Using amphiphilic pseudo amino acid composition to predict enzyme subfamily classes

MOTIVATION: With protein sequences entering into databanks at an explosive pace, the early determination of the family or subfamily class for a newly found enzyme molecule becomes important because this is directly related to the detailed information about which specific target it acts on, as well as to its catalytic process and biological function. Unfortunately, it is both time-consuming and costly to do so by experiments alone. In a previous study, the covariant-discriminant algorithm was introduced to identify the 16 subfamily classes of oxidoreductases. Although the results were quite encouraging, the entire prediction process was based on the amino acid composition alone without including any sequence-order information. Therefore, it is worthy of further investigation. RESULTS: To incorporate the sequence-order effects into the predictor, the 'amphiphilic pseudo amino acid composition' is introduced to represent the statistical sample of a protein. The novel representation contains 20 + 2lambda discrete numbers: the first 20 numbers are the components of the conventional amino acid composition; the next 2lambda numbers are a set of correlation factors that reflect different hydrophobicity and hydrophilicity distribution patterns along a protein chain. Based on such a concept and formulation scheme, a new predictor is developed. It is shown by the self-consistency test, jackknife test and independent dataset tests that the success rates obtained by the new predictor are all significantly higher than those by the previous predictors. The significant enhancement in success rates also implies that the distribution of hydrophobicity and hydrophilicity of the amino acid residues along a protein chain plays a very important role to its structure and function.     

Evolutionary dynamics of the ABCA chromosome 17q24 cluster genes in vertebrates

ABCA is a subfamily of ATP-binding-cassette (ABC) transporter genes. In this subfamily, it was found that five ABCA genes cluster in a head-to-tail pattern in the human and mouse genomes, but only one was found in fish. To understand better the evolution of this cluster of genes, we screened 11 vertebrate genome sequences and newly identified 28 ABCA cluster genes. Comparative genomic analysis reveals that the ABCA5 gene is relatively evolutionarily conserved. In contrast, the repertoires of the other ABCA genes in this cluster diverge tremendously among species, which is due mainly to postspeciation duplications. In addition, maximum likelihood analysis reveals that positive selection is acting on the paralogous genes ABCA6 and Abca8a, suggesting that these two genes have possibly acquired new functions after duplication. Because most eukaryotic ABC proteins integrate into the cytoplasmic membrane and transport a wide range of substrates across it, we conjecture that newly duplicated ABCA cluster genes are under diversifying selection for the ability to recognize a diverse array of substrates.     

Sequence fingerprint and structural analysis of the SCOR enzyme A3DFK9 from Clostridium thermocellum

We have identified a highly conserved fingerprint of 40 residues in the TGYK subfamily of the short‐chain oxidoreductase enzymes. The TGYK subfamily is defined by the presence of an N‐terminal TGxxxGxG motif and a catalytic YxxxK motif. This subfamily contains more than 12,000 members, with individual members displaying unique substrate specificities. The 40 fingerprint residues are critical to catalysis, cofactor binding, protein folding, and oligomerization but are substrate independent. Their conservation provides critical insight into evolution of the folding and function of TGYK enzymes. Substrate specificity is determined by distinct combinations of residues in three flexible loops that make up the substrate‐binding pocket. Here, we report the structure determinations of the TGYK enzyme A3DFK9 from Clostridium thermocellum in its apo form and with bound NAD⁺ cofactor. The function of this protein is unknown, but our analysis of the substrate‐binding loops putatively identifies A3DFK9 as a carbohydrate or polyalcohol metabolizing enzyme. C. thermocellum has potential commercial applications because of its ability to convert biomaterial into ethanol. A3DFK9 contains 31 of the 40 TGYK subfamily fingerprint residues. The most significant variations are the substitution of a cysteine (Cys84) for a highly conserved glycine within a characteristic VNNAG motif, and the substitution of a glycine (Gly106) for a highly conserved asparagine residue at a helical kink. Both of these variations occur at positions typically participating in the formation of a catalytically important proton transfer network. An alternate means of stabilizing this proton wire was observed in the A3DFK9 crystal structures. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

A conserved lysine residue in the crenarchaea-specific loop is important for the crenarchaeal splicing endonuclease activity

