conserved

among all CP12 homologs (including those from eukaryotes), while the Gln41 and Phe57



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residues following the “AWD_VEEL” sequence that have previously been noted as specific to

cyanobacteria (Groben et al. 2010) are not strongly conserved among all cyanobacterial

variants (Fig 2A).

In contrast to any known plant or algal CP12 homologs, a large (92) subset of

cyanobacterial CP12 homologs have a CBS domain fused to the N-terminus (Figs 1 and 2A).

These make up three additional classes of cyanobacterial CP12-like proteins: CP12-N/C-CBS

(14 sequences), CP12-N-CBS (55 sequences), CP12-0-CBS (23 sequences). One of the CP12-

N-CBS proteins was previously described as an ORF4/CP12 fusion in Anabaena variabilis

(Pohlmeyer et al. 1996; Masepohl et al. 1997). As discussed below, CBS domains are typically

associated with regulatory functions and are found in all phylogenetic domains of life (Bateman

1997).

Phylogenetic Distribution of CP12 Types

Phylogenetic analysis of the eight different CP12 types revealed that the distribution of

any individual variant differs among cyanobacterial genomes (Fig. 4). Some genomes have

multiple copies of a given CP12 variant (e.g. Synechococcus sp. PCC 7335 has two copies of

CP12-0 and of CP12-N-CBS). The entire group of marine picoplanktonic cyanobacteria only

possess the CP12-C(M) variant (lacking the “AWD_VEEL” core sequence), occasionally in

multiple copies. With the exception of the marine picoplanktonic cyanobacteria, every genome

in Figure 4 contains at least one copy of the canonical CP12-N/C. A phylogenetic tree of the 274

cyanobacterial CP12 sequences shows each CP12 type clustering in dispersed groups (Fig. 5,

Fig. S2). The primordial cyanobacterium, Gloeobacter violaceus, contains a single canonical

CP12-N/C gene which clusters with the anomalous CP12-C(M)s. The plant and algal eukaryotic

CP12 sequences form two clades that group with the cyanobacterial canonical CP12-N/C type

(Fig. S3). The plant CP12-3 variant forms a cluster distal from the plant CP12-1 and -2 types

and is closer to a distinct group of cyanobacterial CP12-N/Cs.

The remaining cyanobacterial CP12 types are present in clusters of varying sizes. The

lack of clustering of the CP12 variants from any one genome suggests that the duplication,

divergence, and fusion events that gave rise to the different types are very ancient. Recent

duplication events are rare but do seem to have occurred in cases where a genome contains

more than one copy of a CP12 type (for example, the two CP12-N-CBS of Cyanothece sp.

ATCC 51472 appear to be the result of a recent duplication (Fig. S2)).



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The Predicted Intrinsic Disorder of the Cyanobacterial CP12 Proteins

The majority of CP12 proteins from all photosynthetic organisms are predicted to be

more than 50% disordered, with a few exceptions found in land plants (Marri et al. 2008). A

PONDR VL-XT analysis of the CP12 proteins from five representative cyanobacterial genomes

predicted the expected high level of disorder (ranging from 57.53% to 80.52%) particularly in the

N-terminus of CP12-0, CP12-C, CP12-N and CP12-N/C proteins (Table II, Fig. S1). CP12-C(M)

displayed the same pattern of highest disorder at the N-terminus but the overall predicted

disorder was somewhat lower at 43%. The CP12 domain of the CP12-CBS fusion proteins is

also predicted to be generally less disordered than CP12 proteins lacking the fusion, ranging

from 40.51% to 61.90%.

The Cyanobacterial CP12 Variants in the Context of the CP12-GAPDH Structure

By a combination of sequence comparison and homology modeling using the

coordinates of the S. elongatus CP12-C model from the crystal structure as a template, we

evaluated the potential of the different cyanobacterial CP12 types to form a complex with their

cognate GAPDH, coordinate copper and bind NAD (Matsumura et al. 2011; PDB 3B1J).

Two types of GAPDH are present in cyanobacteria: type 1 contains the residues

identified by Matsumura et al. 2011 as being important for CP12 binding (Thr37, Ser38, Asp39,

Arg80, His182, Arg189, Ser194, His195, Arg196 and the copper-interacting Cys155, Thr156,

His182). A second type (type 2) of GAPDH is also found in cyanobacteria; this paralog contains

the copper-interacting residues, but not the other CP12-binding residues. Each cyanobacterial

genome encodes one copy of type 1 GAPDH and one or two copies of type 2 with two

exceptions: Cyanothece sp. BH68, ATCC 51142 has two type 2 GAPDHs (without the CP12

binding residues) and no type 1 GAPDH and Acaryochloris sp CCMEE 5410 has two of each

GAPDH paralog. There is no apparent correlation between the number of CP12 variants and

GAPDH type or copy number within a given genome.

Homology models for all of the cyanobacterial CP12 variants and the corresponding (i.e.

from the same organism) type1 GAPDH were built, except for CP12-0-CBS (the low sequence

homology between it and the template precluded construction of a homology model). Inter-

protein interactions between the CP12 variant and GAPDH models were examined (Fig. 3).

The CP12-N/C, CP12-C, CP12-C(M), and CP12-C/N-CBS (the variants with the C-

terminal cysteine pair) all have the C-terminal loop residues required for interaction with



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GAPDH, copper and NAD+ (Tyr 73, Asp74, Asp75; Table I). Homology models of

representatives of each of these types show some conformational variation in the disposition of

this three-residue C-terminal loop relative to the solved structure (Fig. 3). At the level of primary

structure, Asp74 and Asp75 are conserved, with a Glu occasionally substituted among some

variants (Fig. 2A). The position of Tyr73 is the most disparate among the models, but flexibility

in the C-terminal loop would likely accommodate positioning compatible with the Tyr73 and

Asp75 NAD and copper interaction. However, modeling with S. elongatus’ other CP12 paralog,

a CP12-N/C, shows substantial differences in the interaction of the C-terminus with GAPDH:

Tyr73 clashes with GAPDH Glu185, and Asp75 is replaced by a Val, suggesting that this

paralog interacts with GAPDH very differently or not at all.

