Structure of Penicillium citrinum alpha 1,2-mannosidase reveals the basis for differences in specificity of the endoplasmic reticulum and Golgi class I enzymes.

Class I alpha1,2-mannosidases (glycosylhydrolase family 47) are key enzymes in the maturation of N-glycans. This protein family includes two distinct enzymatically active subgroups. Subgroup 1 includes the yeast and human endoplasmic reticulum (ER) alpha1,2-mannosidases that primarily trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomer B whereas subgroup 2 includes mammalian Golgi alpha1,2-mannosidases IA, IB, and IC that trim Man(9)GlcNAc(2) to Man(5)GlcNAc(2) via Man(8)GlcNAc(2) isomers A and C. The structure of the catalytic domain of the subgroup 2 alpha1,2-mannosidase from Penicillium citrinum has been determined by molecular replacement at 2.2-A resolution. The fungal alpha1,2-mannosidase is an (alphaalpha)(7)-helix barrel, very similar to the subgroup 1 yeast (Vallée, F., Lipari, F., Yip, P., Sleno, B., Herscovics, A., and Howell, P. L. (2000) EMBO J. 19, 581-588) and human (Vallée, F., Karaveg, K., Herscovics, A., Moremen, K. W., and Howell, P. L. (2000) J. Biol. Chem. 275, 41287-41298) ER enzymes. The location of the conserved acidic residues of the catalytic site and the binding of the inhibitors, kifunensine and 1-deoxymannojirimycin, to the essential calcium ion are conserved in the fungal enzyme. However, there are major structural differences in the oligosaccharide binding site between the two alpha1,2-mannosidase subgroups. In the subgroup 1 enzymes, an arginine residue plays a critical role in stabilizing the oligosaccharide substrate. In the fungal alpha1,2-mannosidase this arginine is replaced by glycine. This replacement and other sequence variations result in a more spacious carbohydrate binding site. Modeling studies of interactions between the yeast, human and fungal enzymes with different Man(8)GlcNAc(2) isomers indicate that there is a greater degree of freedom to bind the oligosaccharide in the active site of the fungal enzyme than in the yeast and human ER alpha1,2-mannosidases.     

Structure and function of Class I alpha 1,2-mannosidases involved in glycoprotein synthesis and endoplasmic reticulum quality control

Class I alpha 1,2-mannosidases (glycosylhydrolase family 47) are conserved through eukaryotic evolution. This protein family comprises three subgroups distinguished by their enzymatic properties. The first subgroup includes yeast (Saccharomyces cerevisiae) and human alpha 1,2-mannosidases of the endoplasmic reticulum that primarily form Man(8)GlcNAc(2) isomer B from Man(9)GlcNAc(2). The second subgroup includes mammalian Golgi alpha 1,2-mannosidases, as well as enzymes from insect cells and from filamentous fungi, that trim Man(9)GlcNAc(2) to Man(8)GlcNAc(2) isomers A and/or C intermediates toward the formation of Man(5)GlcNAc(2). Yeast and mammalian proteins of the third subgroup have no enzyme activity with Man(9)GlcNAc(2) as substrate. The members of subgroups 1 and 3 participate in endoplasmic reticulum quality control and promote proteasomal degradation of misfolded glycoproteins. The yeast endoplasmic reticulum alpha 1,2-mannosidase has served as a model for structure-function studies of this family. Its structure was determined by X-ray crystallography as an enzyme-product complex. It consists of a novel (alpha alpha)(7) barrel containing the active site that includes essential acidic residues and calcium. The structures of the subgroup 1 human endoplasmic reticulum alpha 1,2-mannosidase and of a subgroup 2 fungal alpha 1,2-mannosidase were determined by molecular replacement. Comparison of the enzyme structures is providing some insight into the reasons for their different specificities.     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

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.     

