Small-molecule metabolism: an enzyme mosaic

Escherichia coli has been a popular organism for studying metabolic pathways. In an attempt to find out more about how these pathways are constructed, the enzymes were analysed by defining their protein domains. Structural assignments and sequence comparisons were used to show that 213 domain families constitute 90% of the enzymes in the small-molecule metabolic pathways. Catalytic or cofactor-binding properties between family members are often conserved, while recognition of the main substrate with change in catalytic mechanism is only observed in a few cases of consecutive enzymes in a pathway. Recruitment of domains across pathways is very common, but there is little regularity in the pattern of domains in metabolic pathways. This is analogous to a mosaic in which a stone of a certain colour is selected to fill a position in the picture.     

Recognition of remotely related structural homologues using sequence profiles of aligned homologous protein structures

In order to bridge the gap between proteins with three-dimensional (3-D) structural information and those without 3-D structures, extensive experimental and computational efforts for structure recognition are being invested. One of the rapid and simple computational approaches for structure recognition makes use of sequence profiles with sensitive profile matching procedures to identify remotely related homologous families. While adopting this approach we used profiles that are generated from structure-based sequence alignment of homologous protein domains of known structures integrated with sequence homologues. We present an assessment of this fast and simple approach. About one year ago, using this approach, we had identified structural homologues for 315 sequence families, which were not known to have any 3-D structural information. The subsequent experimental structure determination for at least one of the members in 110 of 315 sequence families allowed a retrospective assessment of the correctness of structure recognition. We demonstrate that correct folds are detected with an accuracy of 96.4% (106/110). Most (81/106) of the associations are made correctly to the specific structural family. For 23/106, the structure associations are valid at the superfamily level. Thus, profiles of protein families of known structure when used with sensitive profile-based search procedure result in structure association of high confidence. Further assignment at the level of superfamily or family would provide clues to probable functions of new proteins. Importantly, the public availability of these profiles from us could enable one to perform genome wide structure assignment in a local machine in a fast and accurate manner.     

Multi-domain protein families and domain pairs: comparison with known structures and a random model of domain recombination

There is a limited repertoire of domain families in nature that are duplicated and combined in different ways to form the set of proteins in a genome. Most proteins in both prokaryote and eukaryote genomes consist of two or more domains, and we show that the family size distribution of multi-domain protein families follows a power law like that of individual families. Most domain pairs occur in four to six different domain architectures: in isolation and in combinations with different partners. We showed previously that within the set of all pairwise domain combinations, most small and medium-sized families are observed in combination with one or two other families, while a few large families are very versatile and combine with many different partners. Though this may appear to be a stochastic pattern, in which large families have more combination partners by virtue of their size, we establish here that all the domain families with more than three members in genomes are duplicated more frequently than would be expected by chance considering their number of neighbouring domains. This duplication of domain pairs is statistically significant for between one and three quarters of all families with seven or more members. For the majority of pairwise domain combinations, there is no known three-dimensional structure of the two domains together, and we term these novel combinations. Novel domain combinations are interesting and important targets for structural elucidation, as the geometry and interaction between the domains will help understand the function and evolution of multi-domain proteins. Of particular interest are those combinations that occur in the largest number of multi-domain proteins, and several of these frequent novel combinations contain DNA-binding domains.Abbreviations:SCOP: Structural Classification of Proteins database, PDB: Protein DataBank, HMM: hidden Markov model     