CP12-N, CP12-0 and CP12-N-CBS lack the C-terminal cysteine pair (Fig. 2A) and

therefore the disulfide bond pinning the alpha helix (the ordered section of CP12), in place. In

addition, these variants are missing at least one key interacting C-terminus residue, with the

negative Asp75 most often replaced by a neutral Val (Fig. 2A). Homology modeling shows that

Tyr73 in both CP12-0 and CP12-N-CBS sterically clashes with GAPDH (both at Glu187).

Additionally, CP12-N-CBS Leu75 clashes with GAPDH Thr213 (Fig. 3). For CP12-N, two

residues clash with GAPDH: CP12-N Tyr73 and GAPDH Arg189, and CP12-N Arg71 and

GAPDH Leu186.

DISCUSSION

This study has revealed a previously unknown range of variation among cyanobacterial

CP12 proteins. These data provide strong evidence for a more diverse regulatory role for CP12

and CP12-like proteins in cyanobacteria - in either input signal or output activity - than

previously thought. Direct support for this comes from analysis of a S. elongatus CP12-C knock-

out mutant that showed inhibited growth under dark/light conditions (Tamoi et al. 2005),

suggesting that the CP12 variant (CP12-N/C) present in this organism could not compensate for

the loss of CP12-C.

Consistent with the growing evidence for a multiplicity of roles for CP12, a survey of

numerous expression studies indicates that the CP12 variants we have identified here are

differentially expressed in cyanobacteria (Table S2). For example, the two S. elongatus CP12

proteins, CP12-C and CP12-N/C, in addition to differences in potential interaction with GAPDH

(Fig. 3) show differences in overall expression under constant light and in response to a shift

from high to low atmospheric CO2 (Vijayan et al. 2011; Scharwz et al. 2011). In Synechocystis



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PCC6803, CP12-C expression is upregulated under sulfate deprivation and osmotic stress

conditions and down-regulated in high light (Table S2; Hihara et al. 2001; Shoumskaya et al.

2005; Zhang et al. 2008). In Synechococcus sp. PCC 7002, the ratio of expression of CP12-N-

CBS under dark oxic conditions relative to standard growth conditions is four-fold higher than

that of CP12-N/C (Ludwig and Bryant 2011). Interestingly, in Microcystis aeruginosa PCC7806

mutants lacking the potential to synthesize the toxin microcystin, two of M. aeruginosa’s four

CP12 proteins show differential accumulation: one of its two CP12-N-CBS proteins accumulates

at three-fold the rate of wildtype under light conditions, and its CP12-N/C-CBS accumulates at a

six-fold high rate than wild type under light conditions and three-fold lower rate in the dark

(Zilliges et al. 2011). This study also shows that microcystin plays a role in resistance to

oxidative stress, possibly in conjunction with CP12. In higher plants, the expression of the

CP12-3 like variant is increased under hypoxic conditions and this may indicate a role for this

form of the protein in mediating the shift in metabolism from aerobic to anaerobic (Singh et al.

2008). A microarray analysis of CP12 antisense plants revealed that in roots the most strongly

induced genes were those shown to be involved in hypoxia responses in plants (Liu et al. 2005;

Howard et al. 2011a).

Evidence of differential Interaction of Cyanobacterial CP12 Variants GAPDH and PRK

Previous analysis has shown that the C-terminal loop of CP12 is critical for the

interaction with GAPDH (Matsumura et al. 2011; Erales et al. 2011; Fermani et al. 2012). Glu69

is thought to be important in interactions with PRK, GAPDH and NAD and is particularly well

conserved among cyanobacterial CP12 proteins (Matsumura et al. 2011). Tyr73, which also

interacts with NAD, is conserved among cyanobacterial CP12 variants while Leu71 is not (Fig.

3). Asp75 is conserved in CP12-N/C, CP12-N, CP12-C(M), and CP12-N/C-CBS (Fig. 3). The

presence of the copper-interacting residue in variants with the C-terminal cysteine pairs is

consistent with the proposal that copper binding catalyzes the formation of the disulfide bonds

(Delobel et al. 2005; Erales et al. 2009c). In contrast, the copper-interacting Asp75 is replaced

by Leu (CP12-N-CBS) or by a Val (CP12-0) in CP12 types that do not contain the C-terminal

cysteine pair. Collectively, our survey of residue conservation (Fig. 2) and homology modeling

(Fig. 3) suggests that cyanobacterial CP12-C, CP12-C(M), CP12-N/C and CP12-N/C-CBS could

likely bind GAPDH. However, it is unlikely that CP12-0, CP12-N, CP12-N-CBS and CP12-0-

CBS are able to bind GAPDH, in part because the structure of each of these may be

significantly different from that of the canonical CP12 due to the lack of the disulfide bond that

orients the helical segment (Fig. 1, Fig. 2).



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In contrast to GAPDH, identifying PRK paralogs in cyanobacterial genomes is difficult.

PRK is very similar to another enzyme, uridine kinase, and annotations, COG and pfam

assignments frequently contradict one another, suggesting that such computational-based

functional assignments for this gene product are unreliable. However, it is clear that among the

CP12 variants, the N-terminus, which has been shown to interact with PRK (Groben et al.

2010), is overall less conserved than the GAPDH-interacting C-terminus (Fig. 2A). The CP12-

N/C group contains a conserved N-terminal segment preceding N-terminal cysteine pair that is

not found in the other cyanobacterial CP12 types (Fig. 2A). The function of the N-terminus is

elusive and the N-terminal cysteine pair is not essential for PRK binding in S. elongatus (Tamoi

et al. 2005).