In Archaea, splicing endonuclease (EndA) recognizes and cleaves precursor RNAs to remove introns. Currently, EndAs are classified into three families according to their subunit structures: homotetramer, homodimer, and heterotetramer. The crenarchaeal heterotetrameric EndAs can be further classified into two subfamilies based on the size of the structural subunit. Subfamily A possesses a structural subunit similar in size to the catalytic subunit, whereas subfamily B possesses a structural subunit significantly smaller than the catalytic subunit. Previously, we solved the crystal structure of an EndA from Pyrobaculum aerophilum. The endonuclease was classified into subfamily B, and the structure revealed that the enzyme lacks an N-terminal subdomain in the structural subunit. However, no structural information is available for crenarchaeal heterotetrameric EndAs that are predicted to belong to subfamily A. Here, we report the crystal structure of the EndA from Aeropyrum pernix, which is predicted to belong to subfamily A. The enzyme possesses the N-terminal subdomain in the structural subunit, revealing that the two subfamilies of heterotetrameric EndAs are structurally distinct. EndA from A. pernix also possesses an extra loop region that is characteristic of crenarchaeal EndAs. Our mutational study revealed that the conserved lysine residue in the loop is important for endonuclease activity. Furthermore, the sequence characteristics of the loops and the positions towards the substrate RNA according to a docking model prompted us to propose that crenarchaea-specific loops and an extra amino acid sequence at the catalytic loop of nanoarchaeal EndA are derived by independent convergent evolution and function for recognizing noncanonical bulge-helix-bulge motif RNAs as substrates.     

Phylogenetic analysis of haloalkane dehalogenases

Haloalkane dehalogenases (HLDs) are enzymes that catalyze the cleavage of carbon-halogen bonds by a hydrolytic mechanism. Although comparative biochemical analyses have been published, no classification system has been proposed for HLDs, to date, that reconciles their phylogenetic and functional relationships. In the study presented here, we have analyzed all sequences and structures of genuine HLDs and their homologs detectable by database searches. Phylogenetic analyses revealed that the HLD family can be divided into three subfamilies denoted HLD-I, HLD-II, and HLD-III, of which HLD-I and HLD-III are predicted to be sister-groups. A mismatch between the HLD protein tree and the tree of species, as well as the presence of more than one HLD gene in a few genomes, suggest that horizontal gene transfers, and perhaps also multiple gene duplications and losses have been involved in the evolution of this family. Most of the biochemically characterized HLDs are found in the HLD-II subfamily. The dehalogenating activity of two members of the newly identified HLD-III subfamily has only recently been confirmed, in a study motivated by this phylogenetic analysis. A novel type of the catalytic pentad (Asp-His-Asp+Asn-Trp) was predicted for members of the HLD-III subfamily. Calculation of the evolutionary rates and lineage-specific innovations revealed a common conserved core as well as a set of residues that characterizes each HLD subfamily. The N-terminal part of the cap domain is one of the most variable regions within the whole family as well as within individual subfamilies, and serves as a preferential site for the location of relatively long insertions. The highest variability of discrete sites was observed among residues that are structural components of the access channels. Mutations at these sites modify the anatomy of the channels, which are important for the exchange of ligands between the buried active site and the bulk solvent, thus creating a structural basis for the molecular evolution of new substrate specificities. Our analysis sheds light on the evolutionary history of HLDs and provides a structural framework for designing enzymes with new specificities.     

Silencing of subfamily I of protein phosphatase 2A catalytic subunits results in activation of plant defense responses and localized cell death

The central importance of protein phosphorylation in plant defense responses has been demonstrated by the isolation of several disease-resistance genes that encode protein kinases. In addition, there are many reports of changes in protein phosphorylation accompanying plant responses to pathogens. In contrast, little is known about the role of protein dephosphorylation in regulating plant defenses. We report that expression of the LePP2Ac1 gene, which encodes a catalytic subunit of the heterotrimeric protein phosphatase 2A (PP2Ac), is rapidly induced in resistant tomato leaves upon inoculation with an avirulent strain of Pseudomonas syringae pv. tomato. By analysis of PP2Ac gene sequences from several plant species, we found that PP2Ac genes cluster into two subfamilies, with LePP2Ac1 belonging to subfamily I. Virus-induced gene silencing (VIGS) in Nicotiana benthamiana was used to suppress expression of genes from subfamily I and not from subfamily II. The PP2Ac-silenced plants had greatly decreased PP2A activity, constitutively expressed pathogenesis-related (PR) genes, and developed localized cell death in stems and leaves. In addition, the plants were more resistant to a virulent strain of P. syringae pv. tabaci and showed an accelerated hypersensitive response (HR) to effector proteins from both P. syringae and the fungal pathogen, Cladosporium fulvum. Thus, catalytic subunits of PP2Ac subfamily I act as negative regulators of plant defense responses likely by de-sensitizing protein phosphorylation cascades.     