A nudix enzyme removes pyrophosphate from dihydroneopterin triphosphate in the folate synthesis pathway of bacteria and plants

Removal of pyrophosphate from dihydroneopterin triphosphate (DHNTP) is the second step in the pterin branch of the folate synthesis pathway. There has been controversy over whether this reaction requires a specific pyrophosphohydrolase or is a metal ion-dependent chemical process. The genome of Lactococcus lactis has a multicistronic folate synthesis operon that includes an open reading frame (ylgG) specifying a putative Nudix hydrolase. Because many Nudix enzymes are pyrophosphohydrolases, YlgG was expressed in Escherichia coli and characterized. The recombinant protein showed high DHNTP pyrophosphohydrolase activity with a K(m) value of 2 microM, had no detectable activity against deoxynucleoside triphosphates or other typical Nudix hydrolase substrates, required a physiological level (approximately 1 mM) of Mg(2+), and was active as a monomer. Essentially no reaction occurred without enzyme at 1 mM Mg(2+). Inactivation of ylgG in L. lactis resulted in DHNTP accumulation and folate depletion, confirming that YlgG functions in folate biosynthesis. We therefore propose that ylgG be redesignated as folQ. The closest Arabidopsis homolog of YlgG (encoded by Nudix gene At1g68760) was expressed in E. coli and shown to have Mg(2+)-dependent DHNTP pyrophosphohydrolase activity. This protein (AtNUDT1) was reported previously to have NADH pyrophosphatase activity in the presence of 5 mM Mn(2+) (Dobrzanska, M., Szurmak, B., Wyslouch-Cieszynska, A., and Kraszewska, E. (2002) J. Biol. Chem. 277, 50482-50486). However, we found that this activity is negligible at physiological levels of Mn(2+) and that, with 1 mM Mg(2+), AtNUDT1 prefers DHNTP and (deoxy) nucleoside triphosphates.     

Isolation of a porcine UDP-GalNAc transferase cDNA mapping to the region of the blood group EAA locus on pig chromosome 1

In our studies of the genes constituting the porcine A0 blood group system, we have characterized a cDNA, encoding an alpha(1,3)N-acetylgalactosaminyltransferase, that putatively represents the blood group A transferase gene. The cDNA has a 1095-bp open reading frame and shares 76.9% nucleotide and 66.7% amino acid identity with the human ABO gene. Using a somatic cell hybrid panel, the cDNA was assigned to the q arm of pig chromosome 1, in the region of the erythrocyte antigen A locus (EAA), which represents the porcine blood group A transferase gene. The RNA corresponding to our cDNA was expressed in the small intestinal mucosae of pigs possessing EAA activity, whereas expression was absent in animals lacking this blood group antigen. The UDP-N-acetylgalactosamine (UDP-GalNAc) transferase activity of the gene product, expressed in Chinese hamster ovary (CHO) cells, was specific for the acceptor fucosyl-alpha(1,2)galactopyranoside; the enzyme did not use phenyl-beta-D-galactopyranoside (phenyl-beta-D-Gal) as an acceptor. Because the alpha(1,3)GalNAc transferase gene product requires an alpha(1,2)fucosylated acceptor for UDP-GalNAc transferase activity, the alpha(1,2)fucosyltransferase gene product is necessary for the functioning of the alpha(1,3)GalNAc transferase gene product. This mechanism underlies the epistatic effect of the porcine S locus on expression of the blood group A antigen. ABBREVIATIONS: CDS: coding sequence; CHO: Chinese Hamster Ovary; EAA: erythrocyte antigen A; FCS: foetal calf serum; Fucalpha(1,2)Gal: fucosyl-alpha(1,2)galactopyranoside; Gal: galactopyranoside; GGTA1: Galalpha(1,3)Gal transferase; PCR: polymerase chain reaction; phenyl-beta-D-Gal: phenyl-beta-D-galactopyranoside; R: Galbeta1-4Glcbeta1-1Cer; UDP-GalNAc: uridine diphosphate N-acetylgalactosamine     

Catalytic zinc site and mechanism of the metalloenzyme PR-AMP cyclohydrolase

The enzyme N(1)-(5'-phosphoribosyl) adenosine-5'-monophosphate cyclohydrolase (PR-AMP cyclohydrolase) is a Zn(2+) metalloprotein encoded by the hisI gene. It catalyzes the third step of histidine biosynthesis, an uncommon ring-opening of a purine heterocycle for use in primary metabolism. A three-dimensional structure of the enzyme from Methanobacterium thermoautotrophicum has revealed that three conserved cysteine residues occur at the dimer interface and likely form the catalytic site. To investigate the functions of these cysteines in the enzyme from Methanococcus vannielii, a series of biochemical studies were pursued to test the basic hypothesis regarding their roles in catalysis. Inactivation of the enzyme activity by methyl methane thiosulfonate (MMTS) or 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) also compromised the Zn(2+) binding properties of the protein inducing loss of up to 90% of the metal. Overall reaction stoichiometry and the potassium cyanide (KCN) induced cleavage of the protein suggested that all three cysteines were modified in the process. The enzyme was protected from DTNB-induced inactivation by inclusion of the substrate N(1)-(5'-phosphoribosyl)adenosine 5'-monophosphate; (PR-AMP), while Mg(2+), a metal required for catalytic activity, enhanced the rate of inactivation. Site-directed mutations of the conserved C93, C109, C116 and the double mutant C109/C116 were prepared and analyzed for catalytic activity, Zn(2+) content, and reactivity with DTNB. Substitution of alanine for each of the conserved cysteines showed no measurable catalytic activity, and only the C116A was still capable of binding Zn(2+). Reactions of DTNB with the C109A/C116A double mutant showed that C93 is completely modified within 0.5 s. A model consistent with these data involves a DTNB-induced mixed disulfide linkage between C93 and C109 or C116, followed by ejection of the active site Zn(2+) and provides further evidence that the Zn(2+) coordination site involves the three conserved cysteine residues. The C93 reactivity is modulated by the presence of the Zn(2+) and Mg(2+) and substantiates the role of this residue as a metal ligand. In addition, Mg(2+) ligand binding site(s) indicated by the structural analysis were probed by site-directed mutagenesis of three key aspartate residues flanking the conserved C93 which were shown to have a functional impact on catalysis, cysteine activation, and metal (zinc) binding capacity. The unique amino acid sequence, the dynamic properties of the cysteine ligands involved in Zn(2+) coordination, and the requirement for a second metal (Mg(2+)) are discussed in the context of their roles in catalysis. The results are consistent with a Zn(2+)-mediated activation of H(2)O mechanism involving histidine as a general base that has features similar to but distinct from those of previously characterized purine and pyrimidine deaminases.     

Pyrococcus furiosus alpha-amylase is stabilized by calcium and zinc

The hyperthermophilic archeon Pyrococcus furiosus produces an extracellular alpha-amylase that belongs to glycosyl hydrolases' family 13. This enzyme is more thermostable than its bacterial and archaeal homologues (e.g., Bacillus licheniformis TAKA-term and Pyrococcus kodakaraensis KOD1 alpha-amylases, respectively) even without adding Ca(2+) ions. Unlike the TAKA-therm amylase that contains no cysteine, the P. furiosus enzyme contains five cysteines (C152, C153, C165, C387, and C430), only four of which (C152, C153, C387, and C430) are conserved in the P. kodakaraensis alpha-amylase. To test the potential function of cysteines in P. furiosus alpha-amylase stability, these five residues were substituted with Ser or Ala-either one-by-one or in sequence-to produce eight mutant enzymes. Mutation C165S dramatically destabilized P. furiosus alpha-amylase. At the same time, the quadruple mutant enzyme C152S/C153S/C387S/C430A (mutant SSCSA) was as thermostable as the wild-type enzyme. Mutant SSCSA and wild-type alpha-amylases were strongly destabilized by dithiothreitol and ethylenediaminetetraacetic acid, suggesting that metal binding can be involved in this enzyme's thermostability. Inductively coupled plasma-atomic emission spectrometry showed the presence of Ca(2+) and Zn(2+) metal ions in P. furiosus alpha-amylase. Although Ca(2+) is known to contribute to alpha-amylase's stability, the absence of two out of the three conserved Ca(2+) ligands in the P. furiosus enzyme suggests that a different set of amino acids is involved in this enzyme's Ca(2+) binding. We also provide evidence suggesting that Cys165 is involved in Zn(2+) binding and that Cys165 is essential for the stability of P. furiosus alpha-amylase at very high temperatures.     

Characterization of the yeast DGK1-encoded CTP-dependent diacylglycerol kinase

The Saccharomyces cerevisiae DGK1 gene encodes a diacylglycerol kinase enzyme that catalyzes the formation of phosphatidate from diacylglycerol. Unlike the diacylglycerol kinases from bacteria, plants, and animals, the yeast enzyme utilizes CTP, instead of ATP, as the phosphate donor in the reaction. Dgk1p contains a CTP transferase domain that is present in the SEC59-encoded dolichol kinase and CDS1-encoded CDP-diacylglycerol synthase enzymes. Deletion analysis showed that the CTP transferase domain was sufficient for diacylglycerol kinase activity. Point mutations (R76A, K77A, D177A, and G184A) of conserved residues within the CTP transferase domain caused a loss of diacylglycerol kinase activity. Analysis of DGK1 alleles showed that the in vivo functions of Dgk1p were specifically due to its diacylglycerol kinase activity. The DGK1-encoded enzyme had a pH optimum at 7.0-7.5, required Ca(2+) or Mg(2+) ions for activity, was potently inhibited by N-ethylmaleimide, and was labile at temperatures above 40 degrees C. The enzyme exhibited positive cooperative (Hill number = 2.5) kinetics with respect to diacylglycerol (apparent K(m) = 6.5 mol %) and saturation kinetics with respect to CTP (apparent K(m) = 0.3 mm). dCTP was both a substrate (apparent K(m) = 0.4 mm) and competitive inhibitor (apparent K(i) = 0.4 mm) of the enzyme. Diacylglycerol kinase activity was stimulated by major membrane phospholipids and was inhibited by CDP-diacylglycerol and sphingoid bases.     

Novel L-cysteine-dependent maleylpyruvate isomerase in the gentisate pathway of Paenibacillus sp. strain NyZ101

Glutathione- and mycothiol-dependent maleylpyruvate isomerases are known to be involved, respectively, in gentisate catabolism in Gram-negative and high G+C Gram-positive strains. In the present study, a low-G+C Gram-positive Paenibacillus sp. strain, NyZ101, was isolated and shown to degrade 3-hydroxybenzoate via gentisate. A 6.5-kb fragment containing a conserved region of gentisate 1,2-dioxygenase genes was cloned and sequenced, and four genes (bagKLIX) were shown to encode the enzymes involved in the catabolism to central metabolites of 3-hydroxybenzoate via gentisate. The Bag proteins share moderate identities with the reported enzymes in the 3-hydroxybenzoate catabolism, except BagL that had no obvious homology with any functionally characterized proteins. Recombinant BagL was purified to homogeneity as a His-tagged protein and likely a dimer by gel filtration. BagL was demonstrated to be a novel thiol-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate with L-cysteine, cysteinylglycine, or glutathione, as its cofactor. The K(m) values of these three thiols for BagL were 15.5, 8.4, and 552 μM, respectively. Since cysteine and coenzyme A were reported to be abundant in low-G+C Gram-positive strains, BagL should utilize L-cysteine as its physiological cofactor in vivo. The addition of Ni(2+) increased BagL activity, and site-directed mutagenesis experiments indicated that three conserved histidines in BagL were associated with binding to Ni(2+) ion and were necessary for its enzyme activity. BagL is the first characterized L-cysteine-dependent catabolic enzyme in microbial metabolism and is likely a new and distinct member of DinB family, with a four-helix-bundle topology, as deduced by sequence analysis and homology modeling.     

Specificity and mechanism of metal ion activation in UDP-galactose:beta -galactoside-alpha -1,3-galactosyltransferase

UDP-galactose:beta-galactosyl-alpha1,3-galactosyltransferase (alpha3GT) catalyzes the synthesis of galactosyl-alpha-1,3-beta-galactosyl structures in mammalian glycoconjugates. In humans the gene for alpha3GT is inactivated, and its product, the alpha-Gal epitope, is the target of a large fraction of natural antibodies. alpha3GT is a member of a family of metal-dependent-retaining glycosyltransferases that includes the histo blood group A and B enzymes. Mn(2+) activates the catalytic domain of alpha3GT (alpha3GTcd), but the affinity reported for this ion is very low relative to physiological levels. Enzyme activity over a wide range of metal ion concentrations indicates a dependence on Mn(2+) binding to two sites. At physiological metal ion concentrations, Zn(2+) gives higher levels of activity and may be the natural cofactor. To determine the role of the cation, metal activation was perturbed by substituting Co(2+) and Zn(2+) for Mn(2+) and by mutagenesis of a conserved D(149)VD(151) sequence motif that is considered to act in cation binding in many glycosyltransferases. The aspartates of this motif were found to be essential for activity, and the kinetic properties of a Val(150) to Ala mutant with reduced activity were determined. The results indicate that the cofactor is involved in binding UDP-galactose and has a crucial influence on catalytic efficiency for galactose transfer and for the low endogenous UDP-galactose hydrolase activity. It may therefore interact with one or more phosphates of UDP-galactose in the Michaelis complex and in the transition state for cleavage of the UDP to galactose bond. The DXD motif conserved in many glycosyltransferases appears to have a key role in metal-mediated donor substrate binding and phosphate-sugar bond cleavage.     

Evidence for a metal-thiolate intermediate in alkyl group transfer from epoxypropane to coenzyme M and cooperative metal ion binding in epoxyalkane:CoM transferase

Epoxyalkane:coenzyme M transferase (EaCoMT) catalyzes the nucleophilic addition of coenzyme M (CoM, 2-mercaptoethanesulfonic acid) to epoxypropane forming 2-hydroxypropyl-CoM. The biochemical properties of EaCoMT suggest that the enzyme belongs to the family of alkyltransferase enzymes for which Zn plays a role in activating an organic thiol substrate for nucleophilic attack on an alkyl-donating substrate. The enzyme has a hexameric (alpha(6)) structure with one zinc atom per subunit. In the present work M(2+) binding and the role of Zn(2+) in EaCoMT have been established through a combination of biochemical, calorimetric, and spectroscopic techniques. A variety of metal ions, including Zn(2+), Co(2+), Cd(2+), and Ni(2+), were capable of activating a Zn-deficient "apo" form of EaCoMT, affording enzymes with various levels of activity. Titration of Co(2+) into apo-EaCoMT resulted in UV-visible spectroscopic changes consistent with the formation of a tetrahedral Co(2+) binding site, with coordination of bound Co(2+) to two thiolate ligands. Quantification of UV-visible spectral changes upon Co(2+) titration into apo-EaCoMT demonstrated that EaCoMT binds Co(2+) cooperatively at six interacting sites. Isothermal titration calorimetric studies of Co(2+) and Zn(2+) binding to EaCoMT also showed cooperativity for metal ion binding among six sites. The addition of CoM to Co(2+)-substituted EaCoMT resulted in UV-visible spectral changes indicative of formation of a new thiol-Co(2+) bond. Co(2+)-substituted EaCoMT exhibited a unique Co(2+) EPR spectrum, and this spectrum was perturbed significantly upon addition of CoM. The presence of a divalent metal ion was required for the release of protons from CoM upon binding to EaCoMT, with Zn(2+), Co(2+), and Cd(2+) each facilitating proton release. The divalent metal ion of EaCoMT is proposed to play a key role in the coordination and deprotonation of CoM, possibly through formation of a metal-thiolate that is activated for attack on the epoxide substrate.     

Functional analysis of ISC1 by site-directed mutagenesis

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.     

Characterization of a novel alpha1,2-fucosyltransferase of Escherichia coli O128:b12 and functional investigation of its common motif

The wbsJ gene from Escherichia coli O128:B12 encodes an alpha1,2-fucosyltransferase responsible for adding a fucose onto the galactose residue of the O-antigen repeating unit via an alpha1,2 linkage. The wbsJ gene was overexpressed in E. coli BL21 (DE3) as a fusion protein with glutathione S-transferase (GST) at its N-terminus. GST-WbsJ fusion protein was purified to homogeneity via GST affinity chromatography followed by size exclusion chromatography. The enzyme showed broad acceptor specificity with Galbeta1,3GalNAc (T antigen), Galbeta1,4Man and Galbeta1,4Glc (lactose) being better acceptors than Galbeta-O-Me and galactose. Galbeta1,4Fru (lactulose), a natural sugar, was furthermore found to be the best acceptor for GST-WbsJ with a reaction rate four times faster than that of lactose. Kinetic studies showed that GST-WbsJ has a higher affinity for lactose than lactulose with apparent Km values of 7.81 mM and 13.26 mM, respectively. However, the kcat/appKm value of lactose (6.36 M(-1) x min(-1)) is two times lower than that of lactulose (13.39 M(-1) x min(-1)). In addition, the alpha1,2-fucosyltransferase activity of GST-WbsJ was found to be independent of divalent metal ions such as Mn2+ or Mg2+. This activity was competitively inhibited by GDP with a Ki value of 1.41 mM. Site-directed mutagenesis and a GDP-bead binding assay were also performed to investigate the functions of the highly conserved motif H152xR154R155xD157. In contrast to alpha1,6-fucosyltransferases, none of the mutants of WbsJ within this motif exhibited a complete loss of enzyme activity. However, residues R154 and D157 were found to play critical roles in donor binding and enzyme activity. The results suggest that the common motif shared by both alpha1,2-fucosyltransferases and alpha1,6-fucosyltransferases have similar functions. Enzymatic synthesis of fucosylated sugars in milligram scale was successfully performed using Galbeta-O-Me and Galbeta1,4Glcbeta-N3 as acceptors.     

Bacterial metabolism of naphthalene: construction and use of recombinant bacteria to study ring cleavage of 1,2-dihydroxynaphthalene and subsequent reactions.

The reactions involved in the bacterial metabolism of naphthalene to salicylate have been reinvestigated by using recombinant bacteria carrying genes cloned from plasmid NAH7. When intact cells of Pseudomonas aeruginosa PAO1 carrying DNA fragments encoding the first three enzymes of the pathway were incubated with naphthalene, they formed products of the dioxygenase-catalyzed ring cleavage of 1,2-dihydroxynaphthalene. These products were separated by chromatography on Sephadex G-25 and were identified by 1H and 13C nuclear magnetic resonance spectroscopy and gas chromatography-mass spectrometry as 2-hydroxychromene-2-carboxylate (HCCA) and trans-o-hydroxybenzylidenepyruvate (tHBPA). HCCA was detected as the first reaction product in these incubation mixtures by its characteristic UV spectrum, which slowly changed to a spectrum indicative of an equilibrium mixture of HCCA and tHBPA. Isomerization of either purified product occurred slowly and spontaneously to give an equilibrium mixture of essentially the same composition. tHBPA is also formed from HCCA by the action of an isomerase enzyme encoded by plasmid NAH7. The gene encoding this enzyme, nahD, was cloned on a 1.95-kb KpnI-BglII fragment. Extracts of Escherichia coli JM109 carrying this fragment catalyzed the rapid equilibration of HCCA and tHBPA. Metabolism of tHBPA to salicylaldehyde by hydration and aldol cleavage is catalyzed by a single enzyme encoded by a 1-kb MluI-StuI restriction fragment. A mechanism for the hydratase-aldolase-catalyzed reaction is proposed. The salicylaldehyde dehydrogenase gene, nahF, was cloned on a 2.75-kb BamHI fragment which also carries the naphthalene dihydrodiol dehydrogenase gene, nahB. On the basis of the identification of the enzymes encoded by various clones, the gene order for the nah operon was shown to be p, A, B, F, C, E, D.     

Mouse phosphoinositide 3-kinase p110alpha gene: cloning, structural organization, and localization to chromosome 3 band B.

Phosphoinositide 3-Kinases (PI3-Kinases) are a family of dual specificity enzymes with a unique lipid kinase activity toward the D-3 position of the inositol ring of phosphoinositides and a less well characterized serine/threonine protein kinase activity. Class IA PI3-Kinases comprise a 110-120 kDa catalytic subunit (usually termed p110) and an 85 kDa or 50 to 55 kDa regulatory subunit (often called p85). cDNAs for three mammalian Class IA PI3-Kinase catalytic subunits designated p110alpha, p110beta, and p110delta have been cloned from several species. A YAC clone for the human p110alpha gene has also been cloned and mapped to chromosome 3q26.3. However, structural organization for any of the PI3-Kinase p110alpha genes has not been reported. Here, we report the cloning, structural organization, and chromosomal localization of the mouse PI3-Kinase p110alpha gene. The translated portion of the mouse p110alpha gene is encoded by 19 exons that span at least 24 kb. Dual color fluorescence in situ hybridization (FISH) was performed to determine the chromosomal localization of the mouse PI3-Kinase p110alpha gene. FISH results and DAPI banding demonstrated localization of the p110alpha gene to band B on mouse chromosome 3, a region syntenic with human chromosome 3q26.3.