When CP12 is bound to GAPDH, five acidic residues face outward, creating a patch of

negative charge proposed to interact with positively charged residues in PRK (Matsumura et al.

2011). Among the CP12 variants identified here, four of the negatively charged residues are

conserved; the fifth, Asp54, is frequently replaced by a Lys in CP12-N/C and in all three CP12-

CBS variants (Fig. 2A). Changing this final position of the helix from a negative to positively

charged residue will reduce the strength of the negatively charged patch posited to interact with

PRK. The prevalence of this substitution across the CP12-CBS fusion variants suggests that

these proteins have a role other than formation of a CP12-GAPDH-PRK complex.

The Role of the CP12-Associated CBS Domain Pair

An additional layer of complexity in the regulatory role of CP12 proteins is evident from

the widespread occurrence of the three types of CP12-CBS domain fusions. Each type contains

a single CBS domain. CBS domains are regulatory modules usually in two or four tandem

copies per protein. They are found in functionally diverse proteins across all kingdoms of life

including channel proteins, kinases, signal transduction proteins, and membrane proteins

(Bateman 1997). The cyanobacterial genomes that contain the CP12-CBS fusions tend to be

enriched in CBS domains (Table IV). All cyanobacterial genomes sequenced to-date contain

between 5 and 30 CBS domain-containing proteins; by comparison E. coli contains only 9 CBS

domain-containing proteins. Among CBS domain sequences available in the NCBI database,

the cyanobacterial CP12 CBS domain is most similar to that found in a signal transduction

protein in Thermus sp. CCB_US3_UF1 (BLAST, E-value 3e-66, 78% identity).

CBS domain pairs assemble in an anti-parallel arrangement to form what is known as a

tight Bateman module (Ignoul and Eggermount 2005). CBS domain pair-containing proteins



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often form homodimers, with two CBS domain pairs assembling into a CBS module with a total

of four CBS domains (Baykov et al. 2011). A key question raised by our observations is whether

the CP12 CBS domain facilitates homodimerization of two CP12-CBS proteins, or whether the

CBS domain in a given CP12 dimerizes with a CBS domain found on some other protein. The

CBS domains found in the three CP12 variants are present in single copies at the N-terminus

and share high degree of similarity, with a typical pairwise alignment between their CBS

domains being 70 to 75% identical. In order to identify prospective interaction partners (besides

via homodimerization) for the CP12-associated CBS domains, phylogenetic profiling was used

to search for genes conserved specifically among the subset of cyanobacterial genomes

containing a CP12-CBS domain fusion. No candidate genes could be identified that were

restricted to genomes containing CP12-0-CBS, CP12-N-CBS, CP12-N/C-CBS or any

combination of the three types. Searching any one genome using the CBS domain sequence

from a CBS-CP12 fusion encoded in that genome finds the most significant non-CP12 CBS hit

to be a predicted signal transduction protein annotated as insine-5-monophosphate

dehydrogenase-related with an aldolase-type TIM barrel fold (seen in Fischerella PCC9605, E-

value of 4e-06, 26% identity, Rivularia sp. PCC 7116, E-value of 2e-06, 32% identity and Nostoc

PCC7524 E-value of 1e-07, 26% identity). The relatively low sequence homology between the

CP12 CBS domain and any other CBS domain containing protein within a given genome

suggests that the CP12 CBS domain most likely homodimerizes with a second copy of a CP12-

CBS protein.

CBS domains have been shown to bind Mg2+, single-stranded DNA and RNA, and

double stranded DNA (Kery et al. 1998; McLean et al. 2004; Scott et al. 2004; Sharpe et al.

2008; Hattori et al. 2007; Hattori et al. 2009; Aguado-Llera et al. 2010; Feng et al. 2010). A

potential clue to the function of the CP12 CBS domain is found in the primary structure of all

three types of CP12-CBS proteins. Most of the CBS domains characterized to-date bind

adenine nucleotides as regulatory elements for sensing cellular energy status, and the result

can be either inhibitory or activating. This may be the role of the CP12 CBS domains; each

contains two copies of the nucleotide ribose-PO4 binding motif Ghx(T/S)x(T/S)D (Day et al.

2007; Fig. 2).

A total of 34 single and double CBS domain-containing proteins are encoded in the

genomes of Arabidopsis thaliana and Oryza salitica (Kushwaha et al. 2009), but to-date there is

no evidence for a CP12-CBS fusion among eukaryotic CP12 homologs. However, a recent

study of chloroplast CBS proteins in higher plants suggests that they are involved in activation



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of thioredoxins in the ferredoxin-thioredoxin system and that binding AMP in the chloroplast

increases this activation, thereby enabling these molecules to exert a regulatory effect on both

the Calvin cycle and H2O2 levels (Yoo et al. 2011). This is interesting because, as in higher

plants, the formation and breakdown of the PRK-GAPDH complex is mediated by the redox

state of CP12, which is in turned mediated by thioredoxin (Howard et al. 2008; Marri et al.

2009).

Higher plants contain a CP12-GAPDH fusion; the fused GAPDH subunit is known as

GapB. The C-terminal extension (CTE) of GapB consists of the GAPDH-binding module of

CP12 (~30 residues from the C-terminal half of CP12), including the C-terminal cysteine pair.

All cyanobacteria, algae and plants contain GAPDH composed of four GapA subunits, but in

higher plants an A2B2 heterotetramer, composed of two GapA and two GapB subunits, is the

major chloroplast form of GAPDH (Petersen et al. 2006; Howard et al. 2011b, Howard et al.

