Purification and properties of synephrinase from Arthrobacter synephrinum

Synephrinase, an enzyme catalyzing the conversion of (-)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (+/-)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 mumol product formed/min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (-)-synephrine, the enzyme acted upon (+/-)-octopamine and beta-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.     

Comparison of the proteome of Methylobacterium extorquens AM1 grown under methylotrophic and nonmethylotrophic conditions

Methylobacterium extorquens AM1 is a facultative methylotrophic bacterium that is capable of growing in the presence of methanol as the sole carbon and energy source, but is also able to grow on a limited number of C(2), C(3), and C(4) compounds, for example succinate. This study provides a proteomic view of the cellular adaptation of M. extorquens AM1 to growth on methanol and succinate, respectively. Cytosolic proteins were separated by two-dimensional gel electrophoresis employing overlapping pH ranges and visualized by silver nitrate or fluorescence staining. A proteomic reference map containing 229 different proteins identified by peptide mass fingerprinting of tryptic fragments was established. Comparative proteome profiling of methanol- and succinate-grown cells led to the identification of 68 proteins that are induced under methylotrophic growth conditions in comparison to growth on succinate. This group includes most proteins known to be directly involved in methanol oxidation to CO(2) and in assimilation of one carbon units by the serine cycle as well as 18 proteins without any assigned function and two proteins with a predicted regulatory function. Furthermore, the proteome analysis revealed putative isoenzymes for formaldehyde-activating enzyme Fae, malyl-CoA lyase, malate-dehydrogenase, and fumarase, that need to be characterized functionally in future studies.     

Identification of a kallikrein-like latent serine protease in human erythrocyte membranes

We have discovered and characterized a kallikrein-like latent serine protease in intact human erythrocytes and ghosts. The enzyme is activatable by trypsin. The solubilized enzyme has esterolytic activity with a pH optimum of 9; but the membrane-associated activity increases almost linearly up to pH 10. The activated enzyme releases kinin from bovine low molecular weight kininogen. Enzyme activity is inhibited by TosLysCH2Cl , phenylmethylsulfonyl fluoride, aprotinin and amiloride, and weakly by soybean or lima bean trypsin inhibitor. It is inhibited by Co2+, Zn2+ and Mn2+ but is stimulated by Fe2+, deoxycholate and phospholipase A2. An erythrocyte membrane protein (Mr = 88,000) with an active site serine residue was identified with [14C]-diisopropylphosphorofluoridate labeling. Consistent with the finding of tryptic activation of the latent erythrocyte serine protease, trypsin treatment reduced the density of labeling of this protein and revealed a lower molecular weight form (Mr = 64,000). Possible relationships between the activity of this newly identified serine protease and events such as erythrocyte membrane ion fluxes might be of interest.     

Purification and properties of 4-hydroxy-4-methyl-2-oxoglutarate aldolase from Pseudomonas ochraceae grown on phthalate

4-Hydroxy-4-methyl-2-oxoglutarate aldolase [4-hydroxy-4-methyl-2-oxoglutarate pyruvate-lyase: EC 4.1.3.17] has been purified to homogeneity (about 770-fold purification, yield 11.4%) from Pseudomonas ochraceae grown on phthalate. The enzyme has a molecular weight of 160,000 (gel filtration on Bio-Gel A-1.5m), a subunit molecular weight of 26,000 (SDS-PAGE) and an isoelectric point of 5.0 (isoelectric focusing). The enzyme requires divalent metal ions such as Mg2+, Mn2+, Co2+, Zn2+, and Cd2+ for activity. The enzyme actively cleaves 4-carboxy-4-hydroxy-2-oxoadipate, a physiological substrate of the enzyme, to give pyruvate and oxaloacetate, but shows much lower affinity for 4-hydroxy-4-methyl-2-oxoglutarate. 4-Hydroxy-2-oxoglutarate is cleaved at a low rate to pyruvate and glyoxylate. The l-isomers of the substrates are preferentially cleaved rather than the d-isomers as determined polarimetrically. The enzyme reactions are reversible: the equilibrium constants (pH 8.0, 25 C) for the HMG and HG cleavage reactions are about 0.07 and 0.03 M, respectively, whereas no equilibrium is observed with CHA due to oxaloacetate beta-decarboxylase activity associated with the enzyme. The enzyme activity is hardly affected by thiols and thiol reagents. The non-enzymatic cleavage reaction caused by various metal ions has also been studied to examine the mechanistic similarity to the enzymatic reaction.     

