Evaluation of features for catalytic residue prediction in novel folds

Structural genomics projects are determining the three-dimensional structure of proteins without full characterization of their function. A critical part of the annotation process involves appropriate knowledge representation and prediction of functionally important residue environments. We have developed a method to extract features from sequence, sequence alignments, three-dimensional structure, and structural environment conservation, and used support vector machines to annotate homologous and nonhomologous residue positions based on a specific training set of residue functions. In order to evaluate this pipeline for automated protein annotation, we applied it to the challenging problem of prediction of catalytic residues in enzymes. We also ranked the features based on their ability to discriminate catalytic from noncatalytic residues. When applying our method to a well-annotated set of protein structures, we found that top-ranked features were a measure of sequence conservation, a measure of structural conservation, a degree of uniqueness of a residue's structural environment, solvent accessibility, and residue hydrophobicity. We also found that features based on structural conservation were complementary to those based on sequence conservation and that they were capable of increasing predictor performance. Using a family nonredundant version of the ASTRAL 40 v1.65 data set, we estimated that the true catalytic residues were correctly predicted in 57.0% of the cases, with a precision of 18.5%. When testing on proteins containing novel folds not used in training, the best features were highly correlated with the training on families, thus validating the approach to nonhomologous catalytic residue prediction in general. We then applied the method to 2781 coordinate files from the structural genomics target pipeline and identified both highly ranked and highly clustered groups of predicted catalytic residues.     

Analysis of catalytic residues in enzyme active sites

We present an analysis of the residues directly involved in catalysis in 178 enzyme active sites. Specific criteria were derived to define a catalytic residue, and used to create a catalytic residue dataset, which was then analysed in terms of properties including secondary structure, solvent accessibility, flexibility, conservation, quaternary structure and function. The results indicate the dominance of a small set of amino acid residues in catalysis and give a picture of a general active site environment. It is hoped that this information will provide a better understanding of the molecular mechanisms involved in catalysis and a heuristic basis for predicting catalytic residues in enzymes of unknown function.     

Sequence and structure conservation in a protein core

In order to study structural aspects of sequence conservation in families of homologous proteins, we have analyzed structurally aligned sequences of 585 proteins grouped into 128 homologous families. The conservation of a residue in a family is defined as the average residue similarity in a given position of aligned sequences. The residue similarities were expressed in the form of log-odd substitution tables that take into account the environments of amino acids in three-dimensional structures. The protein core is defined as those residues that have less then 7% solvent accessibility. The density of a protein core is described in terms of atom packing, which is investigated as a criterion for residue substitution and conservation. Although there is no significant correlation between sequence conservation and average atom packing around nonpolar residues such as leucine, valine and isoleucine, a significant correlation is observed for polar residues in the protein core. This may be explained by the hydrogen bonds in which polar residues are involved; the better their protection from water access the more stable should be the structure in that position. Proteins 33:358–366, 1998. © 1998 Wiley-Liss, Inc.     

Prediction of catalytic residues in enzymes based on known tertiary structure,stability profile,and sequence conservation

The catalytic or functionally important residues of a protein are known to exist in evolutionarily constrained regions. However, the patterns of residue conservation alone are sometimes not very informative, depending on the homologous sequences available for a given query protein. Here, we present an integrated method to locate the catalytic residues in an enzyme from its sequence and structure. Mutations of functional residues usually decrease the activity, but concurrently often increase stability. Also, catalytic residues tend to occupy partially buried sites in holes or clefts on the molecular surface. After confirming these general tendencies by carrying out statistical analyses on 49 representative enzymes, these data together with amino acid conservation were evaluated. This novel method exhibited better sensitivity in the prediction accuracy than traditional methods that consider only the residue conservation. We applied it to some so-called "hypothetical" proteins, with known structures but undefined functions. The relationships among the catalytic, conserved, and destabilizing residues in enzymatic proteins are discussed.     