2011c). GapA and GapB form complexes in response to light, with the CTE playing the

regulatory role of CP12 (Wedel and Soll 1998; Scheibe et al. 2002; Trost et al. 2006). Outside of

the Streptophyta, GapB has only been found in the green alga Ostreococcus: the Ostreococcus

genome does not encode any other CP12, suggesting that the CTE function can replace CP12

redox-regulated activity (Robbens et al. 2007).

CONCLUSION

This study has revealed an unexpected diversity in cyanobacterial CP12 proteins; they

can be classified into 8 distinct types based on primary structural features. The presence of

CBS domain fusions as well as variation in the distribution of key structural features and in

expression patterns among these types, suggest a heretofore unknown layer of regulatory

complexity in CP12 function. The CP12-CBS domain fusions in cyanobacteria functionally

connect CBS domains and CP12. This finding raises the question of whether CBS proteins in

plants, together with CP12, play a redox relay type role acting as metabolic switches. In

cyanobacteria the diversity of CP12 proteins and the CBS-CP12 fusions may have evolved to

cope with the need to trigger different metabolic processes in response to rapidly fluctuating

environments. Perhaps as in other intrinsically disordered proteins (Tompa et al. 2005), some

CP12 variants may “moonlight,” interacting with multiple proteins in different conformations for

multiple functions.



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MATERIALS AND METHODS

Bioinformatics

The Function Search tool in IMG ER (http://img.jgi.doe.gov) was used to survey all

available cyanobacterial genomes for the CP12 pfam (pfam02672). The “blastp” program at the

National Center for Biotechnology website (Altschul et al. 1990; Altschul et al. 1997;

http://www.ncbi.nlm.nih.gov/), was also used to verify the results, using the Synechococcus

PCC6801 and Synechococcus PCC7942 CP12 amino acid sequences as queries.

All CP12 amino acid sequences were aligned using the MUltiple Sequence Comparison

by Log-Expectation (MUSCLE; Edgar 2004). The multiple sequence editor JalView (Waterhouse

et al. 2009) was used to manually curate the alignment and categorize sequences into types

based on primary structure features: the presence or absence of an N-terminal cysteine pair, C-

terminal cysteine pair, a core “AWD_VEEL” sequence, and an N-terminal CBS domain fusion.

Using the MUSCLE alignments, Hidden Markov Model profiles for each variant were

built using hmmer3.0:HMMBUILD (Eddy 1998; Eddy 2008; http://mobyle.pasteur.fr), with the

Henikoff position-based weighting scheme and no effective sequence weighting (enone).

LogoMat-M (Schuster-Böckler et al. 2004) was used to visualize Hidden Markov Model logos for

each variant (http://www.sanger.ac.uk, X-axis = contribution, Y-axis = relative entropy).

The PONDR (Predictor of Naturally Disordered Proteins) server (Romero et al. 1997; Li

et al. 1999, Romero et al. 2001; www.pondr.com) was used to predict the degree of disorder in

the following CP12 proteins: geneOID 2509710708, 2509434278, 2508607866, 2509434317,

2509434904, 2503740304, 637231127, 2509436117, 637231794, 650129895, and 650131651

(Table S1).

The CP12 CBS domain consensus sequence was obtained from loading the MUSCLE

alignment of all cyanobacterial CP12 sequences into JalView (Waterhouse et al. 2009). This

consensus sequence was used as a query to search the Research Collaboratory for Structural

Bioinformatics Protein Data Bank (RCSB PDB) (www.pdb.org; Berman et al. 2000). The CP12

CBS domain consensus sequence was entered into the “blastp” program at the National Center

for Biotechnology website (http://www.ncbi.nlm.nih.gov/), searching the non-redundant protein

database excluding cyanobacterial genomes (Altschul et al. 1990; Altschul et al. 1997). The



16

CBS domain from the following genes was used as a query sequence to search its own genome

using the IMG BLAST tool at IMG ER (http://img.jgi.doe.gov): Fischerella PCC9605, geneOID

2509464408 ; Rivularia sp. PCC 7116, geneOID 2510088188; Nostoc PCC7524, geneOID

2509809301.

Phylogenetic profiles were constructed using the “Phylogenetic Profiler: Single Gene”

function in IMG ER (http://img.jgi.doe.gov). Searches were conducted for genes unique to

cyanobacterial genomes possessing all three variants of CP12-CBS fusion proteins, any two

variants of CP12-CBS fusion proteins, or any single variant of CP12-CBS fusion protein.

Cyanobacterial GAPDH and PRK sequences were obtained by using the

Synechococcus PCC7942 GAPDH and PRK amino acid sequences as queries in the IMG

BLAST tool in IMG ER (http://img.jgi.doe.gov; Table S3). The Function Search tool in IMG ER

was also used to search for the GAPDH COG0057.

A phylogenetic tree was constructed of all cyanobacterial CP12s by loading the core

CP12 section of the MUSCLE alignment (the final 84 amino acids) into the “A la carte” analysis

on at www.phylogeny.fr (Dereeper et al. 2008; Dereeper et al. 2010). The alignment was

curated to remove positions with gaps, and a maximum likelihood tree was constructed by

PhyML. The tree generated was visualized using the Interactive Tree of Life (http://itol.embl.de/;

Letunic and Bork 2006; Letunic and Bork 2011).

Expression data was obtained in part via CyanoExpress

(http://cyanoexpress.sysbiolab.eu/; Hernandex-Prieto M and Futschik M 2012).

Structural Comparisons

ESyPred3D Web Server 1.0 (http://www.fundp.ac.be) was used to create homology

models of the following proteins using PDB 3B1J_C as a template: geneOID 641611274,

641610209, 637231127, 637231794, 637231269, 637798767, 2509508362, 2508607866,

2504134271, 637448241 (Lambert et al. 2009; Table S1). PDB 2QH1_A was used as a

template to create a homology model of 2509508362. Homology models were visualized using

The PyMOL Molecular Graphics System, Version 1.5.0.4 Schrödinger, LLC (DeLano WL 2002).