Purification and properties of a citrate-binding transport component, the C protein of Salmonella typhimurium.

Salmonella typhimurium was shown to contain a citrate-binding protein (C protein) which was purified to homogeneity from the periplasmic fraction released by cold osmotic shock. The protein is dimeric, has an apparent molecular weight of 28 000 and an isoelectric point of 6.1. Sodium ions were required for optimum substrate binding, however, the divalent cations Zn2+, Mg2+, and Co2+ were inhibitory. The C protein was relatively stable but sensitive to various detergents and chaotropic agents. Approximately one citrate molecule was bound per molecule of protein and citrate binding (Kd = 1-2.6 microM) was strongly competitively inhibited by DL-isocitrate and DL-fluorocitrate but not by other carboxylates. Neither succinate, glutamate, nor acetate were bound to the C protein. No apparent enzyme activity was associated with this protein. A concomitant reduction in the level of binding protein and in citrate transport activity occurred in osmotically shocked cells as well as with L-malate- or succinate-grown cells. Fluorocitrate-resistant mutants were simultaneously defective in citrate transport, citrate binding, and production of cross-reacting material. One transport-defective mutant did produce citrate binding protein.     

Methylamine metabolism in a pseudomonas species

The mechanism by which a nonphotosynthetic bacterium Pseudomonas sp. (Shaw Strain MA) grows on the one-carbon source, methylamine, was investigated by comparing enzyme levels of cells grown on methylamine, to cells grown on acetate or succinate. Cells grown on methylamine have elevated levels of the enzymes serine hydroxymethyl transferase, serine dehydratase, malic enzyme, glycerate dehydrogenase and malate lyase (CoA acetylating ATP-cleaving). These enzymes, in conjunction with a constitutive glyoxylate transaminase, can account for the net conversion of two one-carbon units into acetyl CoA. Cells grown on acetate or methylamine, but not succinate, contain the enzyme isocitrate lyase; while cells grown on acetate or succinate, but not methylamine, contain significant levels of malate synthetase. These findings suggest that the acetyl CoA derived from one-carbon units in methylamine grown cells, condenses with oxalacetate to yield citrate and then isocitrate, followed by cleavage to succinate and glyoxylate. Thus, growth on methylamine is accomplished by the net synthesis of succinate from two molecules of methyamine and two molecules of CO₂.     

2,4-dichlorophenoxyacetate/alpha-ketoglutarate dioxygenases from Burkholderia cepacia 2a and Ralstonia eutropha JMP134

2,4-Dichlorophenoxyacetate (2,4-D)/alpha-ketoglutarate (alpha-KG) dioxygenase has been purified to apparent homogeneity from Burkholderia cepacia strain 2a, which utilizes 2,4-D as sole carbon source. The enzyme required ferrous ions, and was a homodimer composed of subunits having an Mr of approximately 32,000. The reaction catalysed consumed one mol each of 2,4-D, alpha-KG and dioxygen, with the production of one mol each of succinate, 2,4-dichlorophenol and glyoxylate. Maximum activity was exhibited at pH 7.8 and 25 degrees C, and reactivity was enhanced by the presence of ascorbate and cysteine. Mn2+, Zn2+, Cu2+, Fe3+ and Co2+ were inhibitory, and chemical modification of the dioxygenase revealed that thiol groups were essential for activity. The enzyme was active towards other substituted phenoxyacetates, but reacted most rapidly with 2,4-D. The apparent Michaelis constants for 2,4-D and alpha-KG were 109 and 8.9 microM, respectively. The properties of this enzyme are compared with those of the 2,4-D/alpha-KG dioxygenase from Ralstonia eutropha JMP134, which exhibits a differing N-terminal amino-acid sequence, and a different temperature 'optimum', pH optimum, substrate specificity and sensitivity to thiol-binding reagents.     

Effect of inhibitors and cations on the proteolytic activity of Bacillus mesentericus

The effect of some inhibitors and bivalent metal cations (Mn2+, Ca2+, Fe2+, Zn2+, Mg2+, Co2+ and Cu2+) on the proteolytic activity of two Bacillus mesentericus strains (strain 8 and strain 64 M-variant) was comparatively studied. The both enzymes were shown to be serine proteinases, but the proteinase of strain 64 was also a metal-dependent enzyme. Metal ions exerted no essential effect on the proteinase of strain 8. Ca2+ and Mg2+ ions stimulated the proteinase activity of strain 64 whereas Fe2+ and Zn2+ ions inhibited it in the case of three substrates. Therefore, the two proteinases are different.     