Genomic structure of murine macrophage inflammatory protein-1 alpha and conservation of potential regulatory sequences with a human homolog, LD78

The gene for a murine macrophage inflammatory cytokine, MIP-1 alpha, belongs to a newly recognized superfamily encoding small, inducible peptides shown to be up-regulated in association with cellular activation or transformation (tentatively designated the scy, or small cytokine, gene family). Secreted scy family peptides as a group, and MIP-1 alpha in particular, have inflammatory and mitogenic activities, and the family has been divided into CXC and CC subfamilies according to the spacing of conserved cysteine residues in the primary amino acid sequences. We have isolated and characterized a genomic clone encoding the CC subfamily member MIP-1 alpha. The organization of the murine MIP-1 alpha gene into three exons interrupted by two introns is identical to that found for other members of the CC subfamily (e.g., huLD78, muJE, huJE/MCP-1, muTCA3, and hul-309), which has been taken as evidence of evolution from a common ancestral gene. With the exception of the ratPF4 gene, which shares the two-intron/three-exon pattern typical of the CC subfamily, sequenced genes encoding CXC subfamily peptides (e.g., hulL-8 and hulP-10) include an additional intervening sequence that creates a fourth exon. Genomic nucleotide sequences 5' of the MIP-1 alpha cap site are highly homologous to corresponding regions of the human gene encoding a CC peptide variously designated as LD78/GOS19/pAT464, including consensus regulatory motifs in common, reinforcing the contention that MIP-1 alpha and LD78 may be interspecies homologs.     

Identification and Phylogenetic Characterization of a New Subfamily of α-Amylase Enzymes from Marine Microorganisms

A gene encoding a starch-hydrolyzing enzyme was isolated from a marine metagenomic library and overexpressed in Escherichia coli. The enzyme, designated AmyP, shows very low similarity to full-length sequences of known α-amylases, although a catalytic domain correlated with the α-amylase superfamily was identified. Based on the range of substrate hydrolysis and the product profile, the protein was clearly defined as a saccharifying-type α-amylase. Sequence comparison indicated that AmyP was related to four putative glycosidases previously identified only in bacterial genome sequences. They were all from marine bacteria and formed a new subfamily of glycoside hydrolase GH13. Moreover, this subfamily was closely related to the probable genuine bacterial α-amylases (GH13_19). The results suggested that the subfamily may be an independent clade of ancestral marine bacterial α-amylases.     

The three-dimensional structure of human transaldolase

The crystal structure of human transaldolase has been determined to 2.45 A resolution. The enzyme folds into an alpha/beta barrel structure and is thus similar in structure to other class I aldolases. Structure-based sequence alignment of available sequences of the transaldolase subfamily reveals that eight active site residues are invariant in the whole subfamily. Other invariant residues are mainly involved in the formation of the hydrophobic core of the enzyme. Noteworthy is a hydrophobic cluster consisting of five invariant residues. Human transaldolase has been implicated as an autoantigen in multiple sclerosis and four immunodominant peptide segments are located at the surface of the enzyme, accessible to autoantibodies.     

