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The universal sesquiterpene precursor, farnesyl diphosphate (FPP), is cyclized in an Mg(2+)-dependent reaction catalyzed by the tetrameric aristolochene synthase from Aspergillus terreus to form the bicyclic hydrocarbon aristolochene and a pyrophosphate anion (PP(i)) coproduct. The 2.1-A resolution crystal structure determined from crystals soaked with FPP reveals the binding of intact FPP to monomers A-C, and the binding of PP(i) and Mg(2+)(B) to monomer D. The 1.89-A resolution structure of the complex with 2-fluorofarnesyl diphosphate (2F-FPP) reveals 2F-FPP binding to all subunits of the tetramer, with Mg(2+)(B)accompanying the binding of this analogue only in monomer D. All monomers adopt open activesite conformations in these complexes, but slight structural changes in monomers C and D of each complex reflect the very initial stages of a conformational transition to the closed state. Finally, the 2.4-A resolution structure of the complex with 12,13-difluorofarnesyl diphosphate (DF-FPP) reveals the binding of intact DF-FPP to monomers A-C in the open conformation and the binding of PP(i), Mg(2+)(B), and Mg(2+)(C) to monomer D in a predominantly closed conformation. Taken together, these structures provide 12 independent "snapshots" of substrate or product complexes that suggest a possible sequence for metal ion binding and conformational changes required for catalysis.

The L-to-D-peptide isomerase from the venom of the platypus (Ornithorhyncus anatinus) is the first such enzyme to be reported for a mammal. In delineating its catalytic mechanism and broader roles in the animal, its substrate specificity was explored. We used N-terminal segments of defensin-like peptides DLP-2 and DLP-4 and natriuretic peptide OvCNP from the venom as substrates. The DLP analogues IMFsrs and ImFsrs (srs is a solubilizing chain; lowercase letters denote D-amino acid) were effective substrates for the isomerase; it appears to recognize the N-terminal tripeptide sequence Ile-Xaa-Phe-. A suite of 26 mutants of these hexapeptides was synthesized by replacing the second residue (Met) with another amino acid, viz. Ala, alpha-aminobutyric acid, Ile, Leu, Lys, norleucine, Phe, Tyr, and Val. It was shown that mutant peptides incorporating norleucine and Phe are substrates and exhibit L- or D-amino acid isomerization, but mutant peptides that contain residues with shorter, beta-branched or long side chains with polar terminal groups, viz. Ala, alpha-aminobutyric acid, Ile, Val, Leu, Lys, and Tyr, respectively, are not substrates. It was demonstrated that at least three N-terminal amino acid residues are absolutely essential for L-to-D-isomerization; furthermore, the third amino acid must be a Phe residue. None of the hexapeptides based on LLH, the first three residues of OvCNP, were substrates. A consistent 2-base mechanism is proposed for the isomerization; abstraction of a proton by 1 base is concomitant with delivery of a proton by the conjugate acid of a second base.

Terpene synthases often catalyze complex cyclization reactions that typically represent the committed step in particular biosynthetic pathways, leading to great interest in their enzymatic mechanisms. We have recently demonstrated that substitution of a specific Ile with Thr was sufficient to "short circuit" the complex cyclization reaction normally catalyzed by ent-kaurene synthases to instead produce ent-pimaradiene. Here we report that the complex cyclization/rearrangement reaction catalyzed by abietadiene synthase can be similarly cut short to produce pimaradienes by an analogous Ser for Ala change, albeit with a slight shift in active site location to accommodate the difference in substrate stereochemistry. This result has mechanistic implications for enzymatic catalysis of abietadiene cyclization, and terpene synthases more broadly. Furthermore, these defined single residue switches may be useful in engineering product outcome in diterpene synthases more generally.

The reaction mechanism of 4-oxalocrotonate tautomerase (4-OT) is studied using the density functional theory method B3LYP. This enzyme catalyzes the isomerisation of unconjugated alpha-keto acids to their conjugated isomers. Two different quantum chemical models of the active site are devised and the potential energy curves for the reaction are computed. The calculations support the proposed reaction mechanism in which Pro-1 acts as a base to shuttle a proton from the C3 to the C5 position of the substrate. The first step (proton transfer from C3 to proline) is shown to be the rate-limiting step. The energy of the charge-separated intermediate (protonated proline-deprotonated substrate) is calculated to be quite low, in accordance with measured pKa values. The results of the two models are used to evaluate the methodology employed in modeling enzyme active sites using quantum chemical cluster models.

Although the formation of disulfide bonds is an essential process in every living organism, only little is known about the mechanisms in multicellular eukaryotic systems. The reason for this uncertainty is that in addition to the well-known key enzyme protein disulfide isomerase (PDI), several PDI-like proteins are present in the ER of metazoans. In total, there are now 18 PDI-family members in the human endoplasmic reticulum, with different domain architectures and active site chemistries. To understand why multicellular organisms express multiple proteins with similarity to the archetypal mammalian PDI, the properties of three PDIs from the nematode C. elegans were investigated. Here the authors demonstrate that PDI-1, PDI-2, and PDI-3 show comparable kinetic properties in catalyzing thiol:disulfide exchange reactions in two simple peptide-based assays. However, the three enzymes exhibited clear differences in their reactivity towards protein substrates. The authors therefore propose that the three PDIs can catalyze similar thiol-disulfide exchange reactions in a substrate, but due to differences in substrate binding, they can direct a folding polypeptide chain onto different folding pathways and hence fulfil distinct and different functions in the organism.