ACKNOWLEDGEMENTS

The authors thank Jeff Cameron and Patrick Shih for helpful discussions and Seth Axen for

helpful discussions and figure preparation.



17

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23

Figure Legends

Figure 1 Schematic representation of the eight cyanobacterial CP12 types.

Figure 2 Sequence analysis of cyanobacterial CP12 variants. A) HMM logos of CP12 variants.

Asterisks indicate key residues detailed in panel B. The CBS domains are denoted by a solid

underline; the putative ribose-PO4 binding site Ghx(T/S)x(T/S)D (Day et al. 2007) are denoted

by a dashed underline. The blue helix indicates the segment corresponding to the predicted

alpha helix. B) Average percent of key interacting residues identified by Matsumura et al. 2011

present in each CP12 variant.

Figure 3 Homology models of representative cyanobacterial CP12 and GAPDH proteins in

complex modeled using PDB 3B1J as template (Matsumura et al. 2011). NAD is shown in blue

sticks, copper as an orange sphere, the template CP12 is magenta, the modeled CP12 is

yellow, the template GAPDH is cyan and the modeled GAPDH is red. Residues in the C-

terminal loop are labeled. A) Synechococcus PCC7002 (Identity to template: CP12-N/C 48.0%,

GAPDH 74.33%) B) Synechococcus PCC7942 (Identity to template: CP12-N/C 52.1%, GAPDH

74.11%) C) Gloeocapsa sp. PCC7428 (Identity to template: CP12-C 47.2%, GAPDH 74.11%)

D) Prochlorococcus MIT9313 (Identity to template: CP12-C(M) 36.0%, GAPDH 70.8%) E)

Leptolyngbya PCC6406 (Identity to template: CP12-N 40.0%, GAPDH 74.11%) F) Geitlerinema

PCC7407 (Identity to template: CP12-0 40%, GAPDH 77.98%) G) Fischerella PCC9339

(Identity to template: CP12-N/C-CBS 46.2%, GAPDH 74.41%) H) Nostoc PCC7120 (Identity to

template: CP12-N-CBS 20.7%, GAPDH 76.19%).

Figure 4 Number and type of CP12 variant found in each cyanobacterial genome displayed

phylogenetically (Tree adapted from Shih et al. 2012).

Figure 5 Maximum likelihood gene tree of all cyanobacterial CP12s.



24

Table I CP12 residue interactions Summary of CP12 residues implicated in formation of the GAPDH-PRK-CP12 complex and their ascribed roles GAPDH NAD Cu2+ PRK Asp54 Electrostatic

attraction Asp59 Electrostatic

attraction Glu63 Electrostatic

attraction Asp66 Ion pair with

GAPDH Asp80 Electrostatic

attraction Glu69 H bond GAPDH

Thr37; Van der Waals GAPDH Ser38, Asp39a

Contacts 2’ hydroxyl

Electrostatic attraction

Cys70 H bond GAPDH His195, Arg196

Leu71 H bond GAPDH His195, Arg196a

Tyr73 H bond GAPDH Thr 156, Thr185, Asp187, Arg200, Thr213a

Interacts with phosphate and ribose group

Asp74 H bond GAPDH Thr 156, Thr185, Arg200, Thr213

Asp75 H bond GAPDH Thr 156, Thr185, Arg200, Thr213b

Interactionc

aGAPDH-binding also seen by Fermani et al. 2012 bImportance for GAPDH binding also suggesting by Erales et al. 2011 cCopper interacting seen by Erales et al. 2009c



25

Table II PONDR VL-XT disorder predictions Percent disorder predictions for the CP12 domain of selected cyanobacterial CP12 sequences Type Gene OID Genome Percent Disorder CP12-N/C 2509710708 Pleurocapsa sp. PCC7319 57.53 CP12-N/C 2509434278 Microcoleus sp. PCC7113 60.00 CP12-N 2508607866 Leptolyngbya sp. PCC6406 80.52 CP12-C 2509434317 Microcoleus sp. PCC7113 58.46 CP12-0 2509434904 Microcoleus sp. PCC7113 60.24 CP12-N/C-CBS 2503740304 Nostoc sp. PCC7120 53.49 CP12-N-CBS 637231127 Nostoc sp. PCC7120 40.51 CP12-N-CBS 2509436117 Microcoleus sp. PCC7113 61.90 CP12-0-CBS 637231794 Nostoc sp. PCC7120 34.62 CP12-C(M) 650129895 Synechococcus sp. CB0101 43.06 CP12-C(M) 650131651 Synechococcus sp. CB0101 43.08



CBS domain CP12

CP12-N/C-CBS

CBS domain CP12

CP12-N-CBS

CBS domain CP12

CP12-0-CBS

CP12

CP12-0

CP12

CP12-N

CP12

CP12-C(M)

CP12

CP12-C

CP12

CP12-N/C

= Cysteine pair

= AWD_VEEL core sequence

Figure 1 Schematic representation of the

eight cyanobacterial CP2 variants.

w

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iologists. All rights reserved.


CP12-0

8 sequences

CP12-N

2 sequences

CP12-C(M)

39 sequences

CP12-C

13 sequences

CP12 N/C

120 sequences

CP12-N-CBS

55sequences

CP12-N/C-CBS

14 sequences

CP12-0-CBS

23 sequences

B

Figure 2 Sequence analysis of

cyanobacterial CP12 proteins. A)

HMM logos of CP12 variants. Asterisks

indicate key residues surveyed in panel

B. The CBS domains are denoted by

solid underline, and the putative

ribose-PO4 binding sites

Ghx(T/S)x(T/S)D (Day et al. 2007) are

denoted by a dashed underline. The

blue helix indicates predicted alpha

helix. B) Average percent of key

interacting residues identified by

Matsumura et al. 2011 in present in

each CP12 variant.