The chemistry of protein catalysis

We report, for the first time, on the statistics of chemical mechanisms and amino acid residue functions that occur in enzyme reaction sequences using the MACiE database of 202 distinct enzyme reaction mechanisms as a knowledge base. MACiE currently holds representatives from each Enzyme Commission sub-subclass where there is an available crystal structure and sufficient evidence in the primary literature for a mechanism. Each catalytic step of every reaction sequence in MACiE is fully annotated, so that it includes the function of the catalytic residues involved in the reaction and the chemical mechanisms by which substrates are transformed into products. We show that the most catalytic amino acid residues are histidine, cysteine and aspartate, which are also the residues whose side-chains are more likely to serve as reactants, and that have the greatest versatility of function. We show that electrophilic reactions in enzymes are very rare, and the majority of enzyme reactions rely upon nucleophilic and general acid/base chemistry. However, although rare, radical (homolytic) reactions are much more common than electrophilic reactions. Thus, the majority of amino acid residues perform stabilisation roles (as spectators) or proton shuttling roles (as reactants). The analysis presented provides a better understanding of the mechanisms of enzyme catalysis and may act as an initial step in the validation and prediction of mechanism in an enzyme active site.     

Mechanism for N₂O generation in bacterial nitric oxide reductase: a quantum chemical study

The catalytic mechanism of reduction of NO to N(2)O in the bacterial enzyme nitric oxide reductase has been investigated using hybrid density functional theory and a model of the binuclear center (BNC) based on the newly determined crystal structure. The calculations strongly suggest a so-called cis:b(3) mechanism, while the commonly suggested trans mechanism is found to be energetically unfavorable. The mechanism suggested here involves a stable cis-hyponitrite, and it is shown that from this intermediate one N-O bond can be cleaved without the transfer of a proton or an electron into the binuclear active site, in agreement with experimental observations. The fully oxidized intermediate in the catalytic cycle and the resting form of the enzyme are suggested to have an oxo-bridged BNC with two high-spin ferric irons antiferromagnetically coupled. Both steps of reduction of the BNC after N(2)O formation are found to be pH-dependent, also in agreement with experiment. Finally, it is found that the oxo bridge in the oxidized BNC can react with NO to give nitrite, which explains the experimental observations that the fully oxidized enzyme reacts with NO, and most likely also the observed substrate inhibition at higher NO concentrations.     

Identification and characterization of Piwi subfamily in insects

As a subfamily of Argonaute proteins, Piwi is poorly understood compared with Ago subfamily until recent discovery of Piwi protein interacting with piRNA. We did a large scale screening of insect genomes to identify piwi-like genes. Full or partial cDNA sequences were obtained by EST elongation and GENSCAN. We found that the exon numbers were totally different between vertebrates and invertebrates, approximately 20 exons in mammals but only 6-9 exons in insects. This infers either intron insertion or loss occurred during evolution. Characterized PAZ, c-terminal PIWI domains exist in almost all predicted Piwi-like proteins. We found six conserved motifs, which contain active catalytic triad "Asp-Asp-His/Lys" required for slicer activity. The expression of siwi1 and siwi2 in Bombyx mori were verified with RT-PCR. Phylogenetic tree inferred by Bayesian algorithm indicates invertebrate Piwi-like proteins are classified into three clades, of which Ago3 clade is closer to mammalian Piwi proteins.     

Evidence for a role of sulfhydryl groups in catalytic activity and subunit interaction of the cyclic GMP-dependent protein kinase from silkworm.

Guanosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from silkworm pupal fat body (Bombyx mori) which is devoid of any adenosine 3':5'-monophosphate-dependent protein kinase. The enzyme displayed catalytic properties which were roughly similar to those described for adenosine 3':5'-monophosphate-dependent protein kinase. This similarity has been found in substrate specificity, optimal Mg2+ dependency, polyamines effects and the lack of dependency upon sulfhydryl compound for activation by cyclic GMP. Treatment of the enzyme with sulfhydryl reagents, N-ethylmaleimide or p-chloromercuribenzoic acid, inhibited the catalytic activity as well as the dissociation of the binding and catalytic activities as shown by means of sucrose-density gradient ultracentrifugation. In the presence of cyclic GMP or histone, the disulfide-linked structure did not dissociate into separate subunits nor did it migrate as the holoenzyme but sedimented as an intermediate form carrying both binding and catalytic activities.     

