Computational Biology Institute, Oak Ridge National Laboratory, PO Box 2008, Oak Ridge, TN 37831, USA 1Present address: Virginia Bioinformatics Institute, Washington Street, Blacksburg, VA 24061, USA
2 To whom correspondence should be addressed. E-mail: yu07_2000{at}yahoo.com or samatovan{at}ornl.gov
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Abstract |
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Keywords: chloroplast evolution/CO2/O2 specificity/photosynthesis/photorespiration/ribulose 1,5-bisphosphate carboxylase/oxygenase/RuBisCo/residue substitution pattern
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Introduction |
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RuBisCo's CO2/O2 substrate binding specificities are measured by enzyme kinetic properties such as CO2/O2 specificity factor (
= KoVc/KcVo) and CO2 affinity factor Kc. These kinetics are highly inherent and vary dramatically in six RuBisCo types (1A, 1B, 1C, 1D, Type II, Type III/IV) (Laing et al., 1974
; Jordan and Ogren, 1984
; Spreitzer and Salvucci, 2002
) and also among different photosynthetic organisms. In green plants, the Type 1B RuBisCo enzyme possesses
values of 7882 and Kc values of 1030 (Jordan and Ogren, 1984
; Read and Tabita, 1994
), as opposed to cyanobacterial Type 1B RuBisCo with a 50% lower
(
= 40) and four times increased Kc values (Kc = 150175) (Larimer and Soper, 1993
; Tabita, 1999
). Type 1D RuBisCos in marine non-green algae confer the highest CO2/O2 specificity but suffer from the lowest carbon affinity among all RuBisCo enzymes tested so far, e.g. an
value of 240 and a Kc value of 5 in Galdieria partita (Igarashi and Kodama, 1996
).
Identification of RuBisCo's CO2/O2 specificity-determining residues is an essential step in elucidating the molecular basis of these inherent differences. For this purpose, a number of experiments have been carried out (Tabita, 1999; Parry et al., 2003
), which essentially suggest that certain regions of residues are likely to affect the RuBisCo substrate specificity (Chen and Spreitzer, 1992
). The regions include the following: (a) the residues in the C-terminal end of loop 6 and adjacent
-helix 6 in the large subunit. These residues affect RuBisCo in both catalysis and specificity [for thorough reviews, see Tabita (1999)
and Spreitzer and Salvucci (2002)
]; (b) the C-terminus residues that work as an additional factor controlling RuBisCo activity; longer C-termini may establish added interactions with the protein surface (Zhu et al., 1998
); (c) a highly conserved loop of the N-terminal domain of the second L-subunit that can interact with loop 6 in the quaternary complex; these two regions, plus the L6-
-helix 6, can significantly alter their spatial relationship to allow substrates or products to access to and from the active site (Newman and Gutteridge, 1993
); and (d) contacting loops in a small subunit that could further influence CO2/O2 specificity as preliminarily supported by site-directed mutagenesis (Read and Tabita, 1992
; Spreitzer et al., 2001
).
Although the above experiments provided valuable insights into molecular mechanisms of RuBisCosubstrate binding, they could not significantly improve the overall performance of the enzyme in terms of CO2/O2 specificity and CO2 affinity (Whitney et al., 2001; Parry et al., 2003
). With site-directed mutagenesis, only single residue substitutions (residues 331342) or simple cumulative substitutions of adjacent residues (residues 338341) have been explored (Ramage et al., 1998
). These studies lend credence to the assumption that the CO2/O2 substrate specificity may involve very complex evolutionary and biochemical processes and residues have to be carefully analyzed for their key role in specificity determination.
Our previous efforts (Yu et al., 2005) in this direction sought to identify specificity-determining surface residue clusters (SDRCs). The method developed, called surface patch ranking (SPR), focuses on identifying spatially co-located surface residues. It is based on the assumption that residues involved in specificity determination are most likely on the protein surface and unique interactions among neighboring residues may account for the specificity (Russell and Barton, 1994
; Lichtarge et al., 1996
).