Catalysing new reactions during evolution: economy of residues and mechanism

The diversity of function in some enzyme superfamilies shows that during evolution, enzymes have evolved to catalyse different reactions on the same structure scaffold. In this analysis, we examine in detail how enzymes can modify their chemistry, through a comparison of the catalytic residues and mechanisms in 27 pairs of homologous enzymes of totally different functions. We find that evolution is very economical. Enzymes retain structurally conserved residues to aid catalysis, including residues that bind catalytic metal ions and modulate cofactor chemistry. We examine the conservation of residue type and residue function in these structurally conserved residue pairs. Additionally, enzymes often retain common mechanistic steps catalyzed by structurally conserved residues. We have examined these steps in the context of their overall reactions.     

Conserved core structure and active site residues in alkaline phosphatase superfamily enzymes.

Cofactor-independent phosphoglycerate mutase (iPGM) has been previously identified as a member of the alkaline phosphatase (AlkP) superfamily of enzymes, based on the conservation of the predicted metal-binding residues. Structural alignment of iPGM with AlkP and cerebroside sulfatase confirmed that all these enzymes have a common core structure and revealed similarly located conserved Ser (in iPGM and AlkP) or Cys (in sulfatases) residues in their active sites. In AlkP, this Ser residue is phosphorylated during catalysis, whereas in sulfatases the active site Cys residues are modified to formylglycine and sulfatated. Similarly located Thr residue forms a phosphoenzyme intermediate in one more enzyme of the AlkP superfamily, alkaline phosphodiesterase/nucleotide pyrophosphatase PC-1 (autotaxin). Using structure-based sequence alignment, we identified homologous Ser, Thr, or Cys residues in other enzymes of the AlkP superfamily, such as phosphopentomutase, phosphoglycerol transferase, phosphonoacetate hydrolase, and GPI-anchoring enzymes (glycosylphosphatidylinositol phosphoethanolamine transferases) MCD4, GPI7, and GPI13. We predict that catalytical cycles of all the enzymes of AlkP superfamily include phosphoenzyme (or sulfoenzyme) intermediates.     

Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity

Abstract Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (cIPNS) to similar non-heme Fe²⁺-containing enzymes from 28 different sources (bacterial, fungal, plant and animals) revealed a homologous region of high sequence conservation containing an invariant histidine residue at position 272 in cIPNS. The importance of this histidine residue in cIPNS was investigated through site-directed mutagenesis by replacing the histidine residue with leucine. The mutated gene was verified by DNA sequence analysis and expressed in Escherichia coli . When analyzed by denaturing gel electrophoresis and immunoblotting, the mutant cIPNS had identical mobility as that of the wild-type enzyme. Enzyme studies on the mutant enzyme showed loss of enzymatic activity indicating that His272 is essential for the catalytic function of cIPNS, possibly as a ligand for iron binding.     

Electrostatics in the active site of an alpha-amylase.

The importance of electrostatics in catalysis has been emphasized in the literature for a large number of enzymes. We examined this hypothesis for the Bacillus licheniformis alpha-amylase by constructing site-directed mutants that were predicted to change the pKa values of the catalytic residues and thus change the pH-activity profile of the enzyme. To change the pKa of the catalytic residues in the active site, we constructed mutations that altered the hydrogen bonding network, mutations that changed the solvent accessibility, and mutations that altered the net charge of the molecule. The results show that changing the hydrogen bonding network near an active site residue or changing the solvent accessibility of an active site residue will very likely result in an enzyme with drastically reduced activity. The differences in the pH-activity profiles for these mutants were modest. pH-activity profiles of mutants which change the net charge on the molecule were significantly different from the wild-type pH-activity profile. The differences were, however, difficult to correlate with the electrostatic field changes calculated. In several cases we observed that pH-activity profiles shifted in the opposite direction compared to the shift predicted from electrostatic calculations. This strongly suggests that electrostatic effects cannot be solely responsible for the pH-activity profile of the B. licheniformis alpha-amylase.     