Using support vector machines to distinguish enzymes: Approached by incorporating wavelet transform

The enzymatic attributes of newly found protein sequences are usually determined either by biochemical analysis of eukaryotic and prokaryotic genomes or by microarray chips. These experimental methods are both time-consuming and costly. With the explosion of protein sequences registered in the databanks, it is highly desirable to develop an automated method to identify whether a given new sequence belongs to enzyme or non-enzyme. The discrete wavelet transform (DWT) and support vector machine (SVM) have been used in this study for distinguishing enzyme structures from non-enzymes. The networks have been trained and tested on two datasets of proteins with different wavelet basis functions, decomposition scales and hydrophobicity data types. Maximum accuracy has been obtained using SVM with a wavelet function of Bior2.4, a decomposition scale j=5, and Kyte-Doolittle hydrophobicity scales. The results obtained by the self-consistency test, jackknife test and independent dataset test are encouraging, which indicates that the proposed method can be employed as a useful assistant technique for distinguishing enzymes from non-enzymes.     

Structural Basis for Dimerization and Catalysis of a Novel Esterase from the GTSAG Motif Subfamily of the Bacterial Hormone-sensitive Lipase Family

Hormone-sensitive lipases (HSLs) are widely distributed in microorganisms, plants, and animals. Microbial HSLs are classified into two subfamilies, an unnamed new subfamily and the GDSAG motif subfamily. Due to the lack of structural information, the detailed catalytic mechanism of the new subfamily is not yet clarified. Based on sequence analysis, we propose to name the new subfamily as the GTSAG motif subfamily. We identified a novel HSL esterase E25, a member of the GTSAG motif subfamily, by functional metagenomic screening, and resolved its structure at 2.05 Å. E25 is mesophilic (optimum temperature at 50 °C), salt-tolerant, slightly alkaline (optimum pH at 8.5) for its activity, and capable of hydrolyzing short chain monoesters (C2–C10). E25 tends to form dimers both in the crystal and in solution. An E25 monomer contains an N-terminal CAP domain, and a classical α/β hydrolase-fold domain. Residues Ser¹⁸⁶, Asp²⁸², and His³¹² comprise the catalytic triad. Structural and mutational analyses indicated that E25 adopts a dimerization pattern distinct from other HSLs. E25 dimer is mainly stabilized by an N-terminal loop intersection from the CAP domains and hydrogen bonds and salt bridges involving seven highly conserved hydrophilic residues from the catalytic domains. Further analysis indicated that E25 also has some catalytic profiles different from other HSLs. Dimerization is essential for E25 to exert its catalytic activity by keeping the accurate orientation of the catalytic Asp²⁸² within the catalytic triad. Our results reveal the structural basis for dimerization and catalysis of an esterase from the GTSAG motif subfamily of the HSL family.     

Inactive enzyme-homologues find new function in regulatory processes

Although the catalytic center of an enzyme is usually highly conserved, there have been a few reports of proteins with substitutions at essential catalytic positions, which convert the enzyme into a catalytically inactive form. Here, we report a large-scale analysis of substitutions at enzymes' catalytic sites in order to gain insight into the function and evolution of inactive enzyme-homologues. Our analysis revealed that inactive enzyme-homologues are not an exception only found in single enzyme families, but that they are represented in a large variety of enzyme families and conserved among metazoan species. Even though they have lost their catalytic activity, they have adopted new functions and are now mainly involved in regulatory processes, as shown by several case studies. This modification of existing modules is an efficient mechanism to evolve new functions. The invention of inactive enzyme-homologues in metazoa has thereby led to an enhancement of complexity of regulatory networks.     

Hybrid and Rogue Kinases Encoded in the Genomes of Model Eukaryotes

The highly modular nature of protein kinases generates diverse functional roles mediated by evolutionary events such as domain recombination, insertion and deletion of domains. Usually domain architecture of a kinase is related to the subfamily to which the kinase catalytic domain belongs. However outlier kinases with unusual domain architectures serve in the expansion of the functional space of the protein kinase family. For example, Src kinases are made-up of SH2 and SH3 domains in addition to the kinase catalytic domain. A kinase which lacks these two domains but retains sequence characteristics within the kinase catalytic domain is an outlier that is likely to have modes of regulation different from classical src kinases. This study defines two types of outlier kinases: hybrids and rogues depending on the nature of domain recombination. Hybrid kinases are those where the catalytic kinase domain belongs to a kinase subfamily but the domain architecture is typical of another kinase subfamily. Rogue kinases are those with kinase catalytic domain characteristic of a kinase subfamily but the domain architecture is typical of neither that subfamily nor any other kinase subfamily. This report provides a consolidated set of such hybrid and rogue kinases gleaned from six eukaryotic genomes–S.cerevisiae, D. melanogaster, C.elegans, M.musculus, T.rubripes and H.sapiens–and discusses their functions. The presence of such kinases necessitates a revisiting of the classification scheme of the protein kinase family using full length sequences apart from classical classification using solely the sequences of kinase catalytic domains. The study of these kinases provides a good insight in engineering signalling pathways for a desired output. Lastly, identification of hybrids and rogues in pathogenic protozoa such as P.falciparum sheds light on possible strategies in host-pathogen interactions.     