In this work, we applied the SPR to RuBisCo groups to extract a set of specificity-determining residue clusters and to study when the residues mutate during the RuBisCo evolution and how they correspond to the changes in specificity. More specifically, we categorize how a consensus residue at a certain position of interest changes during the evolutionary path from cyanobacteria to green plants and marine non-green algae. The approach is based on the observation that the enzymes in three RuBisCo groups differ significantly in CO2/O2 specificity and CO2 affinity (Jordan and Ogren, 1984; Read and Tabita, 1992
; Larimer and Soper, 1993
; Tabita, 1999
) and marine non-green algal and green plant RuBisCos are acquired from different endo-symbiotic events (Curtis and Clegg, 1984
; Douglas et al., 1991
; Delwiche et al., 1995
; Stiller and Hall, 1997
; Barbrook et al., 1998
; Turmel et al., 1999
; Whitney et al., 2001
).
From the perspective of the evolutionary path, marine non-green algal RuBisCos were acquired in an earlier endo-symbiotic event and diverged considerably from the lineage of the green plants, fungi and animals (http://www.geocities.com/we_evolve/Plants/chloroplast.html) and have the highest CO2/O2 specificities. Green plant RuBisCos are close to cyanobacterial RuBisCos and yet their specificities are much higher than those of cyanobacterial RuBisCos. Accordingly, we assume that there exist different levels of CO2/O2 specificity-determining factors that correspond to the evolutionary events and specificity levels. Whereas marine non-green algal RuBisCos probably went through a series of considerable residue substitutions in changing substrate specificity conformations, green plant RuBisCo seem to be very limited in such changes. We argue that the proper categorization of residue substitution patterns will reveal the essential role of residue positions in determining protein structures and functionalities. Thus, the categorization will facilitate conducting a successful site-directed mutagenesis in enhancing CO2/O2 specificity and improving the overall performance of RuBisCo enzymes.
Our analysis of RuBisCo evolutionary history and development of RuBisCo specificities strongly suggests that the categorization based on residue substitution pattern will identify residue positions important in enhancing specificity. We found a number of residue clusters that are likely to account for specificities in marine non-green algae and green plant RuBisCos. Furthermore, some residue clusters were successfully mapped to the RuBisCo enzymatic functions and the CO2/O2 specificity, demonstrating that our discoveries are closely consistent with previous experimental results.
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Methods and materials |
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Protein sequences for RuBisCo large subunits of cyanobacteria, green plants and marine non-green algae were extracted by keyword search from protein sequence records in Entrez Proteins (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein). The protein sequences were manually checked to ensure that all the extracted sequences corresponded precisely to the intended function and species. A final set of 131 RuBisCo proteins (35 cyanobacteria, 46 green plants and 50 marine non-green algae) was selected with a sequence filtering procedure (Yu et al., 2005). The filtering procedure was aimed at providing a certain level of sequence homology and evolutionary diversity for building biologically more meaningful multiple sequence alignments and identifying evolutionarily conserved sequence residues. The selected sequences were aligned with ClustalW (Thompson et al., 1994
) and a hidden Markov model (HMM) was built with HMMER (Eddy, 1998
) using a default set of parameters.
Identifying SDRCs in RuBisCo large subunits
To identify the substrate specificity determinants in RuBisCo, we applied surface patch ranking (SPR) scheme developed in our previous work (Yu et al., 2005). SPR is based on the following observations. First, when a functional difference is observed between evolutionarily related proteins, it is often due to mutations on or near surface residues (Lichtarge et al., 1996
). Second, these residues, which determine the functional specificity, are often clustered near each other and act cooperatively for optimal conformations (Gobel et al., 1994
; Neher, 1994
; Russell and Barton, 1994
; Taylor and Hatrick, 1994
). Thus, SPR focuses on identifying a minimum set of spatially co-located surface residues that can discriminate between two classes of functional sub-types with respect to certain enzyme substrate specificity. We named these groups of residues specificity-determining surface residue clusters (SDRCs). By minimum, we mean that the number of residues within the clusters is the smallest sufficient to classify the functional sub-types either individually (an SDRC of single highly conserved residue) or complementarily (an SDRC of multiple non-conserved residues).
SPR sifts SDRCs by examining all possible residue clusters within each surface patch. A surface patch is defined as a set of surface residues that are within a certain radius from a reference residue. For an exhaustive search for SDRCs, we construct a surface patch around every surface residue. Then from each surface patch, SPR iteratively extracts SDRCs of different sizes (smaller to larger); it first identifies and removes single-residue SDRCs from the surface patch, two-residue SDRCs and so on. For the sake of convenience, we named them as singleton, doublet, triplet, etc. Evaluation for an SDRC is made by measuring its discriminating power. We particularly use the classification accuracy of support vector machine (SVM) (Vapnik, 1998) and decision tree (C4.5) (Quinlan, 1993
) for this purpose. Feature vectors for a cluster are obtained from the corresponding HMM scores (see Database of protein sequences section) in the multiple sequence alignments (MSA) of the protein family under consideration.
Identifying categories of specificity-determining factors
Once the specificity-determining residue clusters (SDRCs) have been identified, we categorize the residues in SDRCs to explain when they mutate during RuBisCo evolution and how they correspond to the changes in specificity. The categorization is based on residue substitution pattern (RSP). The RSP defines how the residue in a certain position evolves in RuBisCo from cyanobacteria to marine non-green algae and green plants (Figure 1). For example, if a position of interest in the MSA has arginine (R), phenylalanine (F) and proline (P), as a consensus residue of the cyanobacteria, green plant and marine non-green algal RuBisCos, respectively, then the RSP will be R/F/P (Figure 1b). Here a consensus residue is defined as one that appears in >90% of sequences in each RuBisCo group. An RSP is called non-consensus when no consensus residues are found in any of the three RuBisCo groups.
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Results |
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Detailed comparative analysis indicates that residue substitutions for substrate specificity changes mostly occur between the marine non-green algal RuBisCos and the RuBisCos from green plants and cyanobacteria (data not shown). Over 70% of singletons (60 out of 82) belong to the category 1, where an enormous specificity difference between the marine non-green algal RuBisCos and the other two types of RuBisCos (data not shown; available on request) is observed. The high correlation between the evolutionary type (e.g. marine non-green algae versus cyanobacteria) and the substrate specificity (high versus low) can explain such dominance of single residue clusters. Many residue substitutions must occur to reflect the substrate conformational changes required for such a specificity difference: three (five) times higher in marine non-green algal RuBisCos than in green plant (cyanobacterial) RuBisCos.
Among category 1 residue substitutions, positions 338, 339, 343 and 346 at the C-terminal end of loop 6 and adjacent -helix 6 of the large subunit are identified as singletons. These regions are critical for the catalytic activity of the RuBisCo enzymes and are potentially important for their CO2/O2 substrate specificity (Figures 2 and 3.VII). Note that Figure 3.VII presents the specificity-determining surface residue group VII displayed in Figure 3 and the same format is used in the following sections to pinpoint other residue groups.
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Category 2 of CO2/O2 substrate specificity-determining factors
This category, defined as residue substitution pattern /ß/
, covers residues that are highly conserved within, but dramatically divergent across, RuBisCo groups. They represent specificity-determining factors potentially contributing to specificity improvements for both green plant and non-green algal RuBisCos. Four singletons, (76), (117), (226) and (351), are identified as putative SDRs (Table I). Singleton (76) is adjacent to the largelarge subunit contacting residues (Figures 2 and 3). Singleton (117) is adjacent to the enzymeinhibitor XDP (D-xylulose-2,2-diol-1,5-bisphosphate) binding sites (not shown in Figure 3). Singleton (226) is the most conserved specificity-determining factor among the interface residues between the large subunits and interacting loop of small subunits (Figures 2 and 3.III). Singleton (351), the most conserved among all singletons in green plant and cyanobacterial RuBisCos, is located in a small loop region right between the
-helix 6 and ß-sheet G, and is therefore a potential hinge residue (Figure 3.V). Hinge residues are often critical in controlling substrate conformation changes in proteinsubstrate interactions (Ma et al., 2001
). An additional SDRC in this category is found at position 340, which is identified as a singleton in non-green algae but in a doublet (339 340) in green plant and cyanobacteria (Figure 3.VII). Site-directed mutagenesis experiments showed that singleton (340) plays an important role in the specificity determination. Residue substitutions at this position generated a small gain in the CO2/O2 specificity: a 12% increase for A340
Y340 and a 13% increase for A340
H340 substitutions (Parry et al., 2003
).
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This category covers two residue substitution patterns (RSP /ß/ß and
/ß/
) that are likely to correspond to a recent evolutionary event and contribute exclusively to the specificity difference between green plant and cyanobacterial RuBisCos. Fourteen residues are identified in this category (Table I): four as singletons, six as doublets and ten as triplets. Singleton (73), along with category 2 singleton (76), is adjacent to the largelarge subunit contacting residues (Figure 3.I). Singletons (255) and (258) are located at the interface between large subunits and the interacting loop of the second small subunit (Figure 3.IV). Singleton (369) corresponds to the largelarge subunit contacting residue (Lundqvist and Schneider, 1991
; Kitano et al., 2001
) (Figure 3.VI). Although 16 multi-residue SDRCs are identified in this category, they mainly cover two surface spots: the interface between large subunits and the interacting loop of the small subunits and the C-terminal end of loop 6 and adjacent
-helix 6 in the large subunit. Both regions are potentially important hotspots for catalytic activity and the CO2/O2 substrate specificity of the RuBisCo enzymes (Tabita, 1999
; Spreitzer and Salvucci, 2002
). Hence the predicted SDRCs in category 3 demonstrate their relevance to RuBisCo substrate specificity and functions.
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Discussion |
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This result is hardly surprising considering the complicated evolutionary changes in RuBisCo enzymes (Curtis and Clegg, 1984; Turmel et al., 1999
) and biochemical enzymatic processes (Spreitzer, 1993
; Cleland et al., 1998
). Although the origin of chloroplasts among photosynthetic species is still a subject of intensive research (http://www.geocities.com/we_evolve/Plants/chloroplast.html) (Lockhart et al., 1992
; Douglas, 1998
; Raymond et al., 2002
; Parry et al., 2003
), some evidence has shown that marine non-green algal RuBisCos were acquired from an earlier endo-symbiotic event. Indeed, sequence analysis has shown that marine non-green algal RuBisCos diverged greatly from green plant and cyanobacterial RuBisCos. In contrast, green plant and cyanobacterial RuBisCos are much closer in evolution. Furthermore, the three RuBisCo groups have dramatically different CO2/O2 specificities. We believe that there exist different levels of CO2/O2 specificity-determining factors that correspond to those evolutionary events and the specificity levels. Whereas marine non-green algal RuBisCos probably go through a series of considerable residue substitutions in changing substrate specificity conformations, green plants RuBisCos may be more limited in such changes.
Inherent differences among the three RuBisCo groups present a unique opportunity for better elucidation and control of RuBisCo-catalyzed reactions. To understand what factors potentially account for the specificity of RuBisCo proteins, we applied a surface patch ranking method to discover putative specificity-determining residue clusters (SDRCs). In addition, we analyzed the residue substitution patterns (RSPs) to determine when the residues in SDRCs mutated during chloroplast evolution and how they correspond to the specificity changes. Based on our RSP analysis, we divided the identified specificity-determining factors into three categories. Each category is associated uniquely with given RuBisCo groups, their evolutionary history and substrate specificity levels. For example, category 1 of the specificity-determining factors is highly unique to marine non-green algal RuBisCos.
We found that residue substitutions during the evolution of substrate specificity mostly occur in the marine non-green algal RuBisCos (category 1). Our analysis indicates that mutations at some of the residue positions probably provoked considerable conformational structural changes. One of the positions is 247. Residue substitution at this position eliminates a disulfide bridge between two adjacent large subunits in the marine non-green algal RuBisCos (Taylor and Hatrick, 1994; Shibata et al., 1996
). In theory, the removal of the disulfide bridge may affect the structural stability of the enzyme providing a special role of the covalent bond in stabilizing protein structures. This may also partially explain the change in specificity value
from 240 to 80 when the thermophilic red alga Galdieria partita was assayed at 25 and 45°C, respectively (Uemura et al., 1997
). Another position is 338 that is part of loop 6a region critical for the enzymatic function and CO2/O2 specificity. While highly conserved between green plant and cyanobacterial RuBisCos with glutamic acid (E) as their consensus residue, this position is replaced by aspartic acid (D). Although both residues are negatively charged, the former has a larger side chain. The small size discrepancy probably has a great affect on the substrate specificity conformation owing to the geological importance of this residue. It is part of the catalytic core for the RuBisCo enzymes (Tabita, 1999
).
Other considerable residue changes in the non-green algal RuBisCos can be found at positions 339 and 351. Position 339, another residue for the catalytic core of RuBisCos, has a positive residue [arginine (R) or lysine (K) in green plants and cyanobacteria] but is replaced by an imino acid (proline) in the marine non-green algal RuBisCos. This substitution may well have influenced the substrate conformation largely owing to the special role of proline in shaping protein structures. It is often found in the bends of folded protein chains and its cyclic structure can significantly influence protein architecture. Likewise, residues at position 351 have different biochemical properties among the three RuBisCo groups. They are similar in green plant (aspartate, D) and cyanobacterial (glutamine, E) RuBisCos. However, they are substantially different in the marine non-green algal RuBisCos (leucine, L or isoleucine, I). The potential hinge position of the introduced aliphatic R group in leucine or isoleucine plays an important role in adjusting protein structures in the non-green algal RuBisCos.
In contrast to the substantial changes in the non-green algal RuBisCos, residue substitutions are less significant in green plant RuBisCos. Some positions, recognized as singletons in the non-green algal RuBisCo, are identified as doublets or triplets in green plant RuBisCo. For example, positions 339 and 340 are found as a doublet (339 340); positions 222 and 223 are included in triplets whereas all of them are identified as singletons in marine non-green algae. It is likely that only subtle changes are required for the specificity differentiation between green plant and cyanobacterial RuBisCos.
In addition, only a few residue substitutions, especially those in category 3, are observed in green plant and cyanobacterial RuBisCos. This result indicates that green plant RuBisCos have limited mutations in changing substrate specificity conformations whereas marine non-green algal RuBisCos probably went through more considerable changes. Hence we predict that site-directed mutagenesis at positions that reflect those changes will probably succeed in enhancing RuBisCo specificity and, thus, improve photosynthesis.
For residues in category 2, it is not easy to determine what the earliest residue ancestors are and how they are substituted from one RuBisCo group to another. However, what is certain is that they must be highly associated with the CO2/O2 specificity. These residues are extremely conserved within individual RuBisCo groups but completely different among them. Therefore, they can also be considered as potentially very important candidates for site-directed mutagenesis.
In summary, we have studied the factors that potentially account for specificity in three RuBisCo groups. Each discovered factor gives a unique insight into how and when a particular set of residues may have been mutated to change specificity during the evolutionary path of RuBisCo. Considering that site-directed mutagenesis suffers from huge combinatorial choices in identifying critical residue positions that convert or enhance specificity, our analysis suggests not only clusters of residues of potential interest, but also the prioritized order of residue selections.
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Acknowledgements |
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Received July 11, 2005; revised August 13, 2005; accepted September 2, 2005.
Edited by Michael Hecht
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