Three-dimensional cluster analysis identifies interfaces and functional residue clusters in proteins

Three-dimensional cluster analysis offers a method for the prediction of functional residue clusters in proteins. This method requires a representative structure and a multiple sequence alignment as input data. Individual residues are represented in terms of regional alignments that reflect both their structural environment and their evolutionary variation, as defined by the alignment of homologous sequences. From the overall (global) and the residue-specific (regional) alignments, we calculate the global and regional similarity matrices, containing scores for all pairwise sequence comparisons in the respective alignments. Comparing the matrices yields two scores for each residue. The regional conservation score (C(R)(x)) defines the conservation of each residue x and its neighbors in 3D space relative to the protein as a whole. The similarity deviation score (S(x)) detects residue clusters with sequence similarities that deviate from the similarities suggested by the full-length sequences. We evaluated 3D cluster analysis on a set of 35 families of proteins with available cocrystal structures, showing small ligand interfaces, nucleic acid interfaces and two types of protein-protein interfaces (transient and stable). We present two examples in detail: fructose-1,6-bisphosphate aldolase and the mitogen-activated protein kinase ERK2. We found that the regional conservation score (C(R)(x)) identifies functional residue clusters better than a scoring scheme that does not take 3D information into account. C(R)(x) is particularly useful for the prediction of poorly conserved, transient protein-protein interfaces. Many of the proteins studied contained residue clusters with elevated similarity deviation scores. These residue clusters correlate with specificity-conferring regions: 3D cluster analysis therefore represents an easily applied method for the prediction of functionally relevant spatial clusters of residues in proteins.     

Identification of catalytic residues using a novel feature that integrates the microenvironment and geometrical location properties of residues

Enzymes play a fundamental role in almost all biological processes and identification of catalytic residues is a crucial step for deciphering the biological functions and understanding the underlying catalytic mechanisms. In this work, we developed a novel structural feature called MEDscore to identify catalytic residues, which integrated the microenvironment (ME) and geometrical properties of amino acid residues. Firstly, we converted a residue's ME into a series of spatially neighboring residue pairs, whose likelihood of being located in a catalytic ME was deduced from a benchmark enzyme dataset. We then calculated an ME-based score, termed as MEscore, by summing up the likelihood of all residue pairs. Secondly, we defined a parameter called Dscore to measure the relative distance of a residue to the center of the protein, provided that catalytic residues are typically located in the center of the protein structure. Finally, we defined the MEDscore feature based on an effective nonlinear integration of MEscore and Dscore. When evaluated on a well-prepared benchmark dataset using five-fold cross-validation tests, MEDscore achieved a robust performance in identifying catalytic residues with an AUC1.0 of 0.889. At a ≤ 10% false positive rate control, MEDscore correctly identified approximately 70% of the catalytic residues. Remarkably, MEDscore achieved a competitive performance compared with the residue conservation score (e.g. CONscore), the most informative singular feature predominantly employed to identify catalytic residues. To the best of our knowledge, MEDscore is the first singular structural feature exhibiting such an advantage. More importantly, we found that MEDscore is complementary with CONscore and a significantly improved performance can be achieved by combining CONscore with MEDscore in a linear manner. As an implementation of this work, MEDscore has been made freely accessible at http://protein.cau.edu.cn/mepi/.     

Accurate Prediction of Protein Catalytic Residues by Side Chain Orientation and Residue Contact Density

Prediction of protein catalytic residues provides useful information for the studies of protein functions. Most of the existing methods combine both structure and sequence information but heavily rely on sequence conservation from multiple sequence alignments. The contribution of structure information is usually less than that of sequence conservation in existing methods. We found a novel structure feature, residue side chain orientation, which is the first structure-based feature that achieves prediction results comparable to that of evolutionary sequence conservation. We developed a structure-based method, Enzyme Catalytic residue SIde-chain Arrangement (EXIA), which is based on residue side chain orientations and backbone flexibility of protein structure. The prediction that uses EXIA outperforms existing structure-based features. The prediction quality of combing EXIA and sequence conservation exceeds that of the state-of-the-art prediction methods. EXIA is designed to predict catalytic residues from single protein structure without needing sequence or structure alignments. It provides invaluable information when there is no sufficient or reliable homology information for target protein. We found that catalytic residues have very special side chain orientation and designed the EXIA method based on the newly discovered feature. It was also found that EXIA performs well for a dataset of enzymes without any bounded ligand in their crystallographic structures.     