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Figure 3 Homology models of representative cyanobacterial

CP12 and GAPDH proteins in complex modeled using PDB 3B1J

as template (Matsumura et al. 2011). NAD is shown in blue

sticks, copper as an orange sphere, the template CP12 is

magenta, the modeled CP12 is yellow, the template GAPDH is

cyan and the modeled GAPDH is red. Residues in the C-terminal

loop are labeled. A) Synechococcus PCC7002 (Identity to

template: CP12-N/C 48.0%, GAPDH 74.33%) B) Synechococcus

PCC7942 (Identity to template: CP12-N/C 52.1%, GAPDH

74.11%) C) Gloeocapsa sp. PCC7428 (Identity to template:

CP12-C 47.2%, GAPDH 74.11%) D) Prochlorococcus MIT9313

(Identity to template: CP12-C(M) 36.0%, GAPDH 70.8%) E)

Leptolyngbya PCC6406 (Identity to template: CP12-N 40.0%,

GAPDH 74.11%) F) Geitlerinema PCC7407 (Identity to template:

CP12-0 40%, GAPDH 77.98%) G) Fischerella PCC9339 (Identity

to template: CP12-N/C-CBS 46.2%, GAPDH 74.41%) H) Nostoc

PCC7120 (Identity to template: CP12-N-CBS 20.7%, GAPDH

76.19%).

E F

A B

C

G

D

H

Tyr73

Asp75

Glu74

Tyr73Asp74

Val75

Tyr73

Val75 Asp74 Asp74

Asp75

Glu74

Tyr73

Glu74

Leu75Tyr73

Glu74

Asp75

Tyr73

Val75Tyr73

Asp74

Asp75

Tyr73

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Figure 4 Number and type of CP12

variant found in each

cyanobacterial genome displayed

phylogenetically (after Shih et al).

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647580722 Synechococcus sp. PCC 7335

2509804294 Leptolyngbya sp. PCC 6306

2508650522 Xenococcus sp. PCC 7305

2505785185 Chroococcidiopsis sp. PCC 6712

2509710708 Pleurocapsa sp. PCC 7319

2510729577 Acaryochloris sp CCMEE 5410

641254515 Acaryochloris marina MBIC11017



2510067143 Chlorogloeopsis sp. PCC 7702

2505788516 Chroococcidiopsis sp. PCC 6712

2506600322 Spirulina sp. PCC 9445


650389537 Arthrospira platensis NIES−39

646127978 Arthrospira platensis Paraca

648390703 Arthrospira sp. PCC 8005

643169309 Arthrospira maxim

a CS−3282508551846 C

yanobium gracile P

CC

6307

637446048 Prochlorococcus sp. W

H8102

647590761 Cyanobium

sp. PC

C 7001

638115981 Prochlorococcus sp. C

C9311

2507493897 Synechococcus sp. W

H 8016

640544467 Prochlo rococcus sp. W

H 7803

639019528 S

ynechococcus sp. W

H7 805

641284585 P

rochlorococcus marinus M

IT 921

1

637688466 Prochlorococcus m

arinus NA

TL2A


arinus NA

TL1A


H5701

63777

2015 Proch

loro coc cus s p. CC

9902

63988 696

4 Syne

ch ococcus sp. B

L107

64783

7284 S

ynech

ococcus sp. WH

8109

637775

111 P

rochlo

rococcus s p. C

C9

6056

3 98 88

0 47 S

ynech

ococcu

s sp. RS

9 916

6389

63483

Syn

echococcus sp. R

S991

763

74406

46 P

roc h lo roc oc c us ma

ri nus m

ar inus C

CM

P1

3756

4094 2

8 32 P

r oc h

l or o

c oc cu

s mar in u

s MIT

9 21 5

6 40 1

5980

7 Pro

c hlo

roc o

cc us m

a rinus M

IT 93

01

640

077

939

Pro

chlo

roco

ccu

s m

arin

us

AS

960

16

4 767

3197

Pro

chlo

roco

ccu s

mar

i nus

MIT

9202

64 0

0799

13

Pro

chlo

roco

ccu s

ma

ri nus

MI T

951

563

744

9214

Pro

c hlo

roc o

ccu

s m

ari n

us

pas t

o ris

CC

MP

1986

637

448

241

Pro

chlo

r oco

ccus

ma

ri nus

MIT

931

36 4

008

5 89

9 P

roch

l oro

cocc

u s m

ar in

us

MI T

930

36

377

756

48 P

roc h

loro

c oc c

us

sp.

CC

9605

647

836

271

Syn

ech

oco

ccus

sp

. WH

810

9

637

445

568

Pro

chlo

roco

ccus

sp.

WH

810

2


arinus MIT 9312

650133041 Synechococcus sp. CB

0205

637445495 Prochlorococcus sp. WH

8102


H7805


H 8016

650131651 Synechococcus sp. CB0205

650129895 Synechococcus sp. CB

0101

640545284 Synechococcus sp. RCC

307

2506609250 Spirulin

a major P

CC 6313

2511038772 Microcystis aeruginosa PCC 7806

2511038918 Micr

ocystis a

eruginosa PCC 7806

641539941 Microcy

stis a

eruginosa NIES−843

2506598949 Spiru

lina sp

. PCC 9445

25036

1032

9 Chr

oococc

idiopsis

therm

alis P

CC 720

3

250577103

0 Fisc

herella

sp. J

SC−11

2509813652 F

ische

rella

sp. P

CC 9431

2509474424 Fisc

herella

sp. P

CC 9339

6377

9865

8 Syn

echo

cocc

us e

longa

tus P

CC 794

2

6376

16925

Syn

echoc

occu

s elon

gatus

PCC 6

301

6405

4614

1 Syn

echo

cocc

us sp

. RCC30

7

2503

6138

10 C

hroo

cocc

idio

psis

ther

mal

is P

CC 7

203

6373

1445

0 Th

erm

osyn

echo

cocc

us e

long

atus

BP−

1

6377

9876

7 Syn

echo

cocc

us e

long

atus

PCC

794

2

6376

1681

5 Syn

echo

cocc

us e

longa

tus

PCC 630

1

2510

4415

03 C

ham

aesip

hon

sp. P

CC 660

5

2509

8058

61 L

epto

lyng

bya

sp. P

CC

630

6

2509

5083

62 O

sci ll

ator

ia s

p. P

CC

108

02

6434

8031

7 C

yano

thec

e sp

. PC

C 7

424

6481

8694

1 C

yano

thec

e sp

. PC

C 7

822

2508

6078

66 L

epto

lyng

bya

sp. P

CC

640

6

2509

4292

53 S

ynec

hoco

ccus

sp.

PC

C 6

312

2511

0414

64 M

icro

cyst

is a

erug

inos

a P

CC

780

6

6415

3861

6 M

icro

cyst

is a

erug

inos

a N

IES

−843

2510

6745

56 P

roch

loro

n di

dem

ni

2509

7107

07 P

leur

ocap

sa s

p. P

CC

731

9

2509

8478

56 L

epto

lyng

bya

sp. P

CC

737

5

2508

6505

21 X

enoc

occu

s sp

. PC

C 7

305

2505

7851

86 C

hroo

cocc

idio

psis

sp.

PC

C 6

712

2503

6093

00 G

eitle

rinem

a sp

. PC

C 7

407

2509

4237

60 O

scill

ator

ia a

cum

inat

a P

CC

630

4

2509

5538

87 D

acty

loco

ccop

sis

salin

a P

CC

830

5

2503

6348

53 H

alot

hec

e sp

. PC

C 7

418

250

8645

337

Glo

eoca

psa

sp.

PC

C 7

3106

6434

7666

7 C

yano

thec

e sp

. P

CC

880

1

6449

8163

7 C

yano

thec

e sp

. PC

C 8

802

2503793553 Gloeocapsa sp. PCC 7428

2508873240 Oscillatoria sp. PCC 6407


2504926136 Tolypothrix sp. PCC 9009

2504097236 Calothrix sp. PCC 6303

2504581014 Pseudanabaena sp. PCC 7429

642600009 Nostoc punctiforme PCC 73102

646566537 Anabaena variabilis ATCC 29413

637233234 Nostoc sp. PCC 7120

2503743890 Nostoc sp. PCC 7107

2506494205 Anabaena sp. PCC 7108


2504133791 Anabaena cylindrica PCC 7122

648049720 Nostoc azollae 0708

2509874034 Synechocystis sp. PCC 6308


646570168 Anabaena variabilis ATCC

29413

637231127 Nostoc sp. P

CC

7120

2504133929 Anabaena cylindrica P

CC

7122

2509464408 Fischerella sp. PCC

9605

2509809301 Nostoc sp. P

CC

7524

2506493419 Anabaena sp. P

CC

7108


647108038 Raphidiopsis brookii D

9

647105437 Cylindrosperm

opsis raciborskii CS

−505

642601150 Nostoc punctiform

e PC

C 73102

2503741006 Nostoc sp. P

CC

7107

2510088188 Rivularia sp. P

CC

7116

2509434317 Microcoleus sp. PC

C 7113

2509768339 Cylindrospermum

stagnale PCC 7417

2509780062 Microchaete sp. PC

C 7126

2505798541 Calothrix sp. PC

C 7507


2510089363 Rivularia sp. PCC 7116


2511040501 Microcystis aeruginosa PCC 7806

2503367351 Cyanobacterium stanieri PCC 7202

2503745212 Cyanobacterium sp. PCC 10605


2506350336 Microcoleus vaginatus FGP−2




643474007 Cyanothece sp. PCC 8801


2509500011 Prochlorothrix hollandica PCC 9006


637877023 Synechococcus sp. JA−2−3B

637872594 Synechococcus sp. JA−3−3Ab

2509422989 Oscillatoria acuminata PCC 6304643482301 Cyanothece sp. PCC 74242508646084 Gloeocapsa sp. PCC 73106637010564 Synechocystis sp. PCC 6803

250980

9056 N

os toc sp. P

CC

7524

64 002

71 22 N

od u

l ari a spum

igen

a CC

Y94

14

2 509

47 446

1 Fi sch

erel la sp. P

CC

9339

2509

81414

8 Fischere

l la sp. P

CC

9431

2509764

42 4 Mas tig

o c ladops is re

pens MO

RA

250946

3547 F

ischerella sp

. PC

C 9

605

2509576032 Pleurocapsa sp. P

CC

7327

2510723879 C

rocosphaera watsonii W

H 0

003

638430536

Cro

cosphaera w

atson

ii WH

8501

647579773 S

ynechococcus sp. PC

C 7335

2509774805 Leptol yngbya sp. PC

C 7104

64 3

5 83 8 1

3 Cya

n ot he ce

s p. PC

C 7 4

25

643

4803

87 Cya

noth

e ce sp. P

CC

742

4

2507

0 858

92 P

seu d

anab

aen

a s p

. PC

C 6

802

65 01

2 93 0

7 Syn e

c hoc o

c cus sp

. CB

010165

0386

020

Art

hro

spir a

pla

tens

is N

IES

−39

646

131

496

Ar t

hro

spir a

pla

tens

is P

arac

a

6483

8833

7 A

r thr

ospi

ra s

p. P

CC

80

05

643

1677

36 A

rthr

osp

ira m

axim

a C

S−3

28

2506

345

482

Mic

roco

leus

vag

inat

us F

GP

−2

2504

0907

57 O

sci ll

ator

ia s

p. P

CC

711

2

2509

7621

96 M

astig

ocla

dops

is r

epen

s M

OR

A

251

009

948 7

Ge

i tler

i ne

ma

sp. P

CC

710

5

2 50 9

473

6 30

Fis

c he

rella

sp.

