Mechanistic insights into the retaining glucosyl-3-phosphoglycerate synthase from mycobacteria

Considerable progress has been made in recent years in our understanding of the structural basis of glycosyl transfer. Yet the nature and relevance of the conformational changes associated with substrate recognition and catalysis remain poorly understood. We have focused on the glucosyl-3-phosphoglycerate synthase (GpgS), a "retaining" enzyme, that initiates the biosynthetic pathway of methylglucose lipopolysaccharides in mycobacteria. Evidence is provided that GpgS displays an unusually broad metal ion specificity for a GT-A enzyme, with Mg(2+), Mn(2+), Ca(2+), Co(2+), and Fe(2+) assisting catalysis. In the crystal structure of the apo-form of GpgS, we have observed that a flexible loop adopts a double conformation L(A) and L(I) in the active site of both monomers of the protein dimer. Notably, the L(A) loop geometry corresponds to an active conformation and is conserved in two other relevant states of the enzyme, namely the GpgS·metal·nucleotide sugar donor and the GpgS·metal·nucleotide·acceptor-bound complexes, indicating that GpgS is intrinsically in a catalytically active conformation. The crystal structure of GpgS in the presence of Mn(2+)·UDP·phosphoglyceric acid revealed an alternate conformation for the nucleotide sugar β-phosphate, which likely occurs upon sugar transfer. Structural, biochemical, and biophysical data point to a crucial role of the β-phosphate in donor and acceptor substrate binding and catalysis. Altogether, our experimental data suggest a model wherein the catalytic site is essentially preformed, with a few conformational changes of lateral chain residues as the protein proceeds along the catalytic cycle. This model of action may be applicable to a broad range of GT-A glycosyltransferases.     

Conformational change of cytochrome P-450 indicated by the measurement of fluorescence-energy transfer

The conformation of cytochrome P-450 was studied in relation to the interaction with its substrate by the measurement of fluorescence energy transfer. Cytochrome P-450 I-c and cytochrome P-450 II-d, both obtained from the hepatic microsome of polychlorinated biphenyl-treated rats, were used as P-450 type and P-448 type, respectively, and benzo[a]pyrene and 7-ethoxycoumarin were used as substrate. The distance between the donor and acceptor, which was described by the F?rster equation, was calculated on the basis of the fluorescence-energy transfer between the substrate as a donor and the heme of the enzyme as an acceptor. The distance was apparently changed by incorporation of the enzyme into phospholipid or by reduction of the heme, indicating that the treatments changed the conformation of the enzyme. Differences in the conformational change were observed between the two enzymes, and the conformational change of cytochrome P-450 II-d using benzo[a]pyrene as a substrate was different from that using 7-ethoxycoumarin. The results suggest that the substrate-binding sites of the two enzymes differ in position toward the heme, and that there are at least two different substrate sites in cytochrome P-450 II-d, one for benzo[a]pyrene and another for 7-ethoxycoumarin.     

Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1- and 1.96-A resolution, respectively. Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate-free active site in PAP resembles that of the substrate-bound region of the mesophilic homolog, with both an active-site tyrosine and a substrate-binding loop displaying a conformation as in the substrate-bound form of the mesophilic proteases. Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 A, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions.     

Induced fit movements and metal cofactor selectivity of class II aldolases: structure of Thermus aquaticus fructose-1,6-bisphosphate aldolase

Fructose-1,6-bisphosphate (FBP) aldolase is an essential glycolytic enzyme that reversibly cleaves its ketohexose substrate into triose phosphates. Here we report the crystal structure of a metallo-dependent or class II FBP aldolase from an extreme thermophile, Thermus aquaticus (Taq). The quaternary structure reveals a tetramer composed of two dimers related by a 2-fold axis. Taq FBP aldolase subunits exhibit two distinct conformational states corresponding to loop regions that are in either open or closed position with respect to the active site. Loop closure remodels the disposition of chelating active site histidine residues. In subunits corresponding to the open conformation, the metal cofactor, Co(2+), is sequestered in the active site, whereas for subunits in the closed conformation, the metal cation exchanges between two mutually exclusive binding loci, corresponding to a site at the active site surface and an interior site vicinal to the metal-binding site in the open conformation. Cofactor site exchange is mediated by rotations of the chelating histidine side chains that are coupled to the prior conformational change of loop closure. Sulfate anions are consistent with the location of the phosphate-binding sites of the FBP substrate and determine not only the previously unknown second phosphate-binding site but also provide a mechanism that regulates loop closure during catalysis. Modeling of FBP substrate into the active site is consistent with binding by the acyclic keto form, a minor solution species, and with the metal cofactor mediating keto bond polarization. The Taq FBP aldolase structure suggests a structural basis for different metal cofactor specificity than in Escherichia coli FBP aldolase structures, and we discuss its potential role during catalysis. Comparison with the E. coli structure also indicates a structural basis for thermostability by Taq FBP aldolase.     