Identification of the sequence motif of glycoside hydrolase 13 family members

A bioinformatics analysis of sequences of enzymes of the glycoside hydrolase (GH) 13 family members such as α-amylase, cyclodextrin glycosyltransferase (CGTase), branching enzyme and cyclomaltodextrinase has been carried out in order to find out the sequence motifs that govern the reactions specificities of these enzymes by using hidden Markov model (HMM) profile. This analysis suggests the existence of such sequence motifs and residues of these motifs constituting the -1 to +3 catalytic subsites of the enzyme. Hence, by introducing mutations in the residues of these four subsites, one can change the reaction specificities of the enzymes. In general it has been observed that α -amylase sequence motif have low sequence conservation than rest of the motifs of the GH13 family members.     

Prediction and evolutionary information analysis of protein solvent accessibility using multiple linear regression

A multiple linear regression method was applied to predict real values of solvent accessibility from the sequence and evolutionary information. This method allowed us to obtain coefficients of regression and correlation between the occurrence of an amino-acid residue at a specific target and its sequence neighbor positions on the one hand, and the solvent accessibility of that residue on the other. Our linear regression model based on sequence information and evolutionary models was found to predict residue accessibility with 18.9% and 16.2% mean absolute error respectively, which is better than or comparable to the best available methods. A correlation matrix for several neighbor positions to examine the role of evolutionary information at these positions has been developed and analyzed. As expected, the effective frequency of hydrophobic residues at target positions shows a strong negative correlation with solvent accessibility, whereas the reverse is true for charged and polar residues. The correlation of solvent accessibility with effective frequencies at neighboring positions falls abruptly with distance from target residues. Longer protein chains have been found to be more accurately predicted than their smaller counterparts.     

Three-dimensional structure of the catalytic core of acetylxylan esterase from Trichoderma reesei: insights into the deacetylation mechanism

Acetylxylan esterase from Trichoderma reesei removes acetyl side groups from xylan. The crystal structure of the catalytic core of the enzyme was solved at 1.9 A resolution. The core has an alpha/beta/alpha sandwich fold, similar to that of homologous acetylxylan esterase from Penicillium purpurogenum and cutinase from Fusarium solani. All three enzymes belong to family 5 of the carbohydrate esterases and the superfamily of the alpha/beta hydrolase fold. Evidently, the enzymes have diverged from a common ancestor and they share the same catalytic mechanism. The catalytic machinery of acetylxylan esterase from T. reesei was studied by comparison with cutinase, the catalytic site of which is well known. Acetylxylan esterase is a pure serine esterase having a catalytic triad (Ser90, His187, and Asp175) and an oxyanion hole (Thr13 N, and Thr13 O gamma). Although the catalytic triad of acetylxylan esterase has been reported previously, there has been no mention of the oxyanion hole. A model for the binding of substrates is presented on the basis of the docking of xylose. Acetylxylan esterase from T. reesei is able to deacetylate both mono- and double-acetylated residues, but it is not able to remove acetyl groups located close to large side groups such as 4-O-methylglucuronic acid. If the xylopyranoside residue is double-acetylated, both acetyl groups are removed by the catalytic triad: first one acetyl group is removed and then the residue is reorientated so that the nucleophilic oxygen of serine can attack the second acetyl group.     

A newly identified, essential catalytic residue in a critical secondary structure element in the integrase family of site-specific recombinases is conserved in a similar element in eucaryotic type IB topoisomerases.

