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Ivan Rayment

Publications and source records attributed to Ivan Rayment.

At least 37 records · Page 2Linked to original sources
PubMed2004

Understanding the importance of protein structure to nature's routes for divergent evolution in TIM barrel enzymes.

It is widely agreed that new enzymes evolve from existing ones through the duplication of genes encoding existing enzymes followed by sequence divergence. While evolution is an inherently random process, studies of divergently related enzymes have shown that the evolution of new enzymes follows one of three general routes in which the substrate specificity, reaction mechanism, or active site architecture of the progenitor enzyme is reused in the new enzyme. Recent developments in structural biology relating to divergently related (beta/alpha)8 enzymes have brought new insight into these processes and have revealed that conserved structural elements play an important role in divergent evolution. These studies have shown that, although evolution occurs as a series of random mutations, stable folds such as the (beta/alpha)8 barrel and structural features of the active sites of enzymes are frequently reused in evolution and adapted for new catalytic purposes.

Binding Sites↗
PubMed2004

Molecular dynamics analysis of structural factors influencing back door pi release in myosin.

The back door has been proposed to be an exit pathway from the myosin active site for phosphate (P(i)) generated by adenosine 5'-triphosphate hydrolysis. We used molecular dynamics simulations to investigate the interaction of P(i) with the back door and the plausibility of P(i) release via this route. Molecular dynamics simulations were performed on the Dictyostelium motor domain with bound Mg.adenosine 5'-diphosphate (ADP) and P(i), modeled upon the Mg.ADP.BeF(x) and Mg.ADP.V(i) structures. Simulations revealed that the relaxation of ADP and free P(i) from their initial positions reduced the diameter of the back door via motions of switch 1 and switch 2 located in the upper and lower 50-kDa subdomains, respectively. In neither simulation could P(i) freely diffuse out the back door. Water molecules, however, could flux through the back door in the Mg.ADP.BeF(x)-based simulation but not in the Mg.ADP.V(i)-based simulation. In neither structure was water observed fluxing through the main (front door) entrance. These observations suggest that the ability of P(i) to leave via the back door is linked tightly to conformational changes between the upper and lower 50-kDa subdomains. The simulations offer structural explanations for (18)O-exchange with P(i) at the active site, and P(i) release being the rate-limiting step in the myosin adenosine 5'-triphosphatase.

Adenosine Diphosphate↗
PubMed2003

Evolution of enzymatic activity in the enolase superfamily: structural and mutagenic studies of the mechanism of the reaction catalyzed by o-succinylbenzoate synthase from Escherichia coli.

o-Succinylbenzoate synthase (OSBS) from Escherichia coli, a member of the enolase superfamily, catalyzes an exergonic dehydration reaction in the menaquinone biosynthetic pathway in which 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylate (SHCHC) is converted to 4-(2'-carboxyphenyl)-4-oxobutyrate (o-succinylbenzoate or OSB). Our previous structural studies of the Mg(2+).OSB complex established that OSBS is a member of the muconate lactonizing enzyme subgroup of the superfamily: the essential Mg(2+) is coordinated to carboxylate ligands at the ends of the third, fourth, and fifth beta-strands of the (beta/alpha)(7)beta-barrel catalytic domain, and the OSB product is located between the Lys 133 at the end of the second beta-strand and the Lys 235 at the end of the sixth beta-strand [Thompson, T. B., Garrett, J. B., Taylor, E. A, Meganathan, R., Gerlt, J. A., and Rayment, I. (2000) Biochemistry 39, 10662-76]. Both Lys 133 and Lys 235 were separately replaced with Ala, Ser, and Arg residues; all six mutants displayed no detectable catalytic activity. The structure of the Mg(2+).SHCHC complex of the K133R mutant has been solved at 1.62 A resolution by molecular replacement starting from the structure of the Mg(2+).OSB complex. This establishes the absolute configuration of SHCHC: the C1-carboxylate and the C6-OH leaving group are in a trans orientation, requiring that the dehydration proceed via a syn stereochemical course. The side chain of Arg 133 is pointed out of the active site so that it cannot function as a general base, whereas in the wild-type enzyme complexed with Mg(2+).OSB, the side chain of Lys 133 is appropriately positioned to function as the only acid/base catalyst in the syn dehydration. The epsilon-ammonium group of Lys 235 forms a cation-pi interaction with the cyclohexadienyl moiety of SHCHC, suggesting that Lys 235 also stabilizes the enediolate anion intermediate in the syn dehydration via a similar interaction.

Amino Acid Substitution↗
PubMed2003

Biomolecular mimicry in the actin cytoskeleton: mechanisms underlying the cytotoxicity of kabiramide C and related macrolides.

This study characterizes the interactions between kabiramide C (KabC) and related macrolides and actin and establishes the mechanisms that underlie their inhibition of actin filament dynamics and cytotoxicity. The G-actin-KabC complex is formed through a two-step binding reaction and is extremely stable and long-lived. Competition-binding studies show that KabC binds to the same site on G-actin as Gelsolin domain 1 and CapG. KabC also binds to protomers within F-actin and results in the severing and capping of the (+) end; these studies suggest that free KabC and related macrolides act as biomimetics of Gelsolin. The G-actin-KabC complex binds to the (+) end of a growing filament, where it functions as a novel, unregulated, (+)-end capper and is largely responsible for the inhibition of motility and cytokinesis in approximately 10 -100 nM KabC-treated cells. KabC and related macrolides are useful probes to study the regulation of the actin filament (+) end and may lead to new therapies to treat diseases of the actin cytoskeleton.

2-Naphthylamine↗
PubMed2003

Structural evidence for a 1,2-enediolate intermediate in the reaction catalyzed by 3-keto-L-gulonate 6-phosphate decarboxylase, a member of the orotidine 5'-monophosphate decarboxylase suprafamily.

3-Keto-L-gulonate 6-phosphate decarboxylase (KGPDC) and orotidine 5'-phosphate decarboxylase (OMPDC) are members of an enzyme suprafamily, the OMPDC suprafamily, because they are homologous enzymes that catalyze mechanistically distinct reactions using different substrates. KGPDC catalyzes the Mg(2+) ion-dependent decarboxylation of 3-keto-L-gulonate 6-phosphate to yield L-xylulose 5-phosphate and CO(2); OMPDC catalyzes the metal ion-independent decarboxylation of OMP to UMP and CO(2). Structural studies have shown that KGPDC and OMPDC share several strictly conserved active site residues that are used differently by each enzyme to catalyze their mechanistically distinct reactions. Although the mechanism of the KGPDC-catalyzed reaction has yet to be elucidated, it is thought to proceed via a Mg(2+) ion-stabilized 1,2-enediolate intermediate. Here we report the crystal structures of KGPDC complexed with L-gulonate 6-phosphate, L-threonohydroxamate 4-phosphate, and L-xylitol 5-phosphate, analogues of the substrate, enediolate intermediate, and product, as well as with the product, L-xylulose 5-phosphate, at 1.2, 1.8, 1.7, and 1.8 A resolution, respectively. These structures support a mechanism that involves the formation of a cis-1,2-enediolate intermediate. Contrary to expectations, the geometry of the intermediate does not involve bidentate coordination of both enediolate oxygen atoms to the Mg(2+) ion but rather involves only the coordination of the oxygen on C2 to the Mg(2+) ion. The oxygen atom on C1 instead forms hydrogen bonds to both Lys64 and Asp67, two strictly conserved active site residues. Lys64 also interacts with the oxygen on C2 and may serve to stabilize a cis conformation of the 1,2-enediolate. These structures also implicate His136 to be the general acid that protonates the 1,2-enediolate intermediate. This study further demonstrates that multiple unrelated enzyme functions can evolve from a single active site architecture without regard for substrate binding affinity or mechanism.

Carboxy-Lyases↗
PubMed2003

Trisoxazole macrolide toxins mimic the binding of actin-capping proteins to actin.

Marine macrolide toxins of trisoxazole family target actin with high affinity and specificity and have promising pharmacological properties. We present X-ray structures of actin in complex with two members of this family, kabiramide C and jaspisamide A, at a resolution of 1.45 and 1.6 A, respectively. The structures reveal the absolute stereochemistry of these toxins and demonstrate that their trisoxazole ring interacts with actin subdomain 1 while the aliphatic side chain is inserted into the hydrophobic cavity between actin subdomains 1 and 3. The binding site is essentially the same as the one occupied by the actin-capping domain of the gelsolin superfamily of proteins. The structural evidence suggests that actin filament severing and capping by these toxins is also analogous to that of gelsolin. Consequently, these macrolides may be viewed as small molecule biomimetics of an entire class of actin-binding proteins.

Actins↗
PubMed2003

The structural determination of phosphosulfolactate synthase from Methanococcus jannaschii at 1.7-A resolution: an enolase that is not an enolase.

Members of the enolase mechanistically diverse superfamily catalyze a wide variety of chemical reactions that are related by a common mechanistic feature, the abstraction of a proton adjacent to a carboxylate group. Recent investigations into the function and mechanism of the phosphosulfolactate synthase encoded by the ComA gene in Methanococcus jannaschii have suggested that ComA, which catalyzes the stereospecific Michael addition of sulfite to phosphoenolpyruvate to form phosphosulfolactate, may be a member of the enolase superfamily. The ComA-catalyzed reaction, the first step in the coenzyme M biosynthetic pathway, likely proceeds via a Mg2+ ion-stabilized enolate intermediate in a manner similar to that observed for members of the enolase superfamily. ComA, however, has no significant sequence similarity to any known enolase. Here we report the x-ray crystal structure of ComA to 1.7-A resolution. The overall fold for ComA is an (alpha/beta)8 barrel that assembles with two other ComA molecules to form a trimer in which three active sites are created at the subunit interfaces. From the positions of two ordered sulfate ions in the active site, a model for the binding of phosphoenolpyruvate and sulfite is proposed. Despite its mechanistic similarity to the enolase superfamily, the overall structure and active site architecture of ComA are unlike any member of the enolase superfamily, which suggests that ComA is not a member of the enolase superfamily but instead acquired an enolase-type mechanism through convergent evolution.

Bacterial Proteins↗
PubMed2003

The structural determination of an insect sterol carrier protein-2 with a ligand-bound C16 fatty acid at 1.35-A resolution.

Yellow fever mosquito sterol carrier protein (SCP-2) is known to bind to cholesterol. We report here the three-dimensional structure of the complex of SCP-2 from Aedes aegypti with a C16 fatty acid to 1.35-A resolution. The protein fold is exceedingly similar to the human and rabbit proteins, which consist of a five-stranded beta-sheet that exhibits strand order 3-2-1-4-5 with an accompanying layer of four alpha-helices that cover the beta-sheet. A large cavity exists at the interface of the layer alpha-helices and the beta-sheet, which serves as the fatty acid binding site. The carboxylate moiety of the fatty acid is coordinated by a short loop that connects the first alpha-helix to the first beta-strand, whereas the acyl chain extends deep into the interior of the protein. Interestingly, the orientation of the fatty acid is opposite to the observed orientation for Triton X-100 in the SCP-2-like domain from the peroxisomal multifunctional enzyme (Haapalainen, A. M., van Aalten, D. M., Merilainen, G., Jalonen, J. E., Pirila, P., Wierenga, R. K., Hiltunen, J. K., and Glumoff, T. (2001) J. Mol. Biol. 313, 1127-1138). The present study suggests that the binding pocket in the SCP-2 family of proteins may exhibit conformational flexibility to allow coordination of a variety of lipids.

Acetyl-CoA C-Acetyltransferase↗
PubMed2003

The three-dimensional structure of the core domain of Naf Y from Azotobacter vinelandii determined at 1.8-A resolution.

The Azotobacter vinelandii NafY protein (nitrogenase accessory factor Y) is able to bind either to the iron molybdenum cofactor (FeMo-co) or to apodinitrogenase and is believed to facilitate the transfer of FeMo-co into apodinitrogenase. The NafY protein has two domains: an N-terminal domain (residues Met1-Leu98) and a C-terminal domain (residues Glu99-Ser232), referred here to as the "core domain." The core domain of NafY is shown here to be capable of binding the FeMo cofactor of nitrogenase but unable to bind to apodinitrogenase in the absence of the first domain. The three-dimensional molecular structure of the core domain of NafY has been solved to 1.8-A resolution, revealing that the protein consists of a mixed five-stranded beta-sheet flanked by five alpha-helices that belongs to the ribonuclease H superfamily. As such, this represents a new fold capable of binding FeMo-co, where the only previous example was that seen in dinitrogenase.

Amino Acid Sequence↗
PubMed2003

Project management system for structural and functional proteomics: Sesame.

A computing infrastructure (Sesame) has been designed to manage and link individual steps in complex projects. Sesame is being developed to support a large-scale structural proteomics pilot project. When complete, the system is expected to manage all steps from target selection to data-bank deposition and report writing. We report here on the design criteria of the Sesame system and on results demonstrating successful achievement of the basic goals of its architecture. The Sesame software package, which follows the client/server paradigm, consists of a framework, which supports secure interactions among the three tiers of the system (the client, server, and database tiers), and application modules that carry out specific tasks. The framework utilizes industry standards. The client tier is written in Java2 and can be accessed anywhere through the Internet. All the development on the server tier is also carried out in Java2 so as to accommodate a wide variety of computer platforms. The database tier employs a commercial database management system. Each Sesame application module consists of a simple user interface in the client tier, corresponding objects in the server tier, and relevant data stored in the centralized database. For security, access to stored data is controlled by access privileges. The system facilitates both local and remote collaborations. Because users interact with the system using Java Web Start or through a web browser, access is limited only by the availability of an Internet connection. We describe several Sesame modules that have been developed to the point where they are being utilized routinely to support steps involved in structural and functional proteomics. This software is available to parties interested in using it and assisting to guide its further development.

Database Management Systems↗
PubMed2002

Evidence for "unseen" transposase--DNA contacts.

In this study, evidence of novel, important interactions between a hyperactive Tn5 transposon recognition end sequence and hyperactive Tn5 transposase (Tnp) are presented. A hyperactive Tn5 end sequence, the mosaic end (ME), was isolated previously. The ME and a wild-type end sequence, the outside end (OE), differ at only three positions, yet transposition on the ME is tenfold higher than on the OE in vivo. Also, transposition on the ME is much more efficient than transposition on the OE in vitro. Here, we show that the decreased activity observed for the OE is caused by a defect in paired ends complex (PEC) formation resulting from the orientation of the A-T base-pair at position 4 of this end. Efficient PEC formation requires an interaction between the C5-methyl group (C5-Me) on the non-transferred strand thymine base at position 4 (T4) and Tnp. PEC formation on nicked substrates is much less affected by the orientation of the A-T base-pair at position 4, indicating that the C5-Me group is important only for steps preceding nicking. A recently determined co-crystal structure of Tn5 Tnp with the ME is discussed and a model explaining possible roles for the base-pair at position 4 is explored.

Bacterial Proteins↗
PubMed2002

Structural studies of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica: the apo, substrate, and product-aldimine complexes.

The evolution of biosynthetic pathways is difficult to reconstruct in hindsight; however, the structures of the enzymes that are involved may provide insight into their development. One enzyme in the cobalamin biosynthetic pathway that appears to have evolved from a protein with different function is L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica, which is structurally similar to histidinol phosphate aminotransferase [Cheong, C. G., Bauer, C. B., Brushaber, K. R., Escalante-Semerena, J. C., and Rayment, I. (2002) Biochemistry 41, 4798-4808]. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. To understand the relationship between this decarboxylase and the aspartate aminotransferase family to which it belongs, the structures of CobD in its apo state, the apo state complexed with the substrate, and its product external aldimine complex have been determined at 1.46, 1.8, and 1.8 A resolution, respectively. These structures show that the enzyme steers the breakdown of the external aldimine toward decarboxylation instead of amino transfer by positioning the carboxylate moiety of the substrate out of the plane of the pyridoxal ring and by placing the alpha-hydrogen out of reach of the catalytic base provided by the lysine that forms the internal aldimine. It would appear that CobD evolved from a primordial PLP-dependent aminotransferase, where the selection was based on similarities between the stereochemical properties of the substrates rather than preservation of the fate of the external aldimine. These structures provide a sequence signature for distinguishing between L-threonine-O-3-phosphate decarboxylase and histidinol phosphate aminotransferases, many of which appear to have been misannotated.

Binding Sites↗
PubMed2002

Capture of a labile substrate by expulsion of water molecules from the active site of nicotinate mononucleotide:5,6-dimethylbenzimidazole phosphoribosyltransferase (CobT) from Salmonella enterica.

Nicotinate mononucleotide (NaMN):5,6-dimethylbenzimidazole (DMB) phosphoribosyltransferase (CobT) from Salmonella enterica plays a central role in the synthesis of alpha-ribazole-5'-phosphate, an intermediate for the lower ligand of cobalamin. In earlier studies it proved difficult to obtain the structure of CobT bound to NaMN because it is hydrolyzed in the crystal lattice in the absence of the second substrate DMB. In an effort to map the reaction pathway of this enzyme, NaMN was captured in the active site with the substrate analogs 4,5-dimethyl-1,2-phenylenediamine, 4-methylcatechol, indole, 3,4-dimethylaniline, 2,5-dimethylaniline, 3,4-dimethylphenol, and 2-amino-p-cresol. Structures of these complexes reveal that they exclude water molecules responsible for the hydrolysis from the active site. These structures, together with the early complexes with alpha-ribazole-5'-phosphate and DMB, provide a complete description of the reaction pathway. They demonstrate that the nicotinate moiety and phosphate do not appear to move significantly between reactants and products but that the aromatic base and ribose moiety each move approximately 1.2 A toward each other in the transformation. This study also reveals that, like many other nucleotide binding proteins, coordination of DMB is accompanied by a disorder-order transition in a surface loop. The structure of apo-CobT is also reported.

Binding Sites↗
PubMed2002

The ATP:Co(I)rrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica requires the 2'-OH group of ATP for function and yields inorganic triphosphate as its reaction byproduct.

The specificity of the ATP:corrinoid adenosyltransferase (CobA) enzyme of Salmonella enterica serovar Typhimurium LT2 for its nucleotide substrate was tested using ATP analogs and alternative nucleotide donors. The enzyme showed broad specificity for the nucleotide base and required the 2'-OH group of the ribosyl moiety of ATP for activity. (31)P NMR spectroscopy was used to identify inorganic triphosphate (PPP(i)) as the byproduct of the reaction catalyzed by the CobA enzyme. Cleavage of triphosphate into pyrophosphate and orthophosphate did not occur, indicating that triphosphate cleavage was not required for release of the adenosylcorrinoid product. Triphosphate was a strong inhibitor of the reaction, with 85% of CobA activity lost when the ATP/PPP(i) ratio present in the reaction mixture was 1:2.5.

Adenosine Triphosphate↗
PubMed2002

Three-dimensional structure of the L-threonine-O-3-phosphate decarboxylase (CobD) enzyme from Salmonella enterica.

The three-dimensional structure of the pyridoxal 5'-phosphate (PLP)-dependent L-threonine-O-3-phosphate decarboxylase (CobD) from Salmonella enterica is described here. This enzyme is responsible for synthesizing (R)-1-amino-2-propanol phosphate which is the precursor for the linkage between the nucleotide loop and the corrin ring in cobalamin. The molecule is a molecular dimer where each subunit consists of a large and small domain. Overall the protein is very similar to the members of the family of aspartate aminotransferases. Indeed, the arrangement of the ligands surrounding the cofactor and putative substrate binding site are remarkably close to that observed in histidinol phosphate aminotransferase, which suggests that this latter enzyme might have been its progenitor. The only significant differences in structure occur at the N-terminus, which is approximately 12 residues shorter in CobD and does not form the same type of interdomain interaction common to other aminotransferases. CobD is unusual since within the aspartate aminotransferase subfamily of PLP-dependent enzymes the chemical transformations are substantially conserved, where the only exceptions are 1-aminocyclopropane-1-carboxylate synthase and CobD. Although there are a large number of PLP-dependent amino acid decarboxylases, these are generally larger and structurally distinct from the members of the aspartate aminotransferase subfamily of enzymes. The structure of CobD suggests that the chemical fate of the external aldimine can be redirected by modifications at the N-terminus of the protein. This study provides insight into the evolutionary history of the cobalamin biosynthetic pathway and raises the question of why most PLP-dependent decarboxylases are considerably larger enzymes.

Amino Acid Sequence↗
PubMed2002

Homologous (beta/alpha)8-barrel enzymes that catalyze unrelated reactions: orotidine 5'-monophosphate decarboxylase and 3-keto-L-gulonate 6-phosphate decarboxylase.

The 3-keto-L-gulonate 6-phosphate decarboxylase (KGPDC) encoded by the ulaD gene in the Escherichia coli genome [Yew, W. S., and Gerlt, J. A. (2002) J. Bacteriol. 184, 302-306] and orotidine 5'-monophosphate decarboxylase (OMPDC) are homologous (derived from a common ancestor) but catalyze different reactions. The metal-independent decarboxylation reaction catalyzed by OMPDC avoids the formation of a vinyl anion intermediate; the Mg2+-dependent decarboxylation reaction catalyzed by KGPDC involves the formation of an enediolate anion intermediate. Based on the available structures of OMPDC, a sequence alignment allows the predictions that (1) KGPDC is a dimer of (beta/alpha)8-barrels, with the active sites located at the dimer interface; (2) KGPDC and OMPDC share an aspartate residue at the end of the first beta-strand and an Asp-x-Lys-x-x-Asp motif at the end of the third beta-strand with OMPDC; but (3) KGPDC has a Glu instead of a Lys at the end of the second beta-strand. The structure of KGPDC has been determined in the presence of Mg2+ and the substrate analogue L-gulonate 6-phosphate and confirms these predictions. The carboxylate functional groups at the ends of the first, second, and third beta-strands in KGPDC are ligands of the Mg2+; in OMPDC, the homologues of these residues participate in a hydrogen-bonded network that facilitates the decarboxylation reaction. The 3-OH group of the substrate analogue is coordinated to the Mg2+, supporting the hypothesis that the mechanism of the decarboxylation catalyzed by KGPDC involves stabilization of an enediolate anion intermediate. These structural studies establish the existence of the OMPDC "suprafamily," in which members catalyze reactions that occur in different metabolic pathways and share no mechanistic relationship. The existence of this suprafamily demonstrates that divergent evolution can be opportunistic, conscripting active site features of a progenitor to catalyze unrelated functions. Accordingly, sequence or structure homology alone cannot be used to infer the functions of new proteins discovered in genome projects.

Amino Acid Sequence↗
PubMed2002

Pentaerythritol propoxylate: a new crystallization agent and cryoprotectant induces crystal growth of 2-methylcitrate dehydratase.

In the search for macromolecular crystallization conditions, the precipitant is probably the most important variable, such that when problematic crystals are encountered there is always the question of whether an alternative precipitant might resolve the problem. During an effort to obtain high-quality crystals of several problematic proteins, two new agents, pentaerythritol propoxylate and pentaerythritol ethoxylate, yielded well ordered quality crystals where more traditional precipitants were unsuccessful. Pentaerythritol propoxylate and pentaerythritol ethoxylate contain a pentaerythritol backbone to which organic polymers are bound, forming a branched polymer. As such, they are larger than small organic precipitants such as low molecular-weight alcohols or 2-methyl-2,4-pentanediol, but behave differently to polyethylene glycols. These compounds have been used to crystallize an enzyme encoded by the Salmonella enterica prpD gene that catalyzes the dehydration of 2-methylcitrate to form 2-methyl-cis-aconitate. While the PrpD protein has crystallized readily under a number of conditions, the resultant crystals were unsuitable for a crystal structure determination. The new crystals obtained with 25-40% pentaerythritol propoxylate belong to the orthorhombic space group C222(1), with unit-cell parameters a = 73.2, b = 216.4, c = 214.3 A, and diffract beyond 2.0 A with synchrotron radiation. A further benefit of this precipitant for crystallization is its ability to function as a cryoprotectant, allowing the crystals to be transferred directly from the mother liquor to the nitrogen stream at 113 K.

Aconitate Hydratase↗