A closed conformation of Bacillus subtilis oxalate decarboxylase OxdC provides evidence for the true identity of the active site

Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659-7669). The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3(10) helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Site-directed mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.     

Functional role for Tyr 31 in the catalytic cycle of chicken dihydrofolate reductase

Despite much work, many key aspects of the mechanism of the dihydrofolate reductase (DHFR) catalyzed reduction of dihydrofolate remain unresolved. In bacterial forms of DHFR both substrate and water access to the active site are controlled by the conformation of the mobile M20 loop. In vertebrate DHFRs only one conformation of the residues corresponding to the M20 loop has been observed. Access to the active site was proposed to be controlled by residue 31. MD simulations of chicken DHFR complexed with substrates and cofactor revealed a closing of the side chain of Tyr 31 over the active site on binding of dihydrofolate. This conformational change was dependent on the presence of glutamate on the para-aminobenzoylamide moiety of dihydrofolate. In its absence, the conformation remained open. Although water could enter the active site and hydrogen bond to N5 of dihydrofolate, indicating the feasibility of water as the proton donor, this was not controlled by the conformation of Tyr 31. The water accessibility of the active site was low for both conformations of Tyr 31. However, when hydride was transferred from NADPH to C6 of dihydrofolate before protonation, the average time during which water was found in hydrogen bonding distance to N5 of dihydrofolate in the active site increased almost fivefold. These results indicated that water can serve as the Broensted acid for the protonation of N5 of dihydrofolate during the DHFR catalyzed reduction.     

Effect of cytidine-5'-monophosphate on peptidyl transferase activity.

The transfer reaction with pA-fMet as a donor substrate is strongly stimulated by CMP, whereas the transfer reaction with CpApCpCpA-acLeu as a donor substrate is inhibited by CMP. These results indicate that the donor site of peptidyl transferase contains specific binding sites for the terminal adenosine and for the cytidylic acid residue in the terminal sequence CpCpA of tRNA and that an attachment of proper nucleotides to the donor site induces a conformational change in peptidyl transferase.     

Beta-1,4-galactosyltransferase and lactose synthase: molecular mechanical devices

Recent structural investigations on the beta-1,4-galactosyltransferase-1 (Gal-T1) and lactose synthase (LS) have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates (i) the oligosaccharide binding cavity; (ii) a protein-protein interacting site for the enzyme's partner, alpha-lactalbumin (LA); and (iii) a metal ion binding site. Only in conformation II do Gal-T1 and LA form the LS complex, enabling Gal-T1 to choose the new substrate glucose. LA holds and puts Glc right in the acceptor binding site of Gal-T1, which then maximizes the interactions with Glc, thereby making it a preferred acceptor for the LS reaction. The interaction of LA with Gal-T1 in conformation II also stabilizes the sugar-nucleotide-enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. The conformational change that masks the sugar nucleotide binding site can also be induced by the acceptor alone, thus making it possible for the protein to act as a specific lectin.     

Prediction of a ligand-induced conformational change in the catalytic core of Cdc25A

The cell cycle control phosphatases Cdc25 are dual specificity phosphatases that dephosphorylate both phosphothreonine and phosphotyrosine residues on their substrate proteins. The determination of the apo-protein structure of Cdc25A revealed that this enzyme has a completely different fold compared to all other phosphatases crystallised to date. The conformation of the active site residues does not seem very suitable for catalysis in this unliganded structure. We have studied some structural features of the Cdc25A apo-structure and a modelled Cdc25A-ligand complex by molecular dynamics simulations. The simulations predict a conformational change in the peptide backbone of the complex, which is not observed in the apo-structure. This ligand-induced conformational change yields a structure that is similar to other protein tyrosine phosphatase-ligand complexes that have been crystallised. The change in conformation takes place in the position between a serine and a glutamic acid residue in the phosphate binding loop. We suggest that this type of conformational change is an important molecular switch in the catalytic process.     