The integrase family of site-specific recombinases catalyzes conservative rearrangements between defined segments of DNA. A highly conserved tetrad (RHRY) of catalytic residues is essential for this process. This tetrad is dispersed in two motifs in the linear sequence, but is configured appropriately in the catalytic pocket to execute the strand cleavage and rejoining reactions. A third conserved motif has been identified in the Xer subgroup of the integrase family. Mutational analysis of 12 conserved residues in this motif in the XerD protein from Salmonella typhimurium led to the identification of an essential fifth catalytic residue (lysine 172) which is implicated in strand cleavage or exchange. This lysine residue occupies part of the turn of an antiparallel beta-hairpin which forms one side of the catalytic cleft in XerD, and is found at similar positions among evolutionarily diverse integrase family members. Related antiparallel beta-hairpins are present in eucaryotic type IB topoisomerase enzymes which also contain a critical lysine residue in the turn of the hairpin. In both the integrase family and eucaryotic type IB topoisomerases, the catalytic lysine residues are in close contact with the substrates and may play similar roles in influencing the reactivity of the phosphotyrosine intermediates formed during reactions catalyzed by both enzymes.     

Cold-active citrate synthase: mutagenesis of active-site residues.

A comparison of the crystal structure of the dimeric enzyme citrate synthase from the psychrophilic Arthrobacter strain DS2-3R with that of the structurally homologous enzyme from the hyperthermophilic Pyrococcus furiosus reveals a significant difference in the accessibility of their active sites to substrates. In this work, we investigated the possible role in cold activity of the greater accessibility of the Arthrobacter citrate synthase. By site-directed mutagenesis, we replaced two alanine residues at the entrance to the active site with an arginine and glutamate residue, respectively, as found in the equivalent positions of the Pyrococcus enzyme Also, we introduced a loop into the active site of the psychrophilic citrate synthase, again mimicking the situation in the hyperthermophilic enzyme. Analysis of the thermoactivity and thermostability of the mutant enzymes reveals that cold activity is not significantly compromised by the mutations, but rather the affinity for one of the substrates, acetyl-CoA, is dramatically increased. Moreover, one mutant (Loop insertion/K313L/A361R) has an increased thermostability but a reduced temperature optimum for catalytic activity. This unexpected relationship between stability and activity is discussed with respect to the nature of the dependence of catalytic activity on temperature.     

Sequence and structural differences between enzyme and nonenzyme homologs

To improve our understanding of the evolution of novel functions, we performed a sequence, structural, and functional analysis of homologous enzymes and nonenzymes of known three-dimensional structure. In most examples identified, the nonenzyme is derived from an ancestral catalytic precursor (as opposed to the reverse evolutionary scenario, nonenzyme to enzyme), and the active site pocket has been disrupted in some way, owing to the substitution of critical catalytic residues and/or steric interactions that impede substrate binding and catalysis. Pairwise sequence identity is typically insignificant, and almost one-half of the enzyme and nonenzyme pairs do not share any similarity in function. Heterooligomeric enzymes comprising homologous subunits in which one chain is catalytically inactive and enzyme polypeptides that contain internal catalytic and noncatalytic duplications of an ancient enzyme domain are also discussed.     

Studies into factors contributing to substrate specificity of membrane-bound 3-ketoacyl-CoA synthases.

We are interested in constructing a model for the substrate-binding site of fatty acid elongase-1 3-ketoacyl CoA synthase (FAE1 KCS), the enzyme responsible for production of very long chain fatty acids of plant seed oils. Arabidopsis thaliana and Brassica napus FAE1 KCS enzymes are highly homologous but the seed oil content of these plants suggests that their substrate specificities differ with respect to acyl chain length. We used in vivo and in vitro assays of Saccharomyces cerevisiae-expressed FAE1 KCSs to demonstrate that the B. napus FAE1 KCS enzyme favors longer chain acyl substrates than the A. thaliana enzyme. Domains/residues responsible for substrate specificity were investigated by determining catalytic activity and substrate specificity of chimeric enzymes of A. thaliana and B. napus FAE1 KCS. The N-terminal region, excluding the transmembrane domain, was shown to be involved in substrate specificity. One chimeric enzyme that included A. thaliana sequence from the N terminus to residue 114 and B. napus sequence from residue 115 to the C terminus had substrate specificity similar to that of A. thaliana FAE1 KCS. However, a K92R substitution in this chimeric enzyme changed the specificity to that of the B. napus enzyme without loss of catalytic activity. Thus, this study was successful in identifying a domain involved in determining substrate specificity in FAE1 KCS and in engineering an enzyme with novel activity.