Aspects of glycine and serine biosynthesis during growth of Pseudomonas AM1 on C1 compounds

1. Methanol or formate can replace serine or glycine as supplements for growth on succinate of the auxotrophic mutants 20S and 82G of Pseudomonas AM1, showing that the organism can synthesize glycine and serine in net fashion from C(1) units. 2. Double mutants of Pseudomonas 20S and 82G have been prepared (20ST-1 and 82GT-1) that are unable to grow on succinate+1mm-glyoxylate, succinate+2mm-methanol or methanol alone. 3. Mutants 20ST-1 and 82GT-1 lacked serine-glyoxylate aminotransferase activity, and revertants to the phenotype of 20S and 82G regained serine-glyoxylate aminotransferase activity. A total revertant of 82GT-1 to wild-type phenotype regained activities of serine hydroxymethyltransferase and serine-glyoxylate aminotransferase. 4. The activity of serine-glyoxylate aminotransferase in methanol-grown Pseudomonas AM1 is eightfold higher than in the succinate-grown organism. 5. The combined results show that in Pseudomonas AM1 serine-glyoxylate aminotransferase is necessary for growth on C(1) compounds and is involved in the conversion of methanol into glycine via glyoxylate. 6. It is suggested that the phosphorylated pathway of serine biosynthesis from phosphoglycerate replenishes the supply of alpha-amino groups necessary for the flow of glyoxylate through the main assimilatory pathway during growth on C(1) compounds.     

Regulation of Enzymes Associated with C-1 Metabolism in Three Facultative Methylotrophs

The levels of the oxidation enzyme methanol dehydrogenase and the serine pathway enzymes, hydroxypyruvate reductase, glycerate kinase, serine transhydroxymethylase, serine-glyoxylate aminotransferase, phosphoenolpyruvate carboxylase, and malyl-coenzyme A lyase, were studied in cells of the facultative methylotrophs Pseudomonas AM1, Pseudomonas 3A2 and Hyphomicrobium X grown on different substrates. Induction and dilution curves for these enzymes suggest they may be regulated coordinately in Hyphomicrobium X, but not in Pseudomonas AM1 or 3A2. Glyoxylate stimulated the serine transhydroxymethylase activity in methanol-grown cells of all three organisms. A secondary alcohol dehydrogenase activity was detected at low levels in Pseudomonas AM1 and Hyphomicrobium X, but not in Pseudomonas 3A2.     

Purification and general properties of pectin methyl esterase from Curvularia inaequalis NRRL 13884 in solid state culture using orange peels as an inducer

Pectin methyl esterase (PME) [E.C.3. 1.1.11] production by Curvularia inaequalis (Shear) Boedijn NRRL 13884 was investigated using solid-state culture. The highest level of extracellular pectin methyl esterase was detected with orange peels as an inducing substrate and as a sole carbon source. The enzyme was partially purified using Sephadex G-100 and DEAE-Cellulose column chromatography. It was purified about 40 fold with optimum activity at pH 4.4 and 45 degrees C. The enzyme was activated by Co++, Mg++, Na+, whereas it was slightly activated in the presence of Cu++, K+, Mn++, Zn++. On the other hand Ag++, Ca++ and Hg++ inhibited the activity of the enzyme. The Km was calculated to be 0.52 mM.     

重组乳酸乳球菌X-脯氨酰二-肽酰基-氨基肽酶的纯化及动力学特性(英文)

重组的乳酸乳球菌X 脯氨酰 二肽酰基 氨基肽酶是一个工具酶 ,它对基因构建的神经肽或活性多肽的转活具有重要意义 .通过细菌细胞的破碎 ,洗涤 ,冷冻离心 ,透析 ,DEAE 纤维素 5 2柱层析等工艺过程达到电泳纯 .该酶比活为 11.92 6U mg ,纯化倍数为 14.37倍和总活性收率为5 5 .5 6% .通过SDS PAGE和凝胶柱层析法 ,测得该二肽酶有单肽链组成 ,分子量 89KD .在该酶的动力学研究中 ,针对特异性底物L 甘氨酰 L 脯氨酰 对 硝基苯胺 ,求得该酶的米氏方程式 1 V =0 0 4 8 [S]+ 0 2 5 66(r =0 994 ) .它的Km 值为 0 1871mmol L ,最大反应速度Vmax为 3 897μmol·L-1·min-1.该酶可被苯甲酰基磺酰氟 ,胰蛋白酶抑制剂和Mn2 +,Ba2 +,Cu2 +andZn2 +等金属离子抑制 ,但可被Mg2 +激活 .进一步试验显示 ,当Cu2 +和Zn2 +浓度增加到 3 72 6mmol L ,抑制作用明显增强 .低浓度的EDTA Na2 (≤ 0 62 12mmol L)不影响酶的活性 .因此 ,该X 脯氨酰 二肽酰基 氨基肽酶是一个金属离子非依赖性的丝氨酸蛋白酶     

Kinetic effects of ATP, divalent metal ions and pH on chicken liver mevalonate 5-diphosphate decarboxylase

The activity of chicken liver mevalonate 5-diphosphate decarboxylase was measured over a wide range of Mg2+ and ATP concentrations. It was found that free ATP activated the enzyme, whereas free Mg2+ had no effect on the enzyme activity. Computed analyses of free species concentrations and pH studies indicated that MgATP2- is the true substrate. The relative efficiencies of Mg2+, Mn2+, Cd2+, and Zn2+ as activating metal ions were evaluated in terms of V/Km for the corresponding (metal-ATP)2- complexes, and the relative ratios were: Mn2+ 100, Cd2+ 37, Mg2+ 14, Zn2+ 1.7. Inhibitory effects were demonstrated for all free divalent cations tested, except for Mg2+, and were in the order Zn2+ greater than Cd2+ greater than Mn2+.     

Studies on alkaline phosphatase, catalase and z-galactosidase in Vibrio el tor under normal and rifampicin resistant conditions

Alkaline phosphate, catalase and beta-galactosidase activities of Vibrio et tor were decreased after acquisition of resistance towards rifampicin. Zn2+, Mn2+ and EDTA inhibited alkaline phosphatase which is most active with p-nitrophenylphosphate as substrate while Mg2+ was found to suppress alkaline phosphatase activity. Removal of EDTA however, restores the original activity. Rifampicin could not induce mutation of lactose nonfermenting Vibrio el for cells allowing them to grow on lactose as sole carbon source, z-galactosidase which is a constitutive enzyme in this case is repressed by glucose. This repression is overcome by cAMP.     

Identification and characterization of the two-enzyme system catalyzing oxidation of EDTA in the EDTA-degrading bacterial strain DSM 9103.

In a gram-negative isolate (DSM 9103) able to grow with EDTA as the sole source of carbon, nitrogen, and energy, the first two steps of the catabolic pathway for EDTA were elucidated. They consisted of the sequential oxidative removal of two acetyl groups, resulting in the formation of glyoxylate. An enzyme complex that catalyzes the removal of two acetyl groups was purified and characterized. In the reaction, ethylenediaminetriacetate (ED3A) was formed as an intermediate and N,N'-ethylenediaminediacetate was the end product. The enzyme complex consisted of two components: component A' (cA'), most likely a monooxygenase, which catalyzes the cleavage of EDTA and ED3A while consuming oxygen and reduced flavin mononucleotide (FMN)-H2, and component B' (cB'), an NADH2:FMN oxidoreductase that provides FMNH2 for cA'. cB' could be replaced by other NADH2:FMN oxidoreductases such as component B of the nitrilotriacetate monooxygenase or the NADH2:FMN oxidoreductase from Photobacterium fischeri. The EDTA-oxidizing enzyme complex accepted EDTA as a substrate only when it was complexed with Mg2+, Zn2+, Mn2+, Co2+, or Cu2+. Moreover, the enzyme complex catalyzed the removal of acetyl groups from several other aminopolycarboxylic acids that possess three or more acetyl groups.     

Microbial Oxidation of Methane and Methanol: Isolation of Methane-Utilizing Bacteria and Characterization of a Facultative Methane-Utilizing Isolate

A methane-utilizing organism capable of growth both on methane and on more complex organic substrates as a sole source of carbon and energy, has been isolated and studied in detail. Suspensions of methane-grown cells of this organism oxidized C-1 compounds (methane, methanol, formaldehyde, formate); hydrocarbons (ethane, propane); primary alcohols (ethanol, propanol); primary aldehydes (acetaldehyde, propionaldehyde); alkenes (ethylene, propylene); dimethylether; and organic acids (acetate, malate, succinate, isocitrate). Suspensions of methanol-or succinate-grown cells did not oxidize methane, ethane, propane, ethylene, propylene, or dimethylether, suggesting that the enzymatic systems required for oxidation of these substrates are induced only during growth on methane. Extracts of methane-grown cells contained a particulate reduced nicotinamide adenine dinucleotide-dependent methane monooxygenase activity. Oxidation of methanol, formaldehyde, and primary alcohols was catalyzed by a phenazine methosulfate-linked, ammonium ion-requiring methanol dehydrogenase. Oxidation of primary aldehydes was catalyzed by a phenazine methosulfate-linked, ammonium ion-independent aldehyde dehydrogenase. Formate was oxidized by a nicotinamide adenine dinucleotide-specific formate dehydrogenase. Extracts of methane-grown, but not succinate-grown, cells contained the key enzymes of the serine pathway, hydroxypyruvate reductase and malate lyase, indicating that the enzymes of C-1 assimilation are induced only during growth on C-1 compounds. Glucose-6-phosphate dehydrogenase was induced during growth on glucose. Extracts of methane-grown cells contained low levels of enzymes of the tricarboxylic acid cycle, including alpha-keto glutarate dehydrogenase, relative to the levels found during growth on succinate.     

Properties of the membrane-bound 5'-nucleotidase and utilization of extracellular ATP in Vibrio parahaemolyticus

Vibrio parahaemolyticus utilized ATP, ADP or AMP as the sole source of carbon. About three times higher activity of membrane-bound 5'-nucleotidase was observed in cells grown in the presence of these nucleotides than in their absence: and therefore the enzyme seems to be inducible. Since the 5'-nucleotidase activity could be measured with whole cells, the active site of this enzyme appears to be outwardly oriented. Both Mg2+ and Cl- were required for activity. Among the divalent cations tested, Mn2+ and Co2+ could replace Mg2+ to some extent, whereas Zn2+ strongly inhibited activity. Among the anions tested, Br-, I- and NO3- could replace Cl-, but SO4(2-) and CH3COO- could not. When cells were grown with ATP, Cl- was indispensable and Zn2+ strongly inhibited growth. Therefore, it is concluded that extracellular ATP and other 5'-nucleotides are cleaved by the membrane-bound 5'-nucleotidase outside the cells and that the adenosine produced is then utilized.     

Serine transhydroxymethylase in methionine biosynthesis in Saccharomyces cerevisiae

Serine transhydroxymethylase appears to be the first enzyme in the synthesis of the methyl group of methionine. Properties of serine transhydroxymethylase activity as assayed by the production of formaldehyde were correlated with properties of cell-free extracts for the methylation of homocysteine deriving the methyl group from the beta-carbon of serine. The reaction required pyridoxal phosphate and tetrahydrofolic acid, and was characterized in cell-free extracts with respect to Michaelis constant, pH optimum, incubation time, and optimal enzyme concentration. The activity was sensitive to inhibition by methionine, and to a much greater extent by S-adenosylmethionine. Serine transhydroxymethylase and the methylation of homocysteine reactions were not repressed by methionine and were stimulated by glycine. The activities of cell-free extracts for these reactions were significantly higher in cells in exponential than in stationary growth. When cells were grown in 10 mm glycine, the activities remained high throughout the culture cycle. The data indicated that glycine rather than methionine is involved in the control of the formation of the enzyme.     

Farnesyl pyrophosphate synthetase from Bacillus subtilis

Farnesyl pyrophosphate synthetase was detected in extracts of Bacillus subtilis and partially purified by Sephadex G-100, hydroxylapatite, and DEAE-Sephadex chromatography. The enzyme catalyzed the exclusive formation of all-trans farnesyl pyrophosphate from isopentenyl pyrophosphate and either dimethylallyl or geranyl pyrophosphate. Mg2+ was essential for the catalytic activity and Mn2+ was less effective. The enzyme was slightly activated by sulfhydryl reagents. This enzyme was markedly stimulated by K+, NH4+, or detergents such as Triton X-100 and Tween 80, unlike the known farnesyl pyrophosphate synthetases from eucaryotes. The molecular weight of the enzyme was estimated by gel filtration to be 67,000. The Michaelis constants for dimethylallyl and geranyl pyrophosphate were 50 microM and 18 microM, respectively.