Cysteine-to-Serine Mutants Dramatically Reorder the Active Site of Human ABO(H) Blood Group B Glycosyltransferase without Affecting Activity: Structural Insights into Cooperative Substrate Binding

A common feature in the structures of GT-A-fold-type glycosyltransferases is a mobile polypeptide loop that has been observed to participate in substrate recognition and enclose the active site upon substrate binding. This is the case for the human ABO(H) blood group B glycosyltransferase GTB, where amino acid residues 177-195 display significantly higher levels of disorder in the unliganded state than in the fully liganded state. Structural studies of mutant enzymes GTB/C80S/C196S and GTB/C80S/C196S/C209S at resolutions ranging from 1.93 to 1.40 Å display the opposite trend, where the unliganded structures show nearly complete ordering of the mobile loop residues that is lost upon substrate binding. In the liganded states of the mutant structures, while the UDP moiety of the donor molecule is observed to bind in the expected location, the galactose moiety is observed to bind in a conformation significantly different from that observed for the wild-type chimeric structures. Although this would be expected to impede catalytic turnover, the kinetics of the transfer reaction are largely unaffected. These structures demonstrate that the enzymes bind the donor in a conformation more similar to the dominant solution rotamer and facilitate its gyration into the catalytically competent form. Further, by preventing active-site closure, these structures provide a basis for recently observed cooperativity in substrate binding. Finally, the mutation of C80S introduces a fully occupied UDP binding site at the enzyme dimer interface that is observed to be dependent on the binding of H antigen acceptor analog.     

Three-way stabilization of the covalent intermediate in amylomaltase, an alpha-amylase-like transglycosylase

Amylomaltases are glycosyl hydrolases belonging to glycoside hydrolase family 77 that are capable of the synthesis of large cyclic glucans and the disproportionation of oligosaccharides. Using protein crystallography, we have generated a flip book movie of the amylomaltase catalytic cycle in atomic detail. The structures include a covalent glycosyl enzyme intermediate and a covalent intermediate in complex with an analogue of a co-substrate and show how the structures of both enzyme and substrate respond to the changes required by the catalytic cycle as it proceeds. Notably, the catalytic nucleophile changes conformation dramatically during the reaction. Also, Gln-256 on the 250s loop is involved in orienting the substrate in the +1 site. The absence of a suitable base in the covalent intermediate structure explains the low hydrolysis activity.     

Insights into diterpene cyclization from structure of bifunctional abietadiene synthase from Abies grandis

Abietadiene synthase from Abies grandis (AgAS) is a model system for diterpene synthase activity, catalyzing class I (ionization-initiated) and class II (protonation-initiated) cyclization reactions. Reported here is the crystal structure of AgAS at 2.3 Å resolution and molecular dynamics simulations of that structure with and without active site ligands. AgAS has three domains (α, β, and γ). The class I active site is within the C-terminal α domain, and the class II active site is between the N-terminal γ and β domains. The domain organization resembles that of monofunctional diterpene synthases and is consistent with proposed evolutionary origins of terpene synthases. Molecular dynamics simulations were carried out to determine the effect of substrate binding on enzymatic structure. Although such studies of the class I active site do lead to an enclosed substrate-Mg²⁺ complex similar to that observed in crystal structures of related plant enzymes, it does not enforce a single substrate conformation consistent with the known product stereochemistry. Simulations of the class II active site were more informative, with observation of a well ordered external loop migration. This “loop-in” conformation not only limits solvent access but also greatly increases the number of conformational states accessible to the substrate while destabilizing the nonproductive substrate conformation present in the “loop-out” conformation. Moreover, these conformational changes at the class II active site drive the substrate toward the proposed transition state. Docked substrate complexes were further assessed with regard to the effects of site-directed mutations on class I and II activities.     