The bile/arsenite/riboflavin transporter (BART) superfamily

Secondary transmembrane transport carriers fall into families and superfamilies allowing prediction of structure and function. Here we describe hundreds of sequenced homologues that belong to six families within a novel superfamily, the bile/arsenite/riboflavin transporter (BART) superfamily, of transport systems and putative signalling proteins. Functional data for members of three of these families are available, and they transport bile salts and other organic anions, the bile acid:Na(+) symporter (BASS) family, inorganic anions such as arsenite and antimonite, the arsenical resistance-3 (Acr3) family, and the riboflavin transporter (RFT) family. The first two of these families, as well as one more family with no functionally characterized members, exhibit a probable 10 transmembrane spanner (TMS) topology that arose from a tandemly duplicated 5 TMS unit. Members of the RFT family have a 5 TMS topology, and are homologous to each of the repeat units in the 10 TMS proteins. The other two families [sensor histidine kinase (SHK) and kinase/phosphatase/synthetase/hydrolase (KPSH)] have a single 5 TMS unit preceded by an N-terminal TMS and followed by a hydrophilic sensor histidine kinase domain (the SHK family) or catalytic domains resembling sensor kinase, phosphatase, cyclic di-GMP synthetase and cyclic di-GMP hydrolase catalytic domains, as well as various noncatalytic domains (the KPSH family). Because functional data are not available for members of the SHK and KPSH families, it is not known if the transporter domains retain transport activity or have evolved exclusive functions in molecular reception and signal transmission. This report presents characteristics of a unique protein superfamily and provides guides for future studies concerning structural, functional and mechanistic properties of its constituent members.     

New lives for old: evolution of pseudoenzyme function illustrated by iRhoms

Large-scale sequencing of genomes has revealed that most enzyme families include inactive homologues. These pseudoenzymes are often well conserved, implying a selective pressure to retain them during evolution, and therefore that they have significant function. Mechanistic insights and evolutionary lessons are now emerging from the study of a broad range of such 'dead' enzymes. The recently discovered iRhoms - inactive homologues of rhomboid proteases - have joined derlins and other members of the rhomboid-like clan in regulating the fate of proteins as they pass through the secretory pathway. There is a strong case that dead enzymes, which have been rather overlooked, may be a rich source of biological regulators.     

Catalytic mechanism of penicillin-binding protein 5 of Escherichia coli

Penicillin-binding proteins (PBPs) and beta-lactamases are members of large families of bacterial enzymes. These enzymes undergo acylation at a serine residue with their respective substrates as the first step in their catalytic events. Penicillin-binding protein 5 (PBP 5) of Escherichia coli is known to perform a dd-carboxypeptidase reaction on the bacterial peptidoglycan, the major constituent of the cell wall. The roles of the active site residues Lys47 and Lys213 in the catalytic machinery of PBP 5 have been explored. By a sequence of site-directed mutagenesis and chemical modification, we individually introduced gamma-thialysine at each of these positions. The pH dependence of kcat/Km and of kcat for the wild-type PBP 5 and for the two gamma-thialysine mutant variants at positions 47 and 213 were evaluated. The pH optimum for the enzyme was at 9.5-10.5. The ascending limb to the pH optimum is due to Lys47; hence, this residue exists in the free-base form for catalysis. The descending limb from the pH optimum is contributed to by both Lys213 and a water molecule coordinated to Lys47. These results have been interpreted as Lys47 playing a key role in proton-transfer events in the course of catalysis during both the acylation and deacylation events. However, the findings for Lys213 argue for a protonated state at the pH optimum. Lys213 serves as an electrostatic anchor for the substrate.     

Genes Encoding Cher-TPR Fusion Proteins Are Predominantly Found in Gene Clusters Encoding Chemosensory Pathways with Alternative Cellular Functions

Chemosensory pathways correspond to major signal transduction mechanisms and can be classified into the functional families flagellum-mediated taxis, type four pili-mediated taxis or pathways with alternative cellular functions (ACF). CheR methyltransferases are core enzymes in all of these families. CheR proteins fused to tetratricopeptide repeat (TPR) domains have been reported and we present an analysis of this uncharacterized family. We show that CheR-TPRs are widely distributed in GRAM-negative but almost absent from GRAM-positive bacteria. Most strains contain a single CheR-TPR and its abundance does not correlate with the number of chemoreceptors. The TPR domain fused to CheR is comparatively short and frequently composed of 2 repeats. The majority of CheR-TPR genes were found in gene clusters that harbor multidomain response regulators in which the REC domain is fused to different output domains like HK, GGDEF, EAL, HPT, AAA, PAS, GAF, additional REC, HTH, phosphatase or combinations thereof. The response regulator architectures coincide with those reported for the ACF family of pathways. Since the presence of multidomain response regulators is a distinctive feature of this pathway family, we conclude that CheR-TPR proteins form part of ACF type pathways. The diversity of response regulator output domains suggests that the ACF pathways form a superfamily which regroups many different regulatory mechanisms, in which all CheR-TPR proteins appear to participate. In the second part we characterize WspC of Pseudomonas putida, a representative example of CheR-TPR. The affinities of WspC-Pp for S-adenosylmethionine and S-adenosylhomocysteine were comparable to those of prototypal CheR, indicating that WspC-Pp activity is in analogy to prototypal CheRs controlled by product feed-back inhibition. The removal of the TPR domain did not impact significantly on the binding constants and consequently not on the product feed-back inhibition. WspC-Pp was found to be monomeric, which rules out a role of the TPR domain in self-association.     

Small molecule regulation of Sir2 protein deacetylases

The Sir2 family of histone/protein deacetylases (sirtuins) is comprised of homologues found across all kingdoms of life. These enzymes catalyse a unique reaction in which NAD+ and acetylated substrate are converted into deacetylated product, nicotinamide, and a novel metabolite O-acetyl ADP-ribose. Although the catalytic mechanism is well conserved across Sir2 family members, sirtuins display differential specificity toward acetylated substrates, which translates into an expanding range of physiological functions. These roles include control of gene expression, cell cycle regulation, apoptosis, metabolism and ageing. The dependence of sirtuin activity on NAD+ has spearheaded investigations into how these enzymes respond to metabolic signals, such as caloric restriction. In addition, NAD+ metabolites and NAD+ salvage pathway enzymes regulate sirtuin activity, supporting a link between deacetylation of target proteins and metabolic pathways. Apart from physiological regulators, forward chemical genetics and high-throughput activity screening has been used to identify sirtuin inhibitors and activators. This review focuses on small molecule regulators that control the activity and functions of this unusual family of protein deacetylases.     

Twenty-five years of nomenclature and classification of proteolytic enzymes

Proteolytic enzymes and their homologues have been classified into clans by comparing the tertiary structures of the peptidase domains, into families by comparing the protein sequences of the peptidase domains, and into protein-species by comparing various attributes including domain architecture, substrate preference, inhibitor interactions, subcellular location, and phylogeny. The results are compared with the earlier classification (Rawlings and Barrett, 1993 [1]). The numbers of sequences, protein-species, families, clans and even catalytic type have substantially increased during the intervening 26 years. The alternative classifications by catalytic type and/or activity are shown not to reflect evolutionary relationships.     

Identification and typing of members of the protein-tyrosine phosphatase gene family expressed in mouse brain

Protein-tyrosine phosphatases (PTPases) form a novel and important class of cell regulatory proteins. We evaluated the expression of PTPases in mouse brain by polymerase chain amplification of cDNA segments that encode the catalytic domains of these enzymes. Degenerate primer pairs devised on the basis of conserved protein motifs were used to generate a series of distinct PCR-derived clones. In this way, murine homologues of the human PTPases LRP, PTP, PTP, PTP and LAR were obtained. Corresponding regions in their catalytic domains were used to reveal the evolutionary relationships between all currently known mammalian PTPase protein family members. Phylogenetic reconstruction displayed considerable differences in mutation rates for closely related PTPases.     

Interaction interfaces of protein domains are not topologically equivalent across families within superfamilies: Implications for metabolic and signaling pathways

Using a data set of aligned protein domain superfamilies of known three-dimensional structure, we compared the location of interdomain interfaces on the tertiary folds between members of distantly related protein domain superfamilies. The data set analyzed is comprised of interdomain interfaces, with domains occurring within a polypeptide chain and those between two polypeptide chains. We observe that, in general, the interfaces between protein domains are formed entirely in different locations on the tertiary folds in such pairs. This variation in the location of interface happens in protein domains involved in a wide range of functions, such as enzymes, adapters, and domains that bind protein ligands, or cofactors. While basic biochemical functionality is preserved at the domain superfamily level, the effect of biochemical function on protein assemblies is different in these protein domains related by superfamily. The divergence between proteins, in most cases, is coupled with domain recruitment, with different modes of interaction with the recruited domain. This is in complete contrast to the observation that in closely related homologous protein domains, almost always the interaction interfaces are topologically equivalent. In a small subset of interacting domains within proteins related by remote homology, we observe that the relative positioning of domains with respect to one another is preserved. Based on the analysis of multidomain proteins of known or unknown structure, we suggest that variation in protein-protein interactions in members within a superfamily could serve as diverging points in otherwise parallel metabolic or signaling pathways. We discuss a few representative cases of diverging pathways involving domains in a superfamily.     

Two cDNAs from the purple sea urchin,<Emphasis Type="Italic"> Strongylocentrotus purpuratus</Emphasis>, encoding mosaic proteins with domains found in factor H,factor I,and complement components C6 and C7

The vertebrate complement system is composed of about 30 serum and cell surface proteins that make up three activation pathways, a lytic pathway, and a set of proteins that regulate complement. Regulatory proteins are required for host protection against autologous complement attack and to control the amplification feedback loop of the alternative pathway. Purple sea urchin, Strongylocentrotus purpuratus, homologues of complement C3 (SpC3) and factor B (SpBf) have been identified, suggesting the presence of an alternative complement pathway. This implies that echinoderms require a complement regulatory system for the same reasons that it is required in higher vertebrates. Two cDNAs, Sp5 and Sp5013, have been characterized from coelomocytes and the deduced structures of the encoded mosaic proteins, SpCRL (S. purpuratus complement related protein, long form) and SpCRS (short form), have domains that are also found in regulatory proteins such as factor H and factor I and the terminal pathway components C6 and C7. These domains include multiple short consensus repeats, a fucolectin domain, Ser/Thr/Pro-rich regions, a Cys-rich region, and a factor I-membrane attack complex domain. The genes are constitutively expressed in all tissues of the sea urchin and are not induced in response to immune challenge. Multiple bands of varying intensity on both genome blots and RNA blots suggest that Sp5 and Sp5013 are members of a small gene family and that they might undergo alternative splicing. Based on the domains present in SpCRL and SpCRS, they might be either examples of complement regulatory proteins or members of the terminal pathway of complement.     

Evolution of protein function, from a structural perspective.

The recent growth in structural data, and ensuing analyses, have revealed the structural and functional versatility of protein families. With respect to enzymes, local active-site mutations, variations in surface loops and recruitment of additional domains accommodate the diverse substrate specificities and catalytic activities observed within several superfamilies. Conversely, some functions have more than one structural solution, having evolved independently several times during evolution. Combined with the existence of multi-functional genes, which have arisen by gene recruitment, these phenomena must be considered in the process of genome annotation.     

Regulation of phospholipase D

Structural studies of plant and bacterial members of the phospholipase D (PLD) superfamily are providing information about the role of the conserved HKD domains in the structure of the catalytic center and the catalytic mechanism of mammalian PLD isozymes (PLD1 and PLD2). Mutagenesis and sequence comparison studies have also defined the presence of pleckstrin homology and phox homology domains in the N-terminus and have demonstrated that a conserved sequence at the C-terminus is required for catalysis. The N- and C-terminal regions of PLD1 also contain interaction sites for protein kinase C, which can directly activate the enzyme through a non-phosphorylating mechanism. Small G proteins of the Rho and ADP-ribosylation factor families also directly regulate the enzyme, with RhoA binding to a sequence in the C-terminus. Certain tyrosine kinases and members of the Ras subfamily of small G proteins can activate the enzyme, but the mechanisms appear to be indirect. The mechanisms by which agonists activate PLD in vivo probably involve multiple pathways.     

Molecular mechanisms of axon guidance

In order to form a functional nervous system, neurones extend axons, often over long distances, to reach their targets. This process is controlled by extracellular receptors and their ligands, several families of which have been identified. These proteins may act to either repel or attract growth cones and a given receptor may transduce either type of signal, depending on the cellular context. In addition to these archetypal axon guidance molecules, it is becoming apparent that molecules previously known for their role in patterning can also direct axonal outgrowth. The growth cone receptors do not act in isolation and combine with members of the same or other families to produce a graded response or even a complete reversal in its polarity. These signals can be further combined and/or modulated by processing of the molecule both directly at the cell surface and by the network of intracellular signalling pathways which are activated. The result is a sophisticated and dynamic set of cues that enable a growth cone to successfully navigate to its destination, modulating its response to changing environmental cues along its pathway.     

The amoebapore superfamily

Amoebapores, synthesized by human protozoan parasites, form ion channels in target cells and artificial lipid membranes. The major pathogenic effect of these proteins is due to their cytolytic capability which results in target cell death. They comprise a coherent family and are homologous to other proteins and protein domains found in eight families. These families include in addition to the amoebapores (1) the saposins, (2) the NK-lysins and granulysins, (3) the pulmonary surfactant proteins B, (4) the acid sphingomyelinases, (5) acyloxyacyl hydrolases and (6) the aspartic proteases. These amoebapore homologues have many properties in common including membrane binding and stability. We note for the first time that a new protein, countin, from the cellular slime mold, Dictyostelium discoideum, comprises the eighth family within this superfamily. All currently sequenced members of these eight families are identified, and the structural, functional and phylogenetic properties of these proteins are discussed.     

Bioinformatic characterization of the 4-Toluene Sulfonate Uptake Permease (TSUP) family of transmembrane proteins

The ubiquitous sequence diverse 4-Toluene Sulfonate Uptake Permease (TSUP) family contains few characterized members and is believed to catalyze the transport of several sulfur-based compounds. Prokaryotic members of the TSUP family outnumber the eukaryotic members substantially, and in prokaryotes, but not eukaryotes, extensive lateral gene transfer occurred during family evolution. Despite unequal representation, homologues from the three taxonomic domains of life share well-conserved motifs. We show that the prototypical eight TMS topology arose from an intragenic duplication of a four transmembrane segment (TMS) unit. Possibly, a two TMS α-helical hairpin structure was the precursor of the 4 TMS repeat unit. Genome context analyses confirmed the proposal of a sulfur-based compound transport role for many TSUP homologues, but functional outliers appear to be prevalent as well. Preliminary results suggest that the TSUP family is a member of a large novel superfamily that includes rhodopsins, integral membrane chaperone proteins, transmembrane electron flow carriers and several transporter families. All of these proteins probably arose via the same pathway: 2→4→8 TMSs followed by loss of a TMS either at the N- or C-terminus, depending on the family, to give the more frequent 7 TMS topology.     

Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases.

Over 80 beta-1,4-glucanases and beta-1,4-xylanases can be classified into one of eight families on the basis of amino acid sequence similarities in their catalytic domains (Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991) Microbiol. Rev. 55, 303-315). As a test of this classification, the stereochemical course of hydrolysis of 10 enzymes representative of five families has been determined using proton NMR. These data, together with published data for six additional enzymes, show that representatives of a given enzyme family have the same stereoselectivity: four families catalyze hydrolysis with retention of anomeric configuration, two with inversion. The results support the hypothesis that family members share a common general fold, active site topology, and catalytic mechanism.