Sequence-based enzyme catalytic domain prediction using clustering and aggregated mutual information content

Characterizing enzyme sequences and identifying their active sites is a very important task. The current experimental methods are too expensive and labor intensive to handle the rapidly accumulating protein sequences and structure data. Thus accurate, high-throughput in silico methods for identifying catalytic residues and enzyme function prediction are much needed. In this paper, we propose a novel sequence-based catalytic domain prediction method using a sequence clustering and an information-theoretic approaches. The first step is to perform the sequence clustering analysis of enzyme sequences from the same functional category (those with the same EC label). The clustering analysis is used to handle the problem of widely varying sequence similarity levels in enzyme sequences. The clustering analysis constructs a sequence graph where nodes are enzyme sequences and edges are a pair of sequences with a certain degree of sequence similarity, and uses graph properties, such as biconnected components and articulation points, to generate sequence segments common to the enzyme sequences. Then amino acid subsequences in the common shared regions are aligned and then an information theoretic approach called aggregated column related scoring scheme is performed to highlight potential active sites in enzyme sequences. The aggregated information content scoring scheme is shown to be effective to highlight residues of active sites effectively. The proposed method of combining the clustering and the aggregated information content scoring methods was successful in highlighting known catalytic sites in enzymes of Escherichia coli K12 in terms of the Catalytic Site Atlas database. Our method is shown to be not only accurate in predicting potential active sites in the enzyme sequences but also computationally efficient since the clustering approach utilizes two graph properties that can be computed in linear to the number of edges in the sequence graph and computation of mutual information does not require much time. We believe that the proposed method can be useful for identifying active sites of enzyme sequences from many genome projects.     

Crystal structure of a tetrameric GDP-D-mannose 4,6-dehydratase from a bacterial GDP-D-rhamnose biosynthetic pathway

d-Rhamnose is a rare 6-deoxy monosaccharide primarily found in the lipopolysaccharide of pathogenic bacteria, where it is involved in host-bacterium interactions and the establishment of infection. The biosynthesis of d-rhamnose proceeds through the conversion of GDP-d-mannose by GDP-d-mannose 4,6-dehydratase (GMD) to GDP-4-keto-6-deoxymannose, which is subsequently reduced to GDP-d-rhamnose by a reductase. We have determined the crystal structure of GMD from Pseudomonas aeruginosa in complex with NADPH and GDP. GMD belongs to the NDP-sugar modifying subfamily of the short-chain dehydrogenase/reductase (SDR) enzymes, all of which exhibit bidomain structures and a conserved catalytic triad (Tyr-XXX-Lys and Ser/Thr). Although most members of this enzyme subfamily display homodimeric structures, this bacterial GMD forms a tetramer in the same fashion as the plant MUR1 from Arabidopsis thaliana. The cofactor binding sites are adjoined across the tetramer interface, which brings the adenosyl phosphate moieties of the adjacent NADPH molecules to within 7 A of each other. A short peptide segment (Arg35-Arg43) stretches into the neighboring monomer, making not only protein-protein interactions but also hydrogen bonding interactions with the neighboring cofactor. The interface hydrogen bonds made by the Arg35-Arg43 segment are generally conserved in GMD and MUR1, and the interacting residues are highly conserved among the sequences of bacterial and eukaryotic GMDs. Outside of the Arg35-Arg43 segment, residues involved in tetrameric contacts are also quite conserved across different species. These observations suggest that a tetramer is the preferred, and perhaps functionally relevant, oligomeric state for most bacterial and eukaryotic GMDs.