PC

C 9

339

250

981

5500

Fi s

che

r ell a

sp

. PC

C 9

431

250

5770

084

Fis

che

rell a

sp

. JS

C−

11

25 1

073

4 60

6 A

c ary

ochl

ori s

sp

CC

ME

E 5

41 0

641

2 56

7 98

Aca

ryo c

h lo r

is m

a rin

a M

BIC

1 10

1 7




2510727575 Crocosphaera watsonii WH 0003638431909 Crocosphaera watsonii WH 85012507500366 Cyanothece sp. BH63E641674631 Cyanothece sp. BH68

640627119 Cyanothece sp. CCY 0110

2506491526 Anabaena sp. PCC 7108

2504129939 Anabaena cylindrica PCC 7122

646570018 Anabaena variabilis ATCC 29413

637231269 Nostoc sp. PCC 7120

2509811702 Nostoc sp. PCC 7524


2509780713 Microchaete sp. PCC 7126


2503743779 Nostoc sp. PCC 7107

2504924457 Tolypothrix sp. PCC 90092509768287 Cylindrospermum stagnale PCC 7417

2505770373 Fischerella sp. JSC−112510088360 Rivularia sp. PCC 7116642604205 Nostoc punctiforme PCC 73102

2509464236 Fischerella sp. PCC 9605

2509434904 Microcoleus sp. PCC 7113

640026284 Nodularia spumigena CCY9414


640017815 Lyngbya sp. CCY 9616

2509503036 Prochlorothrix hollandica PCC 9006


2503638412 Halothece sp. PCC 74182509571905 Pleurocapsa sp. PCC 7327

2507504240 Cyanothece sp. BH63E641672289 Cyanothece sp. BH68


2503797385 Gloeocapsa sp. PCC 7428








6475

7213

5 M

icro

cole

us c

htho

nopl

aste

s P

CC

742

0

2503

7420

70 N

osto

c sp

. PC

C 7

107

2503

7403

04 N

osto

c sp

. PC

C 7

107

2509

5102

65 O

scill

ator

ia s

p. P

CC

108

02

2503

6079

28 G

eitle

rinem

a sp

. PC

C 7

407

2506

7482

72 S

ynec

hoco

ccus

sp.

PC

C 7

336

2505785349 Chrooco

ccidiopsis

sp. P

CC 6712

2508874986 Oscil

latoria sp. P

CC 6407

648859217 Oscill

atoria sp. P

CC 6506

25095753

98 Pleuro

capsa

sp. P

CC 7327

2509419962 Osc

illatoria

acuminata P

CC 6304

646569446 Anabaena variabilis

ATCC 29413

637231794 Nostoc sp. PCC 7120

2509808759 Nostoc sp. PCC 7524

2509468491 Fischerella sp. PCC 9605

2507477234 Calothrix sp. P

CC 7103

25097

7773

4 Le

ptolyn

gbya

sp. P

CC 710

4

2505

7675

32 F

ischer

ella sp

. JSC−11

250950

9590

Oscilla

toria

sp. P

CC 10802

2509

8172

85 F

ische

rella

sp. P

CC 943

1

2509

4739

96 F

ische

rella

sp. P

CC 933

9

2509

7822

41 M

icro

chae

te s

p. P

CC 712

6

2505

8044

17 C

alot

hrix

sp. P

CC 750

725

0957

3890

Ple

uroc

apsa

sp.

PC

C 7

327

2503

6349

75 H

alot

hece

sp.

PCC

741

8

2504

1342

71 A

naba

ena

cylin

drica

PCC

712

2

6435

8701

7 Cya

noth

ece

sp. P

CC 7

425

2509

7717

73 C

ylin

dros

perm

um s

tagn

ale

PCC

741

7

2504

9259

43 T

olyp

othr

ix s

p. P

CC

900

9

6426

0040

3 N

osto

c pu

nctif

orm

e PC

C 7

3102

2509

5545

35 D

acty

loco

ccop

sis

salin

a P

CC

830

5

2508

8762

35 O

scil l

ator

ia s

p. P

CC

640

7

6488

5643

1 O

scill

ator

ia s

p. P

CC

650

6

6480

5171

4 N

osto

c az

olla

e 07

08 2509712443 Pleurocapsa sp. P

CC

73192503801366 Stanieria cyanosphaera PCC 7437

2508654911 Synechocystis sp. PCC 7509638108373 Trichodesmium erythraeum IMS1012509509498 Oscillatoria sp. PCC 10802647108260 Raphidiopsis brookii D9640014376 Lyngbya sp. CCY 96162506607707 Spirulina major PCC 6313641611274 Synechococcus sp. PCC 7002

2510103538 Geitlerinema sp. PCC 7105

2504683747 Crinalium epipsammum PCC 9333

2508648941 Xenococcus sp. PCC 7305

2505771342 Fischerella sp. JSC−11


2508608509 Leptolyngbya sp. PCC 6406637461112 Gloeobacter violaceus PCC 7421


640625655 Cyanothece sp. CCY 0110

2507500829 Cyanothece sp. BH63E

641675096 Cyanothece sp. BH68

2503609757 Geitlerinema sp. PCC 7407

2509706763 Pleurocapsa sp. PCC 7319


2510673562 Prochloron didemni

647566986 Microcoleus chthonoplastes PCC 7420

647105539 Cylindrospermopsis raciborskii CS−505

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Documents

Cyanobacterial CP12 Proteins Journal research area … · Biochemical Processes and Macromolecular ... different types based on primary structure ... GAPDH can undergo a thiol-disulfide