Thematic minireview series on enzyme evolution in the post-genomic era

The emergence of genomics; ongoing computational advances; and the development of large-scale sequence, structural, and functional databases have created important new interdisciplinary linkages between molecular evolution, molecular biology, and enzymology. The five minireviews in this series survey advances and challenges in this burgeoning field from complementary perspectives. The series has three major themes. The first is the evolution of enzyme superfamilies, in which members exhibit increasing sequence, structural, and functional divergence with increasing time of divergence from a common ancestor. The second is the evolutionary role of promiscuous enzymes, which, in addition to their primary function, have adventitious secondary activities that frequently provide the starting point for the evolution of new enzymes. The third is the importance of in silico approaches to the daunting challenge of assigning and predicting the functions of the many uncharacterized proteins in the large-scale sequence and structural databases that are now available. A recent computational advance, the use of protein similarity networks that map functional data onto proteins clustered by similarity, is presented as an approach that can improve functional insight and inference. The three themes are illustrated with several examples of enzyme superfamilies, including the amidohydrolase, metallo-β-lactamase, and enolase superfamilies.     

New Insights about Enzyme Evolution from Large Scale Studies of Sequence and Structure Relationships

Understanding how enzymes have evolved offers clues about their structure-function relationships and mechanisms. Here, we describe evolution of functionally diverse enzyme superfamilies, each representing a large set of sequences that evolved from a common ancestor and that retain conserved features of their structures and active sites. Using several examples, we describe the different structural strategies nature has used to evolve new reaction and substrate specificities in each unique superfamily. The results provide insight about enzyme evolution that is not easily obtained from studies of one or only a few enzymes.     

Potent inhibitors of a shikimate pathway enzyme from Mycobacterium tuberculosis: combining mechanism- and modeling-based design

Tuberculosis remains a serious global health threat, with the emergence of multidrug-resistant strains highlighting the urgent need for novel antituberculosis drugs. The enzyme 3-deoxy-d-arabino-heptulosonate 7-phosphate synthase (DAH7PS) catalyzes the first step of the shikimate pathway for the biosynthesis of aromatic compounds. This pathway has been shown to be essential in Mycobacterium tuberculosis, the pathogen responsible for tuberculosis. DAH7PS catalyzes a condensation reaction between P-enolpyruvate and erythrose 4-phosphate to give 3-deoxy-d-arabino-heptulosonate 7-phosphate. The enzyme reaction mechanism is proposed to include a tetrahedral intermediate, which is formed by attack of an active site water on the central carbon of P-enolpyruvate during the course of the reaction. Molecular modeling of this intermediate into the active site reported in this study shows a configurational preference consistent with water attack from the re face of P-enolpyruvate. Based on this model, we designed and synthesized an inhibitor of DAH7PS that mimics this reaction intermediate. Both enantiomers of this intermediate mimic were potent inhibitors of M. tuberculosis DAH7PS, with inhibitory constants in the nanomolar range. The crystal structure of the DAH7PS-inhibitor complex was solved to 2.35 Å. Both the position of the inhibitor and the conformational changes of active site residues observed in this structure correspond closely to the predictions from the intermediate modeling. This structure also identifies a water molecule that is located in the appropriate position to attack the re face of P-enolpyruvate during the course of the reaction, allowing the catalytic mechanism for this enzyme to be clearly defined.     

Genome-wide structural analysis reveals novel membrane binding properties of AP180 N-terminal homology (ANTH) domains

An increasing number of cytosolic proteins are shown to interact with membrane lipids during diverse cellular processes, but computational prediction of these proteins and their membrane binding behaviors remains challenging. Here, we introduce a new combinatorial computation protocol for systematic and robust functional prediction of membrane-binding proteins through high throughput homology modeling and in-depth calculation of biophysical properties. The approach was applied to the genomic scale identification of the AP180 N-terminal homology (ANTH) domain, one of the modular lipid binding domains, and prediction of their membrane binding properties. Our analysis yielded comprehensive coverage of the ANTH domain family and allowed classification and functional annotation of proteins based on the differences in local structural and biophysical features. Our analysis also identified a group of plant ANTH domains with unique structural features that may confer novel functionalities. Experimental characterization of a representative member of this subfamily confirmed its unique membrane binding mechanism and unprecedented membrane deforming activity. Collectively, these studies suggest that our new computational approach can be applied to genome-wide functional prediction of other lipid binding domains.     

Directed evolution of a thermostable quorum-quenching lactonase from the amidohydrolase superfamily

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-l-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å. Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh β-strands. This E101N/R230I mutant had an increased value of k_cat/K_m of 72-fold toward 3-oxo-N-dodecanoyl-l-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-l-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the β-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.     

Effect of codon message on xylanase thermal activity

Because the genetic codon is known for degeneracy, its effect on enzyme thermal property is seldom investigated. A dataset was constructed for GH10 xylanase coding sequences and optimal temperatures for activity (T(opt)). Codon contents and relative synonymous codon usages were calculated and respectively correlated with the enzyme T(opt) values, which were used to describe the xylanase thermophilic tendencies without dividing them into two thermophilic and mesophilic groups. After analyses of codon content and relative synonymous codon usages were checked by the Bonferroni correction, we found five codons, with three (AUA, AGA, and AGG) correlating positively and two (CGU and AGC) correlating negatively with the T(opt) value. The three positive codons are purine-rich codons, and the two negative codons have A-ends. The two negative codons are pyridine-rich codons, and one has a C-end. Comparable with the codon C- and A-ending features, C- and A-content within mRNA correlated negatively and positively with the T(opt) value, respectively. Thereby, codons have effects on enzyme thermal property. When the issue is analyzed at the residual level, the effect of codon message is lost. The codons relating to enzyme thermal property are selected by thermophilic force at nucleotide level.     

Toward a systems biology perspective on enzyme evolution

Large superfamilies of enzymes derived from a common progenitor have emerged by duplication and divergence of genes encoding metabolic enzymes. Division of the functions of early generalist enzymes enhanced catalytic power and control over metabolic fluxes. Later, novel enzymes evolved from inefficient secondary activities in specialized enzymes. Enzymes operate in the context of complex metabolic and regulatory networks. The potential for evolution of a new enzyme depends upon the collection of enzymes in a microbe, the topology of the metabolic network, the environmental conditions, and the net effect of trade-offs between the original and novel activities of the enzyme.     

Origins of specificity and promiscuity in metabolic networks

How enzymes have evolved to their present form is linked to the question of how pathways emerged and evolved into extant metabolic networks. To investigate this mechanism, we have explored the chemical diversity present in a largely unbiased data set of catalytic reactions processed by modern enzymes across the tree of life. In order to get a quantitative estimate of enzyme chemical diversity, we measure enzyme multispecificity or promiscuity using the reaction molecular signatures. Our main finding is that reactions that are catalyzed by a highly specific enzyme are shared by poorly divergent species, suggesting a later emergence of this function during evolution. In contrast, reactions that are catalyzed by highly promiscuous enzymes are more likely to appear uniformly distributed across species in the tree of life. From a functional point of view, promiscuous enzymes are mainly involved in amino acid and lipid metabolisms, which might be associated with the earliest form of biochemical reactions. In this way, results presented in this paper might assist us with the identification of primeval promiscuous catalytic functions contributing to life's minimal metabolism.     

Chimeric Cellulase Matrix for Investigating Intramolecular Synergism between Non-hydrolytic Disruptive Functions of Carbohydrate-binding Modules and Catalytic Hydrolysis

The conversion of renewable cellulosic biomass is of considerable interest for the production of biofuels and materials. The bottleneck in the efficient conversion is the compactness and resistance of crystalline cellulose. Carbohydrate-binding modules (CBMs), which disrupt crystalline cellulose via non-hydrolytic mechanisms, are expected to overcome this bottleneck. However, the lack of convenient methods for quantitative analysis of the disruptive functions of CBMs have hindered systematic studies and molecular modifications. Here we established a practical and systematic platform for quantifying and comparing the non-hydrolytic disruptive activities of CBMs via the synergism of CBMs and a catalytic module within designed chimeric cellulase molecules. Bioinformatics and computational biology were also used to provide a deeper understanding. A convenient vector was constructed to serve as a cellulase matrix into which heterologous CBM sequences can be easily inserted. The resulting chimeric cellulases were suitable for studying disruptive functions, and their activities quantitatively reflected the disruptive functions of CBMs on crystalline cellulose. In addition, this cellulase matrix can be used to construct novel chimeric cellulases with high hydrolytic activities toward crystalline cellulose.     

The crystal complex of phosphofructokinase-2 of Escherichia coli with fructose-6-phosphate: kinetic and structural analysis of the allosteric ATP inhibition

Substrate inhibition by ATP is a regulatory feature of the phosphofructokinases isoenzymes from Escherichia coli (Pfk-1 and Pfk-2). Under gluconeogenic conditions, the loss of this regulation in Pfk-2 causes substrate cycling of fructose-6-phosphate (fructose-6-P) and futile consumption of ATP delaying growth. In the present work, we have broached the mechanism of ATP-induced inhibition of Pfk-2 from both structural and kinetic perspectives. The crystal structure of Pfk-2 in complex with fructose-6-P is reported to a resolution of 2 Å. The comparison of this structure with the previously reported inhibited form of the enzyme suggests a negative interplay between fructose-6-P binding and allosteric binding of MgATP. Initial velocity experiments show a linear increase of the apparent K_0.5 for fructose-6-P and a decrease in the apparent k_cat as a function of MgATP concentration. These effects occur simultaneously with the induction of a sigmoidal kinetic behavior (n_H of approximately 2). Differences and resemblances in the patterns of fructose-6-P binding and the mechanism of inhibition are discussed for Pfk-1 and Pfk-2, as an example of evolutionary convergence, because these enzymes do not share a common ancestor.     

Structural and functional insights into (S)-ureidoglycine aminohydrolase, key enzyme of purine catabolism in Arabidopsis thaliana

The ureide pathway has recently been identified as the metabolic route of purine catabolism in plants and some bacteria. In this pathway, uric acid, which is a major product of the early stage of purine catabolism, is degraded into glyoxylate and ammonia via stepwise reactions of seven different enzymes. Therefore, the pathway has a possible physiological role in mobilization of purine ring nitrogen for further assimilation. (S)-Ureidoglycine aminohydrolase enzyme converts (S)-ureidoglycine into (S)-ureidoglycolate and ammonia, providing the final substrate to the pathway. Here, we report a structural and functional analysis of this enzyme from Arabidopsis thaliana (AtUGlyAH). The crystal structure of AtUGlyAH in the ligand-free form shows a monomer structure in the bicupin fold of the β-barrel and an octameric functional unit as well as a Mn(2+) ion binding site. The structure of AtUGlyAH in complex with (S)-ureidoglycine revealed that the Mn(2+) ion acts as a molecular anchor to bind (S)-ureidoglycine, and its binding mode dictates the enantioselectivity of the reaction. Further kinetic analysis characterized the functional roles of the active site residues, including the Mn(2+) ion binding site and residues in the vicinity of (S)-ureidoglycine. These analyses provide molecular insights into the structure of the enzyme and its possible catalytic mechanism.     

Crystal Structure of the Ectoine Hydroxylase,a Snapshot of the Active Site

Ectoine and its derivative 5-hydroxyectoine are compatible solutes that are widely synthesized by bacteria to cope physiologically with osmotic stress. They also serve as chemical chaperones and maintain the functionality of macromolecules. 5-Hydroxyectoine is produced from ectoine through a stereo-specific hydroxylation, an enzymatic reaction catalyzed by the ectoine hydroxylase (EctD). The EctD protein is a member of the non-heme-containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily and is evolutionarily well conserved. We studied the ectoine hydroxylase from the cold-adapted marine ultra-microbacterium Sphingopyxis alaskensis (Sa) and found that the purified SaEctD protein is a homodimer in solution. We determined the SaEctD crystal structure in its apo-form, complexed with the iron catalyst, and in a form that contained iron, the co-substrate 2-oxoglutarate, and the reaction product of EctD, 5-hydroxyectoine. The iron and 2-oxoglutarate ligands are bound within the EctD active site in a fashion similar to that found in other members of the dioxygenase superfamily. 5-Hydroxyectoine, however, is coordinated by EctD in manner different from that found in high affinity solute receptor proteins operating in conjunction with microbial import systems for ectoines. Our crystallographic analysis provides a detailed view into the active site of the ectoine hydroxylase and exposes an intricate network of interactions between the enzyme and its ligands that collectively ensure the hydroxylation of the ectoine substrate in a position- and stereo-specific manner.     

Divergence and convergence in enzyme evolution: parallel evolution of paraoxonases from quorum-quenching lactonases

We discuss the basic features of divergent versus convergent evolution and of the common scenario of parallel evolution. The example of quorum-quenching lactonases is subsequently described. Three different quorum-quenching lactonase families are known, and they belong to three different superfamilies. Their key active-site architectures have converged and are strikingly similar. Curiously, a promiscuous organophosphate hydrolase activity is observed in all three families. We describe the structural and mechanistic features that underline this converged promiscuity and how this promiscuity drove the parallel divergence of organophosphate hydrolases within these lactonase families by either natural or laboratory evolution.     

Analysis and functional prediction of reactive cysteine residues

Cys is much different from other common amino acids in proteins. Being one of the least abundant residues, Cys is often observed in functional sites in proteins. This residue is reactive, polarizable, and redox-active; has high affinity for metals; and is particularly responsive to the local environment. A better understanding of the basic properties of Cys is essential for interpretation of high-throughput data sets and for prediction and classification of functional Cys residues. We provide an overview of approaches used to study Cys residues, from methods for investigation of their basic properties, such as exposure and pK(a), to algorithms for functional prediction of different types of Cys in proteins.     

Structural and Functional Evolution of Positively Selected Sites in Pine Glutathione S-Transferase Enzyme Family

Phylogenetic analyses have identified positive selection as an important driver of protein evolution, both structural and functional. However, the lack of appropriate combined functional and structural assays has generally hindered attempts to elucidate patterns of positively selected sites and their effects on enzyme activity and substrate specificity. In this study we investigated the evolutionary divergence of the glutathione S-transferase (GST) family in Pinus tabuliformis, a pine that is widely distributed from northern to central China, including cold temperate and drought-stressed regions. GSTs play important roles in plant stress tolerance and detoxification. We cloned 44 GST genes from P. tabuliformis and found that 26 of the 44 belong to the largest (Tau) class of GSTs and are differentially expressed across tissues and developmental stages. Substitution models identified five positively selected sites in the Tau GSTs. To examine the functional significance of these positively selected sites, we applied protein structural modeling and site-directed mutagenesis. We found that four of the five positively selected sites significantly affect the enzyme activity and specificity; thus their variation broadens the GST family substrate spectrum. In addition, positive selection has mainly acted on secondary substrate binding sites or sites close to (but not directly at) the primary substrate binding site; thus their variation enables the acquisition of new catalytic functions without compromising the protein primary biochemical properties. Our study sheds light on selective aspects of the functional and structural divergence of the GST family in pine and other organisms.     

Crystal Structure of Reduced MsAcg,a Putative Nitroreductase from Mycobacterium smegmatis and a Close Homologue of Mycobacterium tuberculosis Acg

This paper presents the structure of MsAcg (MSMEG_5246), a Mycobacterium smegmatis homologue of Mycobacterium tuberculosis Acg (Rv2032) in its reduced form at 1.6 Å resolution using x-ray crystallography. Rv2032 is one of the most induced genes under the hypoxic model of tuberculosis dormancy. The Acg family turns out to be unusual flavin mononucleotide (FMN)-binding proteins that have probably arisen by gene duplication and fusion from a classical homodimeric nitroreductase such that the monomeric protein resembles a classical nitroreductase dimer but with one active site deleted and the other active site covered by a unique lid. The FMN cofactor is not reduced by either NADH or NADPH, but the chemically reduced enzyme is capable of reduction of nitro substrates, albeit at no kinetic advantage over free FMN. The reduced enzyme is rapidly oxidized by oxygen but without any evidence for a radical state commonly seen in oxygen-sensitive nitroreductases. The presence of the unique lid domain, the lack of reduction by NAD(P)H, and the slow rate of reaction of the chemically reduced protein raises a possible alternative function of Acg proteins in FMN storage or sequestration from other biochemical pathways as part of the bacteria''s adaptation to a dormancy state.     

Crystal structure and inhibition studies of transglutaminase from Streptomyces mobaraense

The crystal structure of the microbial transglutaminase (MTGase) zymogen from Streptomyces mobaraense has been determined at 1.9-Å resolution using the molecular replacement method based on the crystal structure of the mature MTGase. The overall structure of this zymogen is similar to that of the mature form, consisting of a single disk-like domain with a deep active cleft at the edge of the molecule. A major portion of the prosequence (45 additional amino acid residues at the N terminus of the mature transglutaminase) folds into an L-shaped structure, consisting of an extended N-terminal segment linked with a one-turn short helix and a long α-helix. Two key residues in the short helix of the prosequence, Tyr-12 and Tyr-16, are located on top of the catalytic triad (Cys-110, Asp-301, and His-320) to block access of the substrate acyl donors and acceptors. Biochemical characterization of the mature MTGase, using N-α-benzyloxycarbonyl-l-glutaminylglycine as a substrate, revealed apparent K_m and k_cat/K_m values of 52.66 mm and 40.42 mm⁻¹ min⁻¹, respectively. Inhibition studies using the partial prosequence SYAETYR and homologous sequence SQAETYR showed a noncompetitive inhibition mechanism with IC₅₀ values of 0.75 and 0.65 mm, respectively, but no cross-linking product formation. Nevertheless, the prosequence homologous oligopeptide SQAETQR, with Tyr-12 and Tyr-16 each replaced with Gln, exhibited inhibitory activity with the formation of the SQAETQR-monodansylcadaverine fluorophore cross-linking product (SQAETQR-C-DNS). MALDI-TOF tandem MS analysis of SQAETQR-C-DNS revealed molecular masses corresponding to those of ^NSQAETQ^C-C-DNS and C-DNS-^NQR^C sequences, suggesting the incorporation of C-DNS onto the C-terminal Gln residue of the prosequence homologous oligopeptide. These results support the putative functional roles of both Tyr residues in substrate binding and inhibition.     

Enzyme Promiscuity: Engine of Evolutionary Innovation

Catalytic promiscuity and substrate ambiguity are keys to evolvability, which in turn is pivotal to the successful acquisition of novel biological functions. Action on multiple substrates (substrate ambiguity) can be harnessed for performance of functions in the cell that supersede catalysis of a single metabolite. These functions include proofreading, scavenging of nutrients, removal of antimetabolites, balancing of metabolite pools, and establishing system redundancy. In this review, we present examples of enzymes that perform these cellular roles by leveraging substrate ambiguity and then present the structural features that support both specificity and ambiguity. We focus on the phosphatases of the haloalkanoate dehalogenase superfamily and the thioesterases of the hotdog fold superfamily.     

Structure and function of APH(4)-Ia, a hygromycin B resistance enzyme

The aminoglycoside phosphotransferase (APH) APH(4)-Ia is one of two enzymes responsible for bacterial resistance to the atypical aminoglycoside antibiotic hygromycin B (hygB). The crystal structure of APH(4)-Ia enzyme was solved in complex with hygB at 1.95 Å resolution. The APH(4)-Ia structure adapts a general two-lobe architecture shared by other APH enzymes and eukaryotic kinases, with the active site located at the interdomain cavity. The enzyme forms an extended hydrogen bond network with hygB primarily through polar and acidic side chain groups. Individual alanine substitutions of seven residues involved in hygB binding did not have significant effect on APH(4)-Ia enzymatic activity, indicating that the binding affinity is spread across a distributed network. hygB appeared as the only substrate recognized by APH(4)-Ia among the panel of 14 aminoglycoside compounds. Analysis of the active site architecture and the interaction with the hygB molecule demonstrated several unique features supporting such restricted substrate specificity. Primarily the APH(4)-Ia substrate-binding site contains a cluster of hydrophobic residues that provides a complementary surface to the twisted structure of the substrate. Similar to APH(2″) enzymes, the APH(4)-Ia is able to utilize either ATP or GTP for phosphoryl transfer. The defined structural features of APH(4)-Ia interactions with hygB and the promiscuity in regard to ATP or GTP binding could be exploited for the design of novel aminoglycoside antibiotics or inhibitors of this enzyme.     

The role of Cys-298 in aldose reductase function

Diabetic tissues are enriched in an "activated" form of human aldose reductase (hAR), a NADPH-dependent oxidoreductase involved in sugar metabolism. Activated hAR has reduced sensitivity to potential anti-diabetes drugs. The C298S mutant of hAR reproduces many characteristics of activated hAR, although it differs from wild-type hAR only by the replacement of a single sulfur atom with oxygen. Isothermal titration calorimetry measurements revealed that the binding constant of NADPH to the C298S mutant is decreased by a factor of two, whereas that of NADP(+) remains the same. Similarly, the heat capacity change for the binding of NADPH to the C298S mutant is twice increased; however, there is almost no difference in the heat capacity change for binding of the NADP(+) to the C298S. X-ray crystal structures of wild-type and C298S hAR reveal that the side chain of residue 298 forms a gate to the nicotinamide pocket and is more flexible for cysteine compared with serine. Unlike Cys-298, Ser-298 forms a hydrogen bond with Tyr-209 across the nicotinamide ring, which inhibits movements of the nicotinamide. We hypothesize that the increased polarity of the oxidized nicotinamide weakens the hydrogen bond potentially formed by Ser-298, thus, accounting for the relatively smaller effect of the mutation on NADP(+) binding. The effects of the mutant on catalytic rate constants and binding constants for various substrates are the same as for activated hAR. It is, thus, further substantiated that activated hAR arises from oxidative modification of Cys-298, a residue near the nicotinamide binding pocket.