Identification of the active conformation and the importance of length of the flexible loop 72-83 in regulating the conformational change of undecaprenyl pyrophosphate synthase

Increasing evidence has shown that intrinsic disorder of proteins plays a key role in their biological functions. In the case of undecaprenyl pyrophosphate synthase (UPPs), which catalyzes the chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate via eight consecutive condensation reactions with isopentenyl pyrophosphate, a highly flexible loop 72-83 was previously linked to protein conformational change required for catalysis [Chen, Y. H., Chen, A. P.-C., Chen, C. T., Wang, A. H.-J., and Liang, P. H., (2002) J. Biol. Chem. 277, 7369-7376]. The crystal structure and fluorescence studies suggested that the alpha3 helix connected to the loop moves toward the active site when the substrate is bound. To identify the active conformation and study the role of the loop for conformational change, the UPPs mutants with amino acids inserted into or deleted from the loop were examined. The inserted mutant with extra Ala residues fails to display the intrinsic fluorescence quenching upon FPP binding, and its crystal structure reveals only the open form. These phenomena appear to be different from the wild-type enzyme in which open and closed conformers were observed and suggest that the extended loop fails to pull the alpha3 helix and/or the extra amino acids in the loop cause steric hindrance on the alpha3 helix movement. The loop-shortening mutants with deletion of V82 and S83 or S72 also adopt an open conformation with the loop stretched, although they show decreased intrinsic fluorescence with FPP bound, similar to that seen in the wild-type enzyme. We conclude that the closed conformation is apparently the active conformation. Change of the length of the loop 72-83 impairs the ability of conformational change and causes remarkably lower activity of UPPs.     

Triggering loops and enzyme function: identification of loops that trigger and modulate movements

Enzyme function often involves a conformational change. There is a general agreement that loops play a vital role in correctly positioning the catalytically important residues. Nevertheless, predicting the functional loops and most importantly their role in enzyme function remains a difficult task. A major reason for this difficulty is that loops that undergo conformational change are frequently not well conserved in their primary sequence. beta1,4-Galactosyltransferase is one such enzyme. There, the amino acid sequence of a long loop that undergoes a large conformational change upon substrate binding is not well conserved. Our molecular dynamics simulations show that the large conformational change in the long loop is brought about by a second, interacting loop. Interestingly, while the structural change of the second loop is much smaller than that of the long loop, its sequence (particularly glycine residues) is highly conserved. We further examine the generality of the proposition that there are loops that trigger movements but nevertheless show little or no structural changes in crystals. We focus on two other enzymes, enolase and lipase. We chose these enzymes, since they too undergo conformational change upon ligand binding, however, they have different folds and different functions. Through multiple sets of simulations we show that the conformational change of the functional loop(s) is brought about through communication of flexibility by triggering loops that have several glycine residues. We further propose that similar to the conservation of common favorable fold types and structural motifs, evolution has also conserved common "skillful" mechanisms. Mechanisms may be conserved across different folds, sequences and functions, with adaptation to specific enzymatic roles.     

Identification of an allosteric anion-binding site on O-acetylserine sulfhydrylase: structure of the enzyme with chloride bound

A new crystal structure of O-acetylserine sulfhydrylase (OASS) has been solved with chloride bound at an allosteric site and sulfate bound at the active site. The bound anions result in a new "inhibited" conformation, that differs from the "open" native or "closed" external aldimine conformations. The allosteric site is located at the OASS dimer interface. The new inhibited structure involves a change in the position of the "moveable domain" (residues 87-131) to a location that differs from that in the open or closed forms. Formation of the external aldimine with substrate is stabilized by interaction of the alpha-carboxyl group of the substrate with a substrate-binding loop that is part of the moveable domain. The inhibited conformation prevents the substrate-binding loop from interacting with the alpha-carboxyl group, and hinders formation of the external Schiff base and thus subsequent chemistry. Chloride may be an analog of sulfide, the physiological inhibitor. Finally, these results suggest that OASS represents a new class of PLP-dependent enzymes that is regulated by small anions.     