An intramolecular proton-transfer mechanism has been proposed for the carbocationic cyclization of farnesyl pyrophosphate (FPP) to (+)-aristolochene catalyzed by aristolochene synthase. This novel mechanism, which is based on results obtained by high-level ab initio molecular orbital and density functional theory calculations, differs from the previous proposal in the key step of carbocation propagation prior to the formation of the bicyclic carbon skeleton. Previously, germacrene A was proposed to be generated as an intermediate by deprotonation of germacryl cation followed by reprotonation of the C6-C7 double bond to yield eudesmane cation. In the mechanism proposed here the direct intramolecular proton transfer has a computed barrier of about 22 kcal/mol, which is further lowered to 16-20 kcal/mol by aristolochene synthase. An alternative pathway is also possible through a proton shuttle via a pyrophosphate-bound water molecule. The mechanism proposed here is consistent with the observation that germacrene A is not a substrate of aristolochene synthase. Furthermore, the modeled substrate-enzyme complex suggests that Trp 334 and Phe 178 play key roles in positioning the substrate in the reactive orientation in the binding pocket. This is consistent with experimental findings that mutations of either residue lead to pronounced generation of aborted cyclization products.

A new method, referred to as the sequential quadratic programming method, is presented for determining minimum energy paths. The method is based on minimizing the points representing the path in the subspace perpendicular to the tangent of the path while using a penalty term to prevent kinks from forming. Rather than taking one full step, the minimization is divided into a number of sequential steps on an approximate quadratic surface. The resulting method can efficiently determine the reaction mechanism, from which transition state can be easily identified and refined with other methods. To improve the resolution of the path close to the transition state, points are clustered close to this region with a reparametrization scheme. The usefulness of the algorithm is demonstrated for the Muller-Brown potential, amide hydrolysis, and an 89 atom cluster taken from the active site of 4-oxalocrotonate tautomerase for the reaction which catalyzes 2-oxo-4-hexenedioate to the intermediate 2-hydroxy-2,4-hexadienedioate.

Analogues of farnesyl diphosphate (FPP, ) containing phenyl substituents in place of methyl groups have been prepared in syntheses that feature use of a Suzuki-Miyaura reaction as a key step. These analogues were found not to act as substrates of the sesquiterpene cyclase aristolochene synthase from Penicillium roqueforti (AS). However, they were potent competitive inhibitors of AS with K(I)-values ranging from 0.8 to 1.2 microM. These results indicate that the diphosphate group contributes the largest part to the binding of the substrate to AS and that the active sites of terpene synthases are sufficiently flexible to accommodate even substrate analogues with large substituents suggesting a potential way for the generation of non-natural terpenoids. Molecular mechanics simulations of the enzyme bound inhibitors suggested that small changes in orientations of active site residues and subtle alterations of the conformation of the backbones of the inhibitors are sufficient to accommodate the phenyl-farnesyl-diphosphates.

The tautomerization of 2-oxo-4E-hexendioate by 4-oxalocrotonate tautomerase has been studied by quantum mechanical/molecular mechanical (QM/MM) methods using three models, A-C, with different substrate orientations. The computed QM/MM energy profiles are rather different. Various energy partitioning analyses indicate the origin of these differences and the role of the active site residues for different substrate orientations. The proposed new model C is preferred over the previously used models A and B because it combines favorable substrate binding geometries with reasonable barriers and is consistent with the experimental evidence from mutation studies concerning the catalytic ability of specific residues in the binding site, especially R11'.

The repeating unit of the glycan chain in the S-layer of the bacterium Aneurinibacillus thermoaerophilus L420-91(T) is composed of four alpha-d-rhamnose molecules and two 3-acetamido-3,6-dideoxy-alpha-d-galactose moieties (abbreviated as Fucp3NAc). Formation of the glycan layer requires nucleotide-activated sugars as the donor molecules. Whereas the enzymes involved in the synthesis of GDP-rhamnose have been well characterized, less is known regarding the structures and enzymatic mechanisms of the enzymes required for the production of dTDP-Fucp3NAc. One of the enzymes involved in the biosynthesis of dTDP-Fucp3NAc is a 3,4-ketoisomerase, hereafter referred to as FdtA. Here we describe the first three-dimensional structure of this sugar isomerase complexed with dTDP and solved to 1.5 A resolution. The FdtA dimer assumes an almost jellyfish-like appearance with the sole alpha-helices representing the tentacles. Formation of the FdtA dimer represents a classical example of domain swapping whereby beta-strands 2 and 3 from one subunit form part of a beta-sheet in the second subunit. The active site architecture of FdtA is characterized by a cluster of three histidine residues, two of which, His(49) and His(51), appear to be strictly conserved in the amino acid sequences deposited to date. Site-directed mutagenesis experiments, enzymatic assays, and x-ray crystallographic analyses suggest that His(49) functions as an active site base.

Mycothiol (MSH) is the major low molecular mass thiols in many Gram-positive bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. The physiological roles of MSH are believed to be equivalent to those of GSH in Gram-negative bacteria, but current knowledge of MSH is limited to detoxification of alkalating chemicals and protection from host cell defense/killing systems. Recently, an MSH-dependent maleylpyruvate isomerase (MDMPI) was discovered from C. glutamicum, and this isomerase represents one example of many putative MSH-dependent enzymes that take MSH as cofactor. In this report, fourteen mutants of MDMPI were generated. The wild type and mutant (H52A) MDMPIs were crystallized and their structures were solved at 1.75 and 2.05 A resolution, respectively. The crystal structures reveal that this enzyme contains a divalent metal-binding domain and a C-terminal domain possessing a novel folding pattern (alphabetaalphabetabetaalpha fold). The divalent metal-binding site is composed of residues His52, Glu144, and His148 and is located at the bottom of a surface pocket. Combining the structural and site-directed mutagenesis studies, it is proposed that this surface pocket including the metal ion and MSH moiety formed the putative catalytic center.

Aristolochene synthase from Aspergillus terreus catalyzes the cyclization of the universal sesquiterpene precursor, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. The 2.2 A resolution X-ray crystal structure of aristolochene synthase reveals a tetrameric quaternary structure in which each subunit adopts the alpha-helical class I terpene synthase fold with the active site in the "open", solvent-exposed conformation. Intriguingly, the 2.15 A resolution crystal structure of the complex with Mg2+3-pyrophosphate reveals ligand binding only to tetramer subunit D, which is stabilized in the "closed" conformation required for catalysis. Tetramer assembly may hinder conformational changes required for the transition from the inactive open conformation to the active closed conformation, thereby accounting for the attenuation of catalytic activity with an increase in enzyme concentration. In both conformations, but especially in the closed conformation, the active site contour is highly complementary in shape to that of aristolochene, and a catalytic function is proposed for the pyrophosphate anion based on its orientation with regard to the presumed binding mode of aristolochene. A similar active site contour is conserved in aristolochene synthase from Penicillium roqueforti despite the substantial divergent evolution of these two enzymes, while strikingly different active site contours are found in the sesquiterpene cyclases 5-epi-aristolochene synthase and trichodiene synthase. Thus, the terpenoid cyclase active site plays a critical role as a template in binding the flexible polyisoprenoid substrate in the proper conformation for catalysis. Across the greater family of terpenoid cyclases, this template is highly evolvable within a conserved alpha-helical fold for the synthesis of terpene natural products of diverse structure and stereochemistry.

Olfaction of insects is currently recognized as the major area of research for developing novel control strategies to prevent mosquito-borne infections. A 3-dimensional model (3D) was developed for the salivary gland odorant-binding protein-2 of the mosquito Culex quinquefasciatus, a major vector of human lymphatic filariasis. A homology modeling method was used for the prediction of the structure. For the modeling, two template proteins were obtained by mGenTHERADER, namely the high-resolution X-ray crystallography structure of a pheromone-binding protein (ASP1) of Apis mellifera L., [1R5R:A] and the aristolochene synthase from Penicillium roqueforti [1DI1:B]. By comparing the template protein a rough model was constructed for the target protein using MODELLER, a program for comparative modelling. The structure of OBP of the mosquito Culex quinquefasciatus resembles the structure of pheromone-binding protein ASP1 of Apis mellifera L., [1R5R:A]. From Ramachandran plot analysis it was found that the portion of residues falling into the most favoured regions was 86.0%. The predicted 3-D model may be further used in characterizing the protein in wet laboratory.

The enzyme S-adenosylmethionine:tRNA ribosyltransferase-isomerase (QueA) is involved in the biosynthesis of the hypermodified tRNA nucleoside queuosine. It is unprecedented in nature as it uses the cofactor S-adenosylmethionine as the donor of a ribosyl group. We have determined the crystal structure of Bacillus subtilis QueA at a resolution of 2.9A. The structure reveals two domains representing a 6-stranded beta-barrel and an alpha beta alpha-sandwich, respectively. All amino acid residues invariant in the QueA enzymes of known sequence cluster at the interface of the two domains indicating the localization of the substrate binding region and active center. Comparison of the B. subtilis QueA structure with the structure of QueA from Thermotoga maritima suggests a high domain flexibility of this enzyme.

The effect of replacing arginine residues (Arg) with citrulline residues (Cit) in the binding site of 4-oxalocrotonate tautomerase (4-OT) was investigated with force field molecular dynamics and hybrid quantum mechanics/molecular mechanics studies. It is found that the Arg61Cit mutation has only minor effects on the k(cat) and K(M) values determined experimentally because of the flexibility of this residue. The decrease in k(cat) and increase in K(M) for the Arg11Cit and Arg39Cit mutations are due to the disruption of the binding site, which arises from repulsive interactions with neighboring residues. The results of this investigation shed new light on the effects of mutations in the binding site of 4-OT and consequently on how the enzyme binds the active substrate.

Many proteins have developed the potential to sequester a client polypeptide chain in its various folding states as a specific intermolecular ligand and, thus, exhibit the properties of a holding chaperone. The resulting complexes can be of a diverse nature in terms of structure and reaction dynamics and are characterized on the basis of various microscopic properties including formation and decay of encounter and Michaelis complexes as well as reactant and product stability. Interpretation of the functional consequences of complex formation in the cell generally tends to be rather complicated, with notable exceptions including complexes formed during the reaction pathways of proteases, protein kinases and protein phosphatases. Peptide bond cis/trans isomerases take up an intermediate position among the poly(oligo)peptide binding proteins because, although the relationship between chain sequestration and catalysis of isomerization can easily be delineated in vitro, it is sometimes difficult to resolve in the cell. Time-resolved studies on interactions involving peptide bond cis/trans isomerases have led to the establishment of generally applicable methods for studying protein-poly(oligo)peptide interactions that are capable of identifying new types of biocatalysis.

Diterpene resin acids are prominent defense compounds against insect pests and pathogens in conifers. Biochemical and molecular analyses in grand fir (Abies grandis), Norway spruce (Picea abies), and loblolly pine (Pinus taeda) have identified two classes of genes and enzymes that generate much of the structural diversity of terpenoid defense compounds: The terpenoid synthases (TPS) and cytochrome P450 monooxgenases (P450). Using a single substrate, geranylgeranyl diphosphate, families of single-product and multi-product diterpene synthases generate an array of cyclic diterpene olefins. These diterpenes are converted to diterpene resin acids by activity of one or more P450 enzymes. A few conifer diterpene synthases have previously been cloned and characterized in grand fir and in Norway spruce. We have also previously shown that the loblolly pine P450 abietadienol/abietadienal oxidase (PtAO) catalyzes multiple oxidations of several diterpene alcohols and aldehydes. Conifer diterpene synthases are thought to function in plastids while P450s can also be localized to plastids or to the endoplasmic reticulum (ER). Here, we show that a loblolly pine cDNA (PtTPS-LAS) encodes a typical multi-product conifer diterpene synthase that forms levopimaradiene, abietadiene, palustradiene, and neoabietadiene similar to the grand fir abietadiene synthase and Norway spruce levopimaradiene/abietadiene synthase. Subcellular targeting of PtTPS-LAS and PtAO to plastids and ER, respectively, was shown with green fluorescent fusion protein expression in tobacco cells. These data suggest that enzymes for conifer diterpene resin acid biosynthesis are localized to at least two different subcellular compartments, plastids and ER, requiring efficient transport of intermediates and secretion of diterpene resin acids into the extracelluar space.

The mechanistic details of the cyclisation of farnesylpyrophosphate (FPP) by aristolochene synthase (AS) from Penicillium roqueforti have only recently begun to emerge, mainly through the analysis of the reaction products generated by AS-mutants. The reaction proceeds through several intermediates including germacrene A and eudesmane cation. Previous work suggested that the side chain of phenylalanine 178 promoted the conversion of eudesmane cation to aristolochene. We now report that the catalytic function of this residue during the conversion of eudesmane cation to aristolochene is mainly due to the large size of its side chain, which facilitates the hydride shift from C2 to C3, rather than its aromatic character. In addition, F178 appears to control the regioselectivity of the final deprotonation step and, together with F112, helps stabilise the developing positive charge on C1 after the expulsion of pyrophosphate from the substrate. These results complete a screen of likely active-site aromatic residues and establish their respective roles in the conversion of FPP to aristolochene.

4-Oxalocrotonate tautomerase (4-OT) and trans-3-chloroacrylic acid dehalogenase (CaaD) are members of the tautomerase superfamily, a group of structurally homologous proteins that share a beta-alpha-beta fold and a catalytic amino-terminal proline. 4-OT, from Pseudomonas putida mt-2, catalyzes the conversion of 2-oxo-4-hexenedioate to 2-oxo-3-hexenedioate through the dienol intermediate 2-hydroxymuconate in a catabolic pathway for aromatic hydrocarbons. CaaD, from Pseudomonas pavonaceae 170, catalyzes the hydrolytic dehalogenation of trans-3-chloroacrylate in the trans-1,3-dichloropropene degradation pathway. Both reactions may involve an arginine-stabilized enediolate intermediate, a capability that may partially account for the low-level CaaD activity of 4-OT. Two active-site residues in 4-OT, Leu-8 and Ile-52, have now been mutated to the positionally conserved and catalytic ones in CaaD, alphaArg-8, and alphaGlu-52. The L8R and L8R/I52E mutants show improved CaaD activity (50- and 32-fold increases in k(cat)/K(m), respectively) and diminished 4-OT activity (5- and 1700-fold decreases in k(cat)/K(m), respectively). The increased efficiency of L8R-4-OT for the CaaD reaction stems primarily from an 8.8-fold increase in k(cat), whereas that of the L8R/I52E mutant is due largely to a 23-fold decrease in K(m). The presence of the additional arginine residue in the active site of L8R-4-OT does not alter the pK(a) of the Pro-1 amino group from that measured for the wild type (6.5 +/- 0.1 versus 6.4 +/- 0.2). Moreover, the crystal structure of L8R-4-OT is comparable to that of the wild type. Hence, the enhanced CaaD activity of L8R-4-OT is likely due to the additional arginine residue that can participate in substrate binding and/or stabilization of the putative enediolate intermediate. The results also suggest that the evolution of new functions within the tautomerase superfamily could be quite facile, requiring only a few strategically placed active-site mutations.

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.

AknH is a small polyketide cyclase that catalyses the closure of the fourth carbon ring in aclacinomycin biosynthesis in Streptomyces galilaeus, converting aklanonic acid methyl ester to aklaviketone. The crystal structure analysis of this enzyme, in complex with substrate and product analogue, showed that it is closely related in fold and mechanism to the polyketide cyclase SnoaL that catalyses the corresponding reaction in the biosynthesis of nogalamycin. Similarity is also apparent at a functional level as AknH can convert nogalonic acid methyl ester, the natural substrate of SnoaL, to auraviketone in vitro and in constructs in vivo. Despite the conserved structural and mechanistic features between these enzymes, the reaction products of AknH and SnoaL are stereochemically distinct. Supplied with the same substrate, AknH yields a C9-R product, like most members of this family of polyketide cyclases, whereas the product of SnoaL has the opposite C9-S stereochemistry. A comparison of high-resolution crystal structures of the two enzymes combined with in vitro mutagenesis studies revealed two critical amino acid substitutions in the active sites, which contribute to product stereoselectivity in AknH. Replacement of residues Tyr15 and Asn51 of AknH, located in the vicinity of the main catalytic residue Asp121, by their SnoaL counter-parts phenylalanine and leucine, respectively, results in a complete loss of product stereoselectivity.

The leptospira cbiC encodes the enzyme catalyzing the methyl rearrangement reaction of the cobalamin biosynthesis pathway. The protein has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The crystal structures have been solved in two crystal forms (P4(2)2(1)2 and P3(1)21) diffracting to 3.0 and 2.3A resolution, respectively. The structures are similar to the precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12.

Protein folding in the endoplasmic reticulum is often associated with the formation of native disulfide bonds, a process which in vivo is one of the rate limiting steps of protein folding and which is facilitated by the enzyme protein disulfide isomerase (PDI). Higher eukaryotes have multiple members of the PDI family, for example, seventeen human PDIs have been reported to date. With multiple members of the same family being present, even within the same cell, the question arises as to what differential functions are they performing? To date there has been no systematic evaluation of the enzymological properties of the different members of the PDI-family. To address the question of whether different PDI family members have differing thioldisulfide chemistry, we have recombinantly expressed and purified six members of the family, PDI, PDIp, ERp57, ERp72, P5, and PDIr from a single organism, human. An examination of the pH-dependence and nature of the rate limiting step for the peptide thiol-disulfide oxidase activity of these enzymes reveals that, with the exception of PDIr, they are all remarkably similar. In the light of this data potential differential functions for these enzymes are discussed.

The combined approaches of rational design and random mutagenesis were applied to generate a sesquiterpene synthase with an altered activity. Due to the lack of a convenient screen for sesquiterpene synthase activity, a high-throughput dual-activity screen was used by fusing (+)-delta-cadinene synthase to chloramphenicol acetyltransferase (CAT). The gene encoding (+)-delta-cadinene synthase was mutagenized using error-prone PCR. The resulting mutant fusion proteins were screened for CAT activity and altered sesquiterpene selectivity. Twenty-one clones producing (+)-delta-cadinene and germacrene D-4-ol in different ratios were isolated from the library. Analysis using a homology model of (+)-delta-cadinene synthase suggested that the G helix plays a very important role in (+)-delta-cadinene formation. Reconstruction of the G helix using site-directed, saturation mutagenesis yielded a mutant, N403P/L405H, that maintained its specific activity and showed higher selectivity to germacrene D-4-ol in vivo (up to 93%).

[reaction: see text] The cyclization of GGPP to taxadiene, catalyzed by taxadiene synthase, has been suggested to proceed through a series of monocyclic isocembrenyl- and bicyclic verticillyl-carbocationic intermediary stages. A set of GGPP analogues with abolished or perturbed pi-nucleophilicity at the delta10 double bond (GGPP numbering) was synthesized and incubated with taxadiene synthase to intercept the cyclization cascade at the monocyclic stage. Each analogue was transformed by taxadiene synthase in vitro to hydrocarbon products in varying yields, and the structures of the major product in each reaction were solved by GCEIMS and one- and two-dimensional (1H and 13C) NMR and found to be 14-membered monocyclic isocembrenyl diterpenes, indicating that the first C-C bond formation catalyzed by taxadiene synthase could be uncoupled from the other subsequent bond formation events by using suitably designed substrate analogues. The formation and isolation of these isocembrenyl diterpene products using taxadiene synthase supports proposals that the isocembrenyl cation is an intermediate in the cyclization of GGPP to taxadiene.

Malonate semialdehyde decarboxylase (MSAD) from Pseudomonas pavonaceae 170 is a tautomerase superfamily member that converts malonate semialdehyde to acetaldehyde by a mechanism utilizing Pro-1 and Arg-75. Pro-1 and Arg-75 have also been implicated in the hydratase activity of MSAD in which 2-oxo-3-pentynoate is processed to acetopyruvate. Crystal structures of MSAD (1.8 A resolution), the P1A mutant of MSAD (2.7 A resolution), and MSAD inactivated by 3-chloropropiolate (1.6 A resolution), a mechanism-based inhibitor activated by the hydratase activity of MSAD, have been determined. A comparison of the P1A-MSAD and MSAD structures reveals little geometric alteration, indicating that Pro-1 plays an important catalytic role but not a critical structural role. The structures of wild-type MSAD and MSAD covalently modified at Pro-1 by 3-oxopropanoate, the adduct resulting from the incubation of MSAD and 3-chloropropiolate, implicate Asp-37 as the residue that activates a water molecule for attack at C-3 of 3-chloropropiolate to initiate a Michael addition of water. The interactions of Arg-73 and Arg-75 with the C-1 carboxylate group of the adduct suggest these residues polarize the alpha,beta-unsaturated acid and facilitate the addition of water. On the basis of these structures, a mechanism for the inactivation of MSAD by 3-chloropropiolate can be formulated along with mechanisms for the decarboxylase and hydratase activities. The results also provide additional evidence supporting the hypothesis that MSAD and trans-3-chloroacrylic acid dehalogenase, a tautomerase superfamily member preceding MSAD in the trans-1,3-dichloropropene degradation pathway, diverged from a common ancestor but retained the key elements for the conjugate addition of water.

Glucosamine-6-phosphate synthase (EC 2.6.1.16) catalyses the first and practically irreversible step in the hexosamine metabolism pathway, the end product of which, uridine 5'-diphospho-N-acetyl D-glucosamine, is an essential substrate for assembly of the cell wall. The isomerase domain, consisting of residues 346-712 (42 kDa), of glucosamine-6-phosphate synthase from Candida albicans has been crystallized. X-ray analysis revealed that the crystals belonged to space group I4, with unit-cell parameters a = b = 149, c = 103 A. Diffraction data were collected to 3.8 A. Preliminary results from molecular replacement using the homologous bacterial monomer reveal that the asymmetric unit contains two monomers that resemble a bacterial dimer. The crystal lattice consists of pairs of such symmetry-related dimers forming elongated tetramers.

Background: Ser/Thr/Tyr kinases (STYKs) commonly found in eukaryotes have been recently reported in many bacterial species. Recent studies elucidating their cellular functions have established their roles in bacterial growth and development. However functions of a large number of bacterial STYKs still remain elusive. The organisation of domains in a large dataset of bacterial STYKs has been investigated here in order to recognise variety in domain combinations which determine functions of bacterial STYKs., Results: Using sensitive sequence and profile search methods, domain organisation of over 600 STYKs from 125 prokaryotic genomes have been examined. Kinase catalytic domains of STYKs tethered to a wide range of enzymatic domains such as phosphatases, HSP70, peptidyl prolyl isomerases, pectin esterases and glycoproteases have been identified. Such distinct preferences for domain combinations are not known to be present in either the Histidine kinase or the eukaryotic STYK families. Domain organisation of STYKs specific to certain groups of bacteria has also been noted in the current anlaysis. For example, Hydrophobin like domains in Mycobacterial STYK and penicillin binding domains in few STYKs of Gram-positive organisms and FHA domains in cyanobacterial STYKs. Homologues of characterised substrates of prokaryotic STYKs have also been identified., Conclusion: The domains and domain architectures of most of the bacterial STYKs identified are very different from the known domain organisation in STYKs of eukaryotes. This observation highlights distinct biological roles of bacterial STYKs compared to eukaryotic STYKs. Bacterial STYKs reveal high diversity in domain organisation. Some of the modular organisations conserved across diverse bacterial species suggests their central role in bacterial physiology. Unique domain architectures of few other groups of STYKs reveal recruitment of functions specific to the species.

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.

Previously, we reported that 2(E)-nonenal, having a low flavor threshold (0.1 ppb) and known as the major contributor to a cardboard flavor (stale flavor) in stored beer, is produced by lipoxygenase-1 and a newly found factor named 9-fatty acid hydroperoxide lyase-like (9-HPL-like) activity in malt. To assess the involvement of 9-HPL-like activity in beer staling, we compared the values of the wort nonenal potential, an index for predicting the staleness of beer, with the lipoxygenase and 9-HPL-like activity of 20 commercial malts. There was a significant correlation between the malt 9-HPL-like activity and the values of wort nonenal potential (r=0.53, P<0.05), while the correlation between malt lipoxygenase activity and the wort nonenal potential was statistically insignificant. Analysis of the partially purified 9-HPL-like activity from embryos of germinating barley seeds indicated that 9-HPL-like activity consisted of fatty acid hydroperoxide lyase and 3Z:2E isomerase.

Anaerobic unsaturated fatty acid synthesis in bacteria occurs through the introduction of a double bond into the growing acyl chain. In the Escherichia coli model system, FabA catalyzes both the dehydration of beta-hydroxydecanoyl-ACP and the isomerization of trans-2-decenoyl-ACP to cis-3-decenoyl-ACP as the essential step. A second dehydratase, FabZ, functions in acyl chain elongation but cannot carry out the isomerization reaction. Enterococcus faecalis has two highly related FabZ homologs. One of these, termed EfFabN, carries out the isomerization reaction in vivo, whereas the other, EfFabZ, does not (Wang, H., and Cronan, J. E. (2004) J. Biol. Chem. 279, 34489-34495). We carried out a series of domain swapping and mutagenesis experiments coupled with in vitro biochemical analyses to define the structural feature(s) that specify the catalytic properties of these two enzymes. Substitution of the beta3 and beta4 strands of EfFabZ with the corresponding strands from EfFabN was necessary and sufficient to convert EfFabZ into an isomerase. These data are consistent with the hypothesis that the isomerase potential of beta-hydroxyacyl-ACP dehydratases is determined by the properties of the beta-sheets that dictate the orientation of the central alpha-helix and thus the shape of the substrate binding tunnel rather than the catalytic machinery at the active site.

The yhcH gene is part of the nan operon in bacteria that encodes proteins involved in sialic acid catabolism. Determination of the crystal structure of YhcH from Haemophilus influenzae was undertaken as part of a structural genomics effort in order to assist with the functional assignment of the protein. The structure was determined at 2.2-A resolution by multiple-wavelength anomalous diffraction. The protein fold is a variation of the double-stranded beta-helix. Two antiparallel beta-sheets form a funnel opened at one side, where a putative active site contains a copper ion coordinated to the side chains of two histidine and two carboxylic acid residues. A comparison to other proteins with a similar fold and analysis of the genomic context suggested that YhcH may be a sugar isomerase involved in processing of exogenous sialic acid.

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in noncovalent association with beta(2)-microglobulin (beta(2)m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the beta(2)m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and beta(2)m, CNX bound to the FcRn H chain before the FcRn H chain association with beta(2)m. However, calreticulin only bound to the FcRn H chain-beta(2)m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for beta(2)m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.

We isolated an Arabidopsis albino and pale green 10 (apg10) mutant which exhibits pale green cotyledons and true leaves at the juvenile stage. We identified a valine to leucine change in BBMII (N'-[(5'-phosphoribosyl)-formimino]-5-aminoimidazole-4-carboxamide ribonucleotide) isomerase involved in histidine biosynthesis. The morphological abnormality of apg10 was recovered by histidine supplementation. The histidine limitation induced by apg10 mutation causes dynamic changes of the free amino acid profile, suggesting the existence of a cross-pathway regulatory mechanism of amino acid biosynthesis in plants. We also revealed that the APG10 knockout mutant exhibited embryo lethality, indicating the essential role of the Arabidopsis BBMII isomerase for plant growth.

The mechanism of the taxadiene synthase-catalyzed cyclization of (E,E,E)-geranylgeranyl diphosphate (GGPP, 7) to taxadiene (5) is proposed to proceed through a verticillen-12-yl carbocation intermediate (8) that undergoes an 11 --> 7 proton transfer leading to formation of the C ring. The substrate analogue 6-fluoroGGPP (17) was synthesized to elucidate the stereochemistry of the putative verticillenyl intermediate. It was expected that the inductive electron-withdrawing effect of the fluoro substituent would prevent the critical proton transfer to the Delta(7) double bond and thereby derail the cyclization at the bicyclic stage. Incubation of the fluoro analogue with recombinant taxadiene synthase yielded a mixture of three major and two minor fluoro diterpenes according to GC/MS analyses. The three major products were identified as the exocyclic, endocyclic, and 4(20)-methylene 7-fluoroverticillenes, i.e., Delta(3,7,12 (18)), Delta(3,7,12), and Delta(4(20),7,11) isomers (22, 23, and 24) on the basis of (1)H NMR analyses and comparisons with the parent bicyclic diterpenes. The H1beta, H11alpha (1S,11R) configurations at the bridgehead positions of 22 were established by means of NOE experiments and CD spectra. The absolute configuration of (+)-verticillol (4) was revised after the anomalous dispersion X-ray analysis of (+)-verticillol p-iodobenzoate. Of particular note, all absolute configurations of verticillane diterpenes in the literature should be reversed. This work affords compelling evidence supporting the H11alpha (11R) stereochemistry of the verticillen-12-yl(+) ion intermediate in the taxadiene synthase-catalyzed reaction and illustrates the capability of vinyl fluoro analogues to intercept complex cyclization cascades.

4-Oxalocrotonate tautomerase (4-OT), a homohexameric enzyme, converts the unconjugated enone, 2-oxo-4-hexenedioate (1), to the conjugated enone, 2-oxo-3-hexenedioate (3), via a dienolic intermediate, 2-hydroxymuconate (2). Pro-1 serves as the general base, and both Arg-11 and Arg-39 function in substrate binding and catalysis in an otherwise hydrophobic active site. Although 4-OT exhibits hyperbolic kinetics and no structural asymmetry either by X-ray or by NMR, inactivation by two affinity labels showed half-site stoichiometry [Stivers, J. T., et al. (1996) Biochemistry 35, 803-813; Johnson, W. H., Jr., et al. (1997) Biochemistry 36, 15724-15732], and titration of the R39Q mutant with cis,cis-muconate showed negative cooperativity [Harris, T. K., et al. (1999) Biochemistry 38, 12343-12357]. To test for anticooperativity during catalysis, 4-OT was titrated with equilibrium mixtures (> or = 81% product) of the reactive dicarboxylate or monocarboxylate intermediates, 2 or 2-hydroxy-2,4-pentadienoate (4), respectively, in three types of NMR experiments: two-dimensional 1H-15N HSQC titrations of backbone NH and of Arg N epsilonH resonances and one-dimensional 15N NMR titrations of Arg N epsilon resonances. All titrations showed substoichiometric binding of the equilibrium mixtures to 3 +/- 1 sites per hexamer with apparent dissociation constants comparable to the Km values of the intermediates. Compound 4 also bound 1 order of magnitude less tightly at another site, suggesting negative cooperativity. Consistent with negative cooperativity, asymmetry of the resulting complexes at saturating levels of 2 and 4 is indicated by splitting of the backbone NH resonances of 11 residues and 10 residues of 4-OT, respectively. The dicarboxylate competitive inhibitor, (2E)-fluoromuconate (5), with a KI of 45 +/- 7 microM, also exhibited substoichiometric binding to 3 +/- 1 sites per hexamer, with a KD of 25 +/- 18 microM, and splitting of the backbone NH resonance of L8. The monocarboxylate inhibitors (2E)- (6) and (2Z)-2-fluoro-2,4-pentadienoate (7) showed much weaker binding (KD = 3.1 +/- 1.3 mM), as well as splitting of two and five backbone NH resonances, respectively, indicating asymmetry of the complexes. The N epsilon resonances of both Arg-11 and Arg-39 were shifted downfield, and that of Pro-1N was broadened by all ligands, consistent with the major catalytic roles of these residues. Structural pathways for the site-site interactions which result in negative cooperativity are proposed on the basis of the X-ray structures of free and affinity-labeled 4-OT. Selective resonance broadenings induced by the binding of inactive analogues and active intermediates indicate residues which may be mobilized during reversible ligand binding and during catalysis, respectively.

In this study, we screened for protein disulfide isomerase (PDI)-binding proteins in bovine liver microsomes under strict salt concentrations, using affinity column chromatography. One main band observed using SDS-PAGE was identified as ERp57 (one of the PDI family proteins) by LC-MS/MS analysis. The K(D) value of PDI binding to ERp57 was calculated as 5.46x10(-6)M with the BIACORE system. The interactions between PDI and ERp57 occurred specifically at their a and b domains, respectively. Interestingly, low concentrations of ERp57 enhanced the chaperone activity of PDI, while high concentrations interfered with chaperone activity. On the other hand, ERp57 did not affect the isomerase activity of PDI. Additionally, following pre-incubation of ERp57 with calreticulin (CRT), decreased interactions were observed between ERp57 and PDI, and vice versa. Based on the data, we propose that once ERp57 binds to PDI or CRT, the resultant complex inhibits further interactions. Therefore, ERp57 selectively forms a protein-folding complex with PDI or CRT in ER.

Peroxisomal beta-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this beta-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4alpha, farnesoid X receptor, and peroxisome proliferator receptor alpha did not appear to be involved in the up-regulation of the transferase.

The substrate specificity of 4-oxalocrotonate tautomerase (4-OT) is characterized by electrostatic interactions between positively charged arginine (Arg) side chains on the enzyme and the dianionic substrate, 4-oxalocrotonate. To generate specific hydrogen-bonding interactions with a monoanionic substrate analogue, we have introduced a urea functional group into the active site by replacing arginine side chains with isosteric citrulline (Cit) residues. This design was based on the complementarity between the urea functionality of citrulline and the uncharged amide function of the substrate, as opposed to the guanidinium-carboxylate electrostatic interaction between the wild-type enzyme and the natural substrate. Indeed, the synthetic (Arg39Cit)4-OT analogue catalyzed the tautomerization of the non-natural monoamide-monoacid substrate while it was a poor catalyst for the natural diacid substrate. The specificity of (Arg39Cit)4-OT for the monoamide-monoacid substrate relative to that of the diacid substrate was found to be 740-fold greater than that of the wild-type enzyme for tautomerization of the non-natural substrate as compared with the natural one. The role of electrostatic interactions in the tautomerization of the monoamide-monoacid substrate was probed in detail with several other Arg to Cit analogues of this enzyme. This study has demonstrated that chemical manipulation of the functional groups within the active site of an enzyme can modify its catalytic activity and substrate specificity in a predictable way, suggesting that the incorporation of noncoded amino acids into proteins has great promise for the development of new enzymatic mechanisms and new binding interactions.

The wide-ranging physiology and large genetic variability observed for prokaryotes is largely attributed, not to the prokaryotic genome itself, but rather to mechanisms of lateral gene transfer. Cassette PCR has been used to sample the integron/gene cassette metagenome from different natural environments without laboratory cultivation of the host organism, and without prior knowledge of any target protein sequence. Since over 90% of cassette genes are unrelated to any sequence in the current databases, it is not clear whether these genes code for folded functional proteins. We have selected a sample of eight cassette-encoded genes with no known homologs; five have been isolated as soluble protein products and shown by biophysical techniques to be folded. In solution, at least three of these proteins organise as stable oligomeric assemblies. The tertiary structure of one of these, Bal32a derived from a contaminated soil site, has been solved by X-ray crystallography to 1.8 A resolution. From the three-dimensional structure, Bal32a is found to be a member of the highly adaptable alpha+beta barrel family of transport proteins and enzymes. In Bal32a, the barrel cavity is unusually deep and inaccessible to solvent. Polar side-chains in its interior are reminiscent of catalytic sites of limonene-1,2-epoxide hydrolase and nogalonic acid methyl ester cyclase. These studies demonstrate the viability of direct sampling of mobile DNA as a route for the discovery of novel proteins.

Vibrational spectroscopy has identified that in many cases, substrate association with enzyme active sites results in significant bond polarization. This bond polarization can be attributed to a combination of desolvation, conformational restriction, and true polarization by the local electric field. Quantum chemical calculations permit the extent of polarization to be quantified both in terms of partial charge and energy. The changes in vibrational frequency that occur during the binding process necessarily result in equilibrium isotope effects. The equilibrium isotope effect on association is one feature that differentiates isotope effects on k(cat) and k(cat)/K(m). An improved chemical understanding of the changes that occur on substrate binding will help elucidate the role of substrate activation in enzyme catalysis.

Glutathione transferases (GSTs) constitute a superfamily of detoxifying enzymes with a major role in protecting cellular macromolecules from reactive electrophilic compounds. A growing body of evidence suggests, however, that at least certain glutathione transferases are involved in other essential cellular processes, such as cellular signaling and anabolic pathways. One of them is the human GST A3-3, which is selectively expressed in steroidogenic organs and which efficiently catalyzes the obligatory isomerization of the Delta(5)-ketosteroid precursors in the biosynthesis of progesterone and testosterone. In this chapter, we summarize the current knowledge on human GST A3-3 and describe methods for heterologous expression and functional characterization of the enzyme.

Enzyme function is much less conserved than anticipated, i.e., the requirement for sequence similarity that implies similarity in enzymatic function is much higher than the requirement that implies similarity in protein structure. This is because the function of an enzyme is an extremely complicated problem that may involve very subtle structural details as well as many other physical chemistry factors. Accordingly, if simply based on the sequence similarity approach, it would hardly get a decent success rate in predicting enzyme sub-class even for a dataset consisting of samples with 50% sequence identity. To cope with such a situation, the GO-PseAA predictor was adopted to identify the sub-class for each of the six main enzyme families. It has been observed that, even for the much more stringent datasets in which none of the enzymes has 25% sequence identity to any others, the overall success rates are 73-95%, suggesting that the GO-PseAA predictor can catch the core features of the statistical samples concerned and may become a useful high throughput tool in proteomics and bioinformatics.

Phenazines produced by Pseudomonas and Streptomyces spp. are heterocyclic nitrogen-containing metabolites with antibiotic, antitumor, and antiparasitic activity. The antibiotic properties of pyocyanin, produced by Pseudomonas aeruginosa, were recognized in the 1890s, although this blue phenazine is now known to be a virulence factor in human disease. Despite their biological significance, the biosynthesis of phenazines is not fully understood. Here we present structural and functional studies of PhzF, an enzyme essential for phenazine synthesis in Pseudomonas spp. PhzF shares topology with diaminopimelate epimerase DapF but lacks the same catalytic residues. The structure of PhzF in complex with its substrate, trans-2,3-dihydro-3-hydroxyanthranilic acid, suggests that it is an isomerase using the conserved glutamate E45 to abstract a proton from C3 of the substrate. The proton is returned to C1 of the substrate after rearrangement of the double-bond system, yielding an enol that converts to the corresponding ketone. PhzF is a dimer that may be bifunctional, providing a shielded cavity for ketone dimerization via double Schiff-base formation to produce the phenazine scaffold. Our proposed mechanism is supported by mass and NMR spectroscopy. The results are discussed in the context of related structures and protein sequences of unknown biochemical function.

Recent studies have implied that the isopropylmalate isomerase small subunit of the hyperthermophilic archaea Pyrococcus horikoshii (PhIPMI-s) functions as isopropylmalate isomerase in the leucine biosynthesis pathway, and as homoaconitase (HACN) in the lysine biosynthesis pathway via alpha-aminoadipic acid. PhIPMI is thus considered a key to understanding the fundamental metabolism of the earliest organisms. We describe for the first time the crystal structure of PhIPMI-s, which displays dual substrate specificity. The crystal structure unexpectedly shows that four molecules create an interlocked assembly with intermolecular disulfide linkages having a skewed 222 point-group symmetry. Although the overall fold of the PhIPMI-s monomer is related closely to domain 4 of the aconitase (ACN), one alpha-helix in the ACN structure is replaced by a short loop with relatively high temperature factor values. Because this region is essential for discriminating the structurally similar substrate based on interactions with its diversified gamma-moiety, the loop structure in the PhIPMI-s must be dependent on the presence of a substrate. The flexibility of the loop region might be a structural basis for recognizing both hydrophobic and hydrophilic gamma-moieties of two distinct substrates, isopropylmalate and homocitrate.

STAT3 has been found constitutively activated in M14 melanoma cell line, as previously found in other melanoma cells. Using EMSA, DNA affinity experiments, and chromatin immunoprecipitation, STAT3 was found in M14 to bind the alpha2-macroglobulin gene enhancer in association with the protein disulfide isomerase isoform ERp57. The two proteins have also been found to be associated when bound to the SIE sequence in HepG2 cells stimulated by IL-6. In both cases an anti-ERp57 antibody hinders the binding of STAT3 to its consensus sequence on DNA, indicating that ERp57 is a necessary component of the DNA-bound STAT3 complex. Considering the functional association of the two proteins, the overexpression of ERp57 observed in a variety of transformed cells might be relevant to the oncogenic properties of STAT3.

The synthesis and kinetic parameters of a comprehensive set of 4-OT analogues with arginine (X = NH2+) to citrulline (X = O) substitutions at positions 11, 39, and 61 are reported. These data suggest that the main contribution of Arg39' ' to catalysis is by electrostatic stabilization of the anionic transition state leading to intermediate 2, and not by hydrogen bonding.