Purification and properties of 3-hexulose phosphate synthase and phospho-3-hexuloisomerase from Methylococcus capsulatus

3-Hexulose phosphate synthase and phospho-3-hexuloisomerase were purified 40- and 150-fold respectively from methane-grown Methylococcus capsulatus. The molecular weights of the enzymes were approximately 310000 and 67000 respectively, as determined by gel filtration. Dissociation of 3-hexulose phosphate synthase into subunits of molecular weight approx. 49000 under conditions of low pH or low ionic strength was observed. Within the range of compounds tested, 3-hexulose phosphate synthase is specific for formaldehyde and d-ribulose 5-phosphate (forward reaction) and d-arabino-3-hexulose 6-phosphate (reverse reaction), and phospho-3-hexuloisomerase is specific for d-arabino-3-hexulose 6-phosphate (forward reaction) and d-fructose 6-phosphate (reverse reaction). A bivalent cation is essential for activity and stability of 3-hexulose phosphate synthase; phospho-3-hexuloisomerase is inhibited by many bivalent cations. The pH optima of the two enzymes are 7.0 and 8.3 respectively and the equilibrium constants are 4.0x10(-5)m and 1.9x10(2)m respectively. The apparent Michaelis constants for 3-hexulose phosphate synthase are: d-ribulose 5-phosphate, 8.3x10(-5)m; formaldehyde, 4.9x10(-4)m; d-arabino-3-hexulose 6-phosphate, 7.5x10(-5)m. The apparent Michaelis constants for phospho-3-hexuloisomerase are: d-arabino-3-hexulose 6-phosphate, 1.0x10(-4)m; d-fructose 6-phosphate, 1.1x10(-3)m.     

Purification and properties of 3-hexulosephosphate synthase from Methylomonas M 15.

3-Hexulosephosphate synthase, the first enzyme of the ribulose monophosphate cycle, was purified 15-fold from methanol-grown Methylomonas M 15. The purification procedure involved chromatography on DEAE-cellulose, Sephadex G-75, and DEAE-Sephadex A-50. The purified enzyme was more than 95% pure as judged by analytical polyacrylamide gel electrophoresis. The molecular weight was calculated to be 43000 from sedimentation equilibrium experiments. Electrophoresis in sodium dodecylsulfate gels gave a single band corresponding to a molecular weight of 22000. The enzyme catalyzes specifically the condensation formaldehyde with ribulose 5-phosphate to yield D-arabino-3-hexulose 6-phosphate. The Km values were found to be 1.1 mM for formaldehyde and 1.6 mM for ribulose 5-phosphate. A bivalent cation is essential for activity and stability of the enzyme, Mg2+ and Mn2+ serve best for this purpose. The optimum of pH for enzyme activity is 7.5--8.0.     

Hexose phosphate synthetase from Methylococcus capsulatus makes d-arabino-3-hexulose phosphate

The product of the reaction catalysed by hexose phosphate synthase prepared from Methylococcus capsulatus was dephosphorylated and the sugar moiety purified. The sugar and derivatives were compared by various chromatographic and other methods with authentic samples of allulose (psicose), d-erythro-l-glycero-3-hexulose and d-erythro-d-glycero-3-hexulose. The sugar is not allulose, as was previously thought on the basis of less extensive evidence (Kemp & Quayle, 1966), but is in fact d-erythro-l-glycero-3-hexulose (d-arabino-3-hexulose). This identification is consistent with recent studies which have shown that hexose phosphate synthase catalyses the condensation of formaldehyde with d-ribulose 5-phosphate rather than with d-ribose 5-phosphate (Kemp, 1972).     

Cloning of D-glucose dehydrogenase with a narrow substrate specificity fromBacillus thuringiensis M15

We have cloned an ORF ofBacillus thuringiensis M15, which encodes a protein sharing high similarity with D-glucose dehydrogenase. A high-expression plasmid (pBtGDH) for the ORF was constructed.Escherichia coli JM 109 transformed with pBtGDH exhibited D-glucose dehydrogenase activity, and the enzyme was purified by 3 chromatographic steps to homogeneity with 6.9 fold and a final yield of 13%. The purified enzyme has highly narrow substrate specificity for glucose and 2-deoxy-D-glucose and showed no activity with any other sugars we tested. The properties of the purified enzyme were similar to those of the D-glucose dehydrogenase (BtGDH) that is mainly produced inB. thuringiensis M15. These results show that the cloned gene encodes BtGDH, as we previously reported. This is the first report to determine the sequence of the enzyme with narrow substrate specificity. BtGDH shows 89% sequence similarity with D-glucose dehydrogenase fromBacillus megaterium IWG3 (GDH-IWG3), which has broad substrate specificity. A comparative analysis between BtGDH and GDH-IWG3 will reveal the differences between them and show the narrow specific activity of BtGDH.     

Catalytic mechanism and substrate specificity of HIF prolyl hydroxylases

This review describes the catalytic mechanism, substrate specificity, and structural peculiarities of alpha-ketoglutarate dependent nonheme iron dioxygenases catalyzing prolyl hydroxylation of hypoxia-inducible factor (HIF). Distinct localization and regulation of three isoforms of HIF prolyl hydroxylases suggest their different roles in cells. The recent identification of novel substrates other than HIF, namely β2-adrenergic receptor and the large subunit of RNA polymerase II, places these enzymes in the focus of drug development efforts aimed at development of isoform-specific inhibitors. The challenges and prospects of designing isoform-specific inhibitors are discussed.     

Enantioselective substrate specificity of 15-lipoxygenase 1

15-Lipoxygenases are lipid-peroxidizing enzymes which have been implicated in the pathogenesis of various diseases, such as inflammation, atherosclerosis, and osteoporosis. Although the crystal structures for several lipoxygenase isoforms have been solved, there is little information on the substrate alignment at the active site and its impact on the catalytic mechanism. Investigating the oxygenation of specifically designed hydroxy fatty acids, we observed a pronounced enantioselectivity of 15-lipoxygenases for substrates carrying the oxygen moiety in close proximity to the site of hydrogen abstraction [16(R/S)-HETE, 17(R/S)-HETE]. To investigate the mechanistic basis for this unexpected behavior, we applied a strategy involving targeted substrate modification, site-directed mutagenesis, and structural modeling of the enzyme-substrate complex. Taken together, our data suggest that an (S)-hydroxy group in 16-HETE may form a hydrogen bridge between the substrate molecule and Gln548, which contributes to proper alignment of the fatty acid derivative at the active site of the enzyme. This interaction, which was not observed with 16(R)-HETE, 18(R)-HETE, or 18(S)-HETE, appears to be a major reason for the high degree of enantioselectivity during lipoxygenation of 16-HETE.     

Catalytic properties of MGAT3, a putative triacylgycerol synthase

Acyl-coenzyme A:monoacylglycerol acyltransferase 3 (MGAT3) is a member of the MGAT family of enzymes that catalyze the synthesis of diacylglycerol (DAG) from monoacylglycerol (MAG), a committed step in dietary fat absorption. Although named after the initial identification of its MGAT activity, MGAT3 shares higher sequence homology with acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) than with other MGAT enzymes, suggesting that MGAT3 may also possess significant DGAT activity. This study compared the catalytic properties of MGAT3 with those of MGAT1 and MGAT2 enzymes using both MAG and DAG as substrates. Our results showed that in addition to the expected MGAT activity, the recombinant MGAT3 enzyme expressed in Sf-9 insect cells displayed a strong DGAT activity relative to that of MGAT1 and MGAT2 enzymes in the order MGAT3 > MGAT1 > MGAT2. In contrast, none of the three MGAT enzymes recognized biotinylated acyl-CoA or MAG as a substrate. Although MGAT3 possesses full DGAT activity, it differs from DGAT1 in catalytic properties and subcellular localization. The MGAT3 activity was sensitive to inhibition by the presence of 1% CHAPS, whereas DGAT1 activity was stimulated by the detergent. Consistent with high sequence homology with DGAT2, the MGAT3 enzyme demonstrated a similar subcellular distribution pattern to that of DGAT2, but not DGAT1, when expressed in COS-7 cells. Our data suggest that MGAT3 functions as a novel triacylglycerol (TAG) synthase that catalyzes efficiently the two consecutive acylation steps in TAG synthesis.     

On the substrate specificity of nitric oxide synthase.

Nitric oxide (.NO) synthase (NOS) activity in subcellular fractions from cultured endothelial cells (EC) and lipopolysaccharide-activated J774.2 monocyte/macrophages was investigated by monitoring the .NO-mediated increase in intracellular cyclic GMP in LLC-PK1 pig kidney epithelial cells. The constitutive NOS in EC (NOSc) was largely membrane-bound, whereas the inducible NOS in J774.2 cells (NOSi) was equally distributed among cytosol and membrane(s). Both the cytosolic NOSc in EC and the membrane-bound NOSi in J774.2 cells were strictly Ca(2+)-dependent, whereas the membrane-bound NOSc in EC and the cytosolic NOSi in J774.2 cells were not. L-Homoarginine and L-arginine-containing small peptides, such as L-arginyl-L-phenylalanine, replaced L-arginine as a substrate for the NOSc in EC and the Ca(2+)-independent NOSi in J774.2 cells, but not the Ca(2+)-dependent NOSi. Thus, irrespective of their intracellular localisation, at least three isoforms of NOS exist, which can be differentiated by their substrate specificity and Ca(2+)-dependency.     

ADAM33 enzyme properties and substrate specificity

ADAM33 is an asthma susceptibility gene recently identified through a genetic study of asthmatic families [van Eerdewegh, et al. (2002) Nature 418, 426-430]. To understand the function of the gene product, the recombinant metalloproteinase domain of human ADAM33 was purified and tested for its substrate cleavage specificity using peptides derived from beta-amyloid precursor protein (APP). A single Ala substitution at the P2 position of a 10-residue APP peptide, YEVHHQKLVF, yielded a 20-fold more efficient substrate. Terminal truncation studies identified a minimal nine-residue core (P5-P4') important for ADAM33 recognition and cleavage. Full positional scanning of the 10-mer peptide using the 19 naturally occurring l-amino acids (excluding Cys) revealed a substrate specificity profile. A strong preference for Val or Ile at P3, Ala at P2, and Gln at P1' was observed. The substrate binding model based on the X-ray structure of the ADAM33-inhibitor complex supported the observed substrate specificity profile. On the basis of this, an improved substrate was designed and a fluorescence resonance energy transfer (FRET) assay was developed using a fluorogenic derivative of this substrate. Kinetic studies confirmed that the best substrate, FRET-P2 [K(Dabcyl)YRVAFQKLAE(Edans)K], was approximately 100-fold more efficient than the wild-type APP peptide substrate, with a k(cat)/K(m) value of (3.6 +/- 0.1) x 10(4) s(-)(1) M(-)(1). Using this substrate and the FRET assay, ADAM33 enzyme activity and thermal stability were characterized. ADAM33 dependence on buffer conditions, detergents, and temperature was examined, and optimal conditions were defined. Accurate K(i) values for tissue inhibitors of metalloproteinase and small molecule compounds were obtained.     

Anomeric and other substrate specificity studies with myo-inositol-1-P synthase

L-myo-Inositol-1-phosphate synthase has been found to have at least a 5-fold preference for the beta-anomer of its natural substrate D-Glc-6-P. The alpha-anomer appears to be an inhibitor of the reaction and may be converted to product as well. As well as showing an enzymatic preference for the equatorial C-1 hydroxyl of D-Glc-6-P, our results suggest that it is the pyranose form of D-Glc-6-P that binds to the enzyme and that ring-opening is an enzymatic step. We have also found D-2-dGlc-6-P, D-2-F-2-dGlc-6-P, and D-Man-6-P each to be both competitive inhibitors and substrates that are converted to inositol phosphates by the synthase. D-Allose-6-P is a weak inhibitor of the enzyme, but not a substrate. D-Gal-6-P is neither substrate nor inhibitor. Thus the specificity of the synthase with respect to single position epimers of D-Glc-6-P increases in the order C1 less than C2 much less than C3 less than C4.     

Deacetoxycephalosporin C synthase isozymes exhibit diverse catalytic activity and substrate specificity

The biosynthesis of cephalosporins involving a thiozolidine ring expansion is catalyzed by deacetoxycephalosporin C synthase (DAOCS). In this study, three DAOCS isozymes were cloned and expressed as active enzymes together with Streptomyces jumonjinensis DAOCS that was newly isolated and partially characterized. The enzymes showed excellent substrate conversion for penicillin G, phenethicillin, ampicillin and carbenicillin, but they were less effective in the ring expansion of penicillin V, amoxicillin and metampicillin. Streptomyces clavuligerus DAOCS was the most active among the recombinant enzymes. The results also showed that truncation of 20 amino acids at the C-terminus of the Acremonium chrysogenum deacetoxy/deacetylcephalosporin C synthase polypeptide did not affect penicillin ring expansion.     

Catalytic cysteine of thymidylate synthase is activated upon substrate binding

The role of Ser 167 of Escherichia coli thymidylate synthase (TS) in catalysis has been characterized by kinetic and crystallographic studies. Position 167 variants including S167A, S167N, S167D, S167C, S167G, S167L, S167T, and S167V were generated by site-directed mutagenesis. Only S167A, S167G, S167T, and S167C complemented the growth of thymidine auxotrophs of E. coli in medium lacking thymidine. Steady-state kinetic analysis revealed that mutant enzymes exhibited k(cat) values 1.1-95-fold lower than that of the wild-type enzyme. Relative to wild-type TS, K(m) values of the mutant enzymes for 2'-deoxyuridylate (dUMP) were 5-90 times higher, while K(m) values for 5,10-methylenetetrahydrofolate (CH(2)H(4)folate) were 1.5-16-fold higher. The rate of dehalogenation of 5-bromo-2'-deoxyuridine 5'-monophosphate (BrdUMP), a reaction catalyzed by TS that does not require CH(2)H(4)folate as cosubstrate, by mutant TSs was analyzed and showed that only S167A and S167G catalyzed the dehalogenation reaction and values of k(cat)/K(m) for the mutant enzymes were decreased by 10- and 3000-fold, respectively. Analysis of pre-steady-state kinetics of ternary complex formation revealed that the productive binding of CH(2)H(4)folate is weaker to mutant TSs than to the wild-type enzyme. Chemical transformation constants (k(chem)) for the mutant enzymes were lower by 1.1-6.0-fold relative to the wild-type enzyme. S167A, S167T, and S167C crystallized in the I2(1)3 space group and scattered X-rays to either 1.7 A (S167A and S167T) or 2.6 A (S167C). The high-resolution data sets were refined to a R(crys) of 19.9%. In the crystals some cysteine residues were derivatized with 2-mercaptoethanol to form S,S-(2-hydroxyethyl)thiocysteine. The pattern of derivatization indicates that in the absence of bound substrate the catalytic cysteine is not more reactive than other cysteines. It is proposed that the catalytic cysteine is activated by substrate binding by a proton-transfer mechanism in which the phosphate group of the nucleotide neutralizes the charge of Arg 126', facilitating the transfer of a proton from the catalytic cysteine to a His 207-Asp 205 diad via a system of ordered water molecules.     

Crystal structure of wild-type tryptophan synthase complexed with the natural substrate indole-3-glycerol phosphate

We used freeze trapping to stabilize the Michaelis complex of wild-type tryptophan synthase and the alpha-subunit substrate indole-3-glycerol phosphate (IGP) and determined its structure to 1. 8 A resolution. In addition, we determined the 1.4 A resolution structure of the complex with indole-3-propanole phosphate (IPP), a noncleavable IGP analogue. The interaction of the 3'-hydroxyl of IGP with the catalytic alphaGlu49 leads to a twisting of the propane chain and to a repositioning of the indole ring compared to IPP. Concomitantly, the catalytic alphaAsp60 rotates resulting in a translocation of the COMM domain [betaGly102-betaGly189, for definition see Schneider et al. (1998) Biochemistry 37, 5394-5406] in a direction opposite to the one in the IPP complex. This results in loss of the allosteric sodium ion bound at the beta-subunit and an opening of the beta-active site, thereby making the cofactor pyridoxal 5'-phosphate (PLP) accessible to solvent and thus serine binding. These findings form the structural basis for the information transfer from the alpha- to the beta-subunit and may explain the affinity increase of the beta-active site for serine upon IGP binding.     

Crystal structure of inositol phosphate multikinase 2 and implications for substrate specificity

Inositol polyphosphates perform essential functions as second messengers in eukaryotic cells, and their cellular levels are regulated by inositol phosphate kinases. Most of these enzymes belong to the inositol phosphate kinase superfamily, which consists of three subgroups, inositol 3-kinases, inositol phosphate multikinases, and inositol hexakisphosphate kinases. Family members share several strictly conserved signature motifs and are expected to have the same backbone fold, despite very limited overall amino acid sequence identity. Sequence differences are expected to play important roles in defining the different substrate selectivity of these enzymes. To investigate the structural basis for substrate specificity, we have determined the crystal structure of the yeast inositol phosphate multikinase Ipk2 in the apoform and in a complex with ADP and Mn(2+) at up to 2.0A resolution. The overall structure of Ipk2 is related to inositol trisphosphate 3-kinase. The ATP binding site is similar in both enzymes; however, the inositol binding domain is significantly smaller in Ipk2. Replacement of critical side chains in the inositolbinding site suggests how modification of substrate recognition motifs determines enzymatic substrate preference and catalysis.     

Bacillaene酮还原酶结构域的异源表达及底物特异性分析

Bacillaene生物合成过程中,聚酮合酶第一个延伸模块的酮还原酶结构域(Bac KR1)既催化α酮基的还原,也催化β酮基的还原,具有天然的底物宽泛性。为进一步研究该结构域的底物特异性,在大肠杆菌中对其进行了异源表达。体外酶学分析表明Bac KR1可以催化聚酮类底物(±)-2-甲基-3-氧代戊酸-乙酰半胱胺硫酯外消旋体的立体选择性还原,仅生成4种非对映异构体中的一种,此外Bac KR1还可以催化环己酮和对氯苯乙酮等非聚酮类底物的还原,暗示了聚酮合酶中酮还原酶结构域作为生物催化剂的潜力。     

Studies on the substrate specificity of clavaminic acid synthase and associated enzymes

Fifteen structural analogues of proclavaminic acid have been synthesised and incubated with enzyme preparations from Streptomyces clavuligerus in order to test the substrate specificity of the enzymes responsible for converting monocyclic β-lactams to bicyclic β-lactams.     

Physiological properties and substrate specificity of a pentachlorophenol-degradingPseudomonas species

A bacterial strain capable of utilizing pentachlorophenol (PCP) as sole source of carbon and energy for growth was isolated from enrichment cultures containing 100 mg/l PCP in a mineral salts medium inoculated with contaminated soil from a lumber treatment waste site. The isolate, designated strain SR3, was identified as a species ofPseudomonas by virtue of its physiological and biochemical characteristics. Mineralization of PCP byPseudomonas sp. strain SR3 was demonstrated by loss of detectable PCP from growth medium, stoichiometry of chloride release (5 equivalents of chloride per mole of PCP), and formation of biomass consistent with the concentration of PCP mineralized. PCP-induced cells of strain SR3 showed elevated rates of oxygen consumption in the presence of PCP, and with different chlorinated phenols, with complete degradation of 2,3,5,6-, 2,3,6-, 2,4,6-, 2,4-, and 2,6-chloro-substituted phenols. Concentrations of PCP up to 175 mg/liter supported growth of this organism, but maximal rates of PCP removal were observed at a PCP concentration of 100 mg/liter. Based on its degradative properties,Pseudomonas sp. strain SR3 appears to have utility in bioremediation of soil and water contaminated with PCP.Abbreviations DCP dichlorophenol - TCP trichlorophenol - TeCP tetrachlorophenol Contribution No. 750 from the United States Environmental Protection Agency Environmental Research Laboratory, Gulf Breeze, FL32561, USA. A preliminary report of this work has appeared in abstract form (Resnick & Chapman 1990; Abstr. Annu Meet Amer Soc Microbiol Q-70, p. 300).     

Multisite phosphorylation of glycogen synthase. Molecular basis for the substrate specificity of glycogen synthase kinase-3 and casein kinase-II (glycogen synthase kinase-5)

Glycogen synthase kinase-3 was isolated from rabbit skeletal muscle by an improved procedure. The purification was estimated to be 67000-fold and 0.2 mg of enzyme was isolated from 5000 g muscle, corresponding to an overall yield of 7%. The preparation was homogeneous by ultracentrifugal and electrophoretic criteria. The enzyme had a relative molecular mass of 47 kDa by sedimentation equilibrium centrifugation and 51 kDa by SDS-polyacrylamide gel electrophoresis. These values demonstrate that glycogen synthase kinase-3 is monomeric. The Stokes radius of 37 nm suggests the molecule to be asymmetric. The activating factor of the Mg-ATP dependent form of protein phosphatase-1 coeluted with glycogen synthase kinase-3 activity at the final step, establishing that these two activities reside in the same protein. Glycogen synthase kinase-3 phosphorylates glycogen synthase at sites-3, while casein kinase-II phosphorylates site-5, just C-terminal to sites-3 (Picton, C., Aitken, A., Bilham, T. and Cohen, P. (1982) Eur. J. Biochem. 124, 37-45). The basis for the substrate specificities of these protein kinases was investigated using chymotryptic peptides that contain the sites phosphorylated by each enzyme. These studies showed that efficient phosphorylation of sites-3, required the presence of phosphate in site-5 and a region of polypeptide more than 20 residues C-terminal to site-5. In contrast, efficient phosphorylation by casein kinase-II does not require this C-terminal region, and the results are consistent with the view that the enzyme recognises acidic residues immediately C-terminal to site-5.     

A comprehensive analysis of the geranylgeranylglyceryl phosphate synthase enzyme family identifies novel members and reveals mechanisms of substrate specificity and quaternary structure organization

Geranylgeranylglyceryl phosphate synthase (GGGPS) family enzymes catalyse the formation of an ether bond between glycerol‐1‐phosphate and polyprenyl diphosphates. They are essential for the biosynthesis of archaeal membrane lipids, but also occur in bacterial species, albeit with unknown physiological function. It has been known that there exist two phylogenetic groups (I and II) of GGGPS family enzymes, but a comprehensive study has been missing. We therefore visualized the variability within the family by applying a sequence similarity network, and biochemically characterized 17 representative GGGPS family enzymes regarding their catalytic activities and substrate specificities. Moreover, we present the first crystal structures of group II archaeal and bacterial enzymes. Our analysis revealed that the previously uncharacterized bacterial enzymes from group II have GGGPS activity like the archaeal enzymes and differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases. The length of the isoprenoid substrate is determined in group II GGGPS enzymes by ‘limiter residues’ that are different from those in group I enzymes, as shown by site‐directed mutagenesis. Most of the group II enzymes form hexamers. We could disrupt these hexamers to stable and catalytically active dimers by mutating a single amino acid that acts as an ‘aromatic anchor’.