Coupling between Catalytic Loop Motions and Enzyme Global Dynamics

Catalytic loop motions facilitate substrate recognition and binding in many enzymes. While these motions appear to be highly flexible, their functional significance suggests that structure-encoded preferences may play a role in selecting particular mechanisms of motions. We performed an extensive study on a set of enzymes to assess whether the collective/global dynamics, as predicted by elastic network models (ENMs), facilitates or even defines the local motions undergone by functional loops. Our dataset includes a total of 117 crystal structures for ten enzymes of different sizes and oligomerization states. Each enzyme contains a specific functional/catalytic loop (10–21 residues long) that closes over the active site during catalysis. Principal component analysis (PCA) of the available crystal structures (including apo and ligand-bound forms) for each enzyme revealed the dominant conformational changes taking place in these loops upon substrate binding. These experimentally observed loop reconfigurations are shown to be predominantly driven by energetically favored modes of motion intrinsically accessible to the enzyme in the absence of its substrate. The analysis suggests that robust global modes cooperatively defined by the overall enzyme architecture also entail local components that assist in suitable opening/closure of the catalytic loop over the active site.     

The crystal structure of feruloyl esterase A from Aspergillus niger suggests evolutive functional convergence in feruloyl esterase family

As a component of the array of enzymes produced by micro-organisms to deconstruct plant cell walls, feruloyl esterases hydrolyze phenolic groups involved in the cross-linking of arabinoxylan to other polymeric structures. This is important for opening the cell wall structure, making material more accessible to glycosyl hydrolases. Here, we describe the first crystal structure of the non-modular type-A feruloyl esterase from Aspergillus niger (AnFaeA) solved at 2.5A resolution. AnFaeA displays an alpha/beta hydrolase fold similar to that found in fungal lipases and different from that reported for other feruloyl esterases. Crystallographic and site-directed mutagenesis studies allow us to identify the catalytic triad (Ser133-His247-Asp194) that forms the catalytic machinery of this enzyme. The active-site cavity is confined by a lid (residues 68-80), on the analogy of lipases, and by a loop (residues 226-244) that confers plasticity to the substrate-binding site. The lid presents a high ratio of polar residues, which in addition to a unique N-glycosylation site stabilises the lid in an open conformation, conferring the esterase character to this enzyme. A putative model for bound 5,5'-diferulic acid-linked arabinoxylan has been built, pointing to the more relevant residues involved in substrate recognition. Comparison with structurally related lipases reveals that subtle amino acid and conformational changes within a highly conserved protein fold may produce protein variants endowed with new enzymatic properties, while comparison with functionally related proteins points to a functional convergence after evolutionary divergence within the feruloyl esterases family.     

Structural basis for the catalytic mechanism of aspartate ammonia lyase

Aspartate ammonia lyases (or aspartases) catalyze the reversible deamination of L-aspartate into fumarate and ammonia. The lack of crystal structures of complexes with substrate, product, or substrate analogues so far precluded determination of their precise mechanism of catalysis. Here, we report crystal structures of AspB, the aspartase from Bacillus sp. YM55-1, in an unliganded state and in complex with L-aspartate at 2.4 and 2.6 ? resolution, respectively. AspB forces the bound substrate to adopt a high-energy, enediolate-like conformation that is stabilized, in part, by an extensive network of hydrogen bonds between residues Thr101, Ser140, Thr141, and Ser319 and the substrate's β-carboxylate group. Furthermore, substrate binding induces a large conformational change in the SS loop (residues G(317)SSIMPGKVN(326)) from an open conformation to one that closes over the active site. In the closed conformation, the strictly conserved SS loop residue Ser318 is at a suitable position to act as a catalytic base, abstracting the Cβ proton of the substrate in the first step of the reaction mechanism. The catalytic importance of Ser318 was confirmed by site-directed mutagenesis. Site-directed mutagenesis of SS loop residues, combined with structural and kinetic analysis of a stable proteolytic AspB fragment, further suggests an important role for the small C-terminal domain of AspB in controlling the conformation of the SS loop and, hence, in regulating catalytic activity. Our results provide evidence supporting the notion that members of the aspartase/fumarase superfamily use a common catalytic mechanism involving general base-catalyzed formation of a stabilized enediolate intermediate.     

Structural features in the KshA terminal oxygenase protein that determine substrate preference of 3-ketosteroid 9α-hydroxylase enzymes

Rieske nonheme monooxygenase 3-ketosteroid 9α-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshA(H37Rv) of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a β sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the β sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 β-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities.