重组球形红细菌5-氨基乙酰丙酸合酶同工酶hemA和hemT的特性

将编码光合细菌Rhodobactersphaeroides 5- 氨基乙酰丙酸合酶(ALAS)的同工酶基因hemA、hemT转入E .coli中进行高表达,并将高表达的同工酶进行分离、纯化.纯化的hemA是可溶的,并具有催化活性,而hemT大部分是不溶的,且在体外条件下无活性.与其它重组ALAS相比,R .sphaeroides的hemA活性表达需PLP作为催化因子,除去PLP或用硼酸钠破坏与PLP的连接,hemA活性下降90 % .hemA PLP的紫外 可见光谱分析表明hemA与PLP之间形成一个醛亚胺键,而hemT与PLP之间未形成该键.hemA对修饰组氨酸、精氨酸、胱氨酸残基的试剂很敏感,对可切割Arg15 1和Ser15 2的类胰蛋白酶也很敏感,PLP也不能阻止该酶的切割作用.抗血清试验表明,hemA、hemT的抗血清均可与小鼠的ALAS杂交,并都有一个抗原决定簇.     

Characterization of the rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes, HemA and HemT, isolated from recombinant Escherichia coli.

The hemA and hemT genes encoding 5-aminolaevulinic acid synthase (ALAS) from the photosynthetic bacterium Rhodobacter sphaeroides, were cloned to allow high expression in Escherichia coli. Both HemA and HemT appeared to be active in vivo as plasmids carrying the respective genes complemented an E. coli hemA strain (glutamyl-tRNA reductase deficient). The over-expressed isoenzymes were isolated and purified to homogeneity. Isolated HemA was soluble and catalytically active whereas HemT was largely insoluble and failed to show any activity ex vivo. Pure HemA was recovered in yields of 5-7 mg x L-1 of starting bacterial culture and pure HemT at 10 mg x L-1 x HemA has a final specific activity of 13 U x mg-1 with 1 unit defined as 1 micromol of 5-aminolaevulinic acid formed per hour at 37 degrees C. The Km values for HemA are 1.9 mM for glycine and 17 microM for succinyl-CoA, with the enzyme showing a turnover number of 430 h-1. In common with other ALASs the recombinant R. sphaeroides HemA requires pyridoxal 5'-phosphate (PLP) as a cofactor for catalysis. Removal of this cofactor resulted in inactive apo-ALAS. Similarly, reduction of the HemA-PLP complex using sodium borohydride led to > 90% inactivation of the enzyme. Ultraviolet-visible spectroscopy with HemA suggested the presence of an aldimine linkage between the enzyme and pyridoxal 5'-phosphate that was not observed when HemT was incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine and cysteine amino acid residues and the enzyme was also highly sensitive to tryptic cleavage between Arg151 and Ser152 in the presence or absence of PLP and substrates. Antibodies were raised to both HemA and HemT but the respective antisera were not only found to bind both enzymes but also to cross-react with mouse ALAS, indicating that all of the proteins have conserved epitopes.     

The oxidized pyrimidine ribonucleotide, 5-hydroxy-CTP,is hydrolyzed efficiently by the Escherichia coli recombinant Orf135 protein

The Escherichia coli orf135 gene encodes a 15.4kDa protein with homology to the MutT family of nucleotide hydrolases. The orf135 gene was cloned within a glutathione S-transferase (GST) fusion protein expression vector, which was used to overproduce the GST-Orf135 fusion protein in E. coli. The fusion protein thus obtained was purified by affinity column chromatography and gel filtration chromatography from the crude extract. The recombinant Orf135 protein was obtained by removing the GST tag from the purified fusion protein. Various oxidized nucleotides were tested as substrates for the recombinant Orf135 protein. As a result, we found a novel 5-hydroxy-CTPase activity of Orf135, but the hydrolyzing activities for the other nucleotides, including 5-hydroxy-dCTP, were very low. The activation constant (K(a)) of Mg(2+) for the 5-hydroxy-CTPase activity was 1.2 mM, and the pH optimum was 8.5. The catalytic efficiency (k(cat)/K(m)) for this activity was 630 s(-1) mM(-1) at 30 degrees C, which was 30-fold higher than that for the CTPase activity. This result indicates that 5-hydroxy-CTP is the best substrate of Orf135 among the nucleotides tested.     

Production, purification, and characterization of a Mg2+-responsive porphobilinogen synthase from Pseudomonas aeruginosa.

During tetrapyrrole biosynthesis the metalloenzyme porphobilinogen synthase (PBGS) catalyzes the condensation of two molecules of 5-aminolevulinic acid to form the pyrrole porphobilinogen. Pseudomonas aeruginosa PBGS was synthesized in Escherichia coli, and the enzyme was purified as a fusion protein with glutathione S-transferase (GST). After removal of GST, a molecular mass of 280 000 +/- 10 000 with a Stokes radius of 57 A was determined for native PBGS, indicating a homooctameric structure of the enzyme. Mg2+ stabilized the oligomeric state but was not essential for octamer formation. Alteration of N-terminal amino acids changed the oligomeric state and reduced the activity of the enzyme, revealing the importance of this region for oligomerization and activity. EDTA treatment severely inhibited enzymatic activity which could be completely restored by the addition of Mg2+ or Mn2+. At concentrations in the micromolar range Co2+, Zn2+, and Ni2+ partially restored EDTA-inhibited enzymatic activity while higher concentrations of Zn2+ inhibited the enzyme. Pb2+, Cd2+, and Hg2+ did not restore activity. A stimulatory effect of monovalent ions was observed. A Km of 0.33 mM for ALA and a maximal specific activity of 60 micromol h-1 mg-1 at the pH optimum of 8.6 in the presence of Mg2+ and K+ were found. pH-dependent kinetic studies were combined with protein modifications to determine the structural basis of two observed pKa values of approximately 7.9 (pKa1) and 9.5 (pKa2). These are postulated respectively as ionization of an active site lysine residue and of free substrate during catalysis. Some PBGS inhibitors were characterized. Finally, we succeeded in obtaining well-ordered crystals of P. aeruginosa PBGS complexed with the substrate analogue levulinic acid.     

Biosynthesis of 2-amino-3-hydroxycyclopent-2-enone moiety of bafilomycin in <Emphasis Type="Italic">Kitasatospora cheerisanensis</Emphasis> KCTC2395

Bafilomycins produced by Kitasatospora cheerisanensis KCTC- 2395 belong to the 16-membered macrolactone family plecomacrolide antibiotics. Bafilomycin B₁ contains 2-amino- 3-hydroxycyclopent-2-enone (C₅N), a five membered ring, which gets condensed via an amide linkage to bafilomycin polyketide. To study the biosynthetic pathway of C₅N during bafilomycin biosynthesis in K. cheerisanensis KCTC2395, we attempted the functional analysis of two putative genes, encoding 5-aminolevulinic acid synthase (ALAS) and acyl- CoA ligase (ACL). The amplified putative genes for ALAS and ACL were cloned into the E. coli expression vector pET- 32a(+) plasmid, following which the soluble recombinant ALAS and ACL proteins were purified through nickel-affinity column chromatography. Through HPLC analysis of the enzyme reaction mixture, we confirmed the products of putative ALAS and ACL reaction as 5-aminolevulinic acid (5-ALA) and 5-ALA-CoA, respectively. The optimal pH for the putative ALAS reaction was 7.5, and for putative ACL reaction was 7.0, as confirmed by the colorimetric assay. Furthermore, pyridoxal 5'-phosphate (PLP) was found to be an essential cofactor in the putative ALAS reaction, and ATP was a cofactor for the putative ACL catalysis. Finally, we also confirmed that the simultaneous treatment of putative ACL and putative ALAS enzymes resulted in the production of C5N compound from 5-ALA.     

New effective sources of the Staphylococcus simulans lysostaphin

The gene encoding Staphylococcus simulans lysostaphin has been cloned into two Escherichia coli expression systems: pET23b+ (Novagen, UK) and pBAD/Thio-TOPO (Invitrogen, USA), which allow the overexpression of a target protein as a fusion protein. The enzyme produced in the pET system contains a cluster of six histidines at the C-terminus, and the protein produced in the pBAD system contains 133 additional amino acid residues at the N-terminus, including thioredoxin, a cluster of six histidines and a recognition site for endoprotease Factor Xa. The recombinant enzymes were purified by metal-affinity chromatography on a Co2+-Sepharose column. Approximately 20 mg of purified recombinant enzyme were obtained in the pET expression system and 39 mg in the pBAD system, from a 1-L culture. The obtained fusion protein from the pET system revealed specific activity that was approximately 10 times higher than that of the fusion protein from the pBAD system (970 U/mg versus 83 U/mg). The purified enzymes displayed maximum activity at close to 45 degrees C and pH 8.0 or 7.5 for the enzyme obtained from pET and pBAD system, respectively. The lysostaphin activity was strongly inhibited by Zn2+ or Cu2+ (2 mM) with a 70-80% decrease. The Ni2+ (2 mM) also inhibited the enzyme with a 60 and 20% activity decrease for enzyme from the pET and pBAD system, respectively. The Co2+ had no impact on enzymatic activity at the 2 mM concentration; however, 30 and 20% activity decreases were observed at the 10mM concentration for the enzyme obtained from the pET and pBAD expression systems, respectively. EDTA, known as a strong inhibitor of the native lysostaphin, had no impact on the antistaphylococcal activity of either recombinant enzyme.     

Expression and characterization of recombinant thermostable alkaline phosphatase from a novel thermophilic bacterium Thermus thermophilus XM

A gene （tap） encoding a thermostable alkaline phosphatase from the thermophilic bacterium Thermus thermophilus XM was cloned and sequenced. It is 1506 bp long and encodes a protein of 501 amino acid residues with a calculated molecular mass of 54.7 kDa. Comparison of the deduced amino acid sequence with other alkaline phosphatases showed that the regions in the vicinity of the phosphorylation site and metal binding sites are highly conserved. The recombinant thermostable alkaline phosphatase was expressed as a His6-tagged fusion protein in Escherichia coli and its enzymatic properties were characterized after purification. The pH and temperature optima for the recombinant thermostable alkaline phosphatases activity were pH 12 and 75 ℃. As expected, the enzyme displayed high thermostability, retaining more than 50% activity after incubating for 6 h at 80 ℃. Its catalytic function was accelerated in the presence of 0.1 mM Co^2＋, Fe^2＋, Mg^2＋, or Mn^2＋ but was strongly inhibited by 2.0 mM Fe^2＋. Under optimal conditions, the Michaelis constant （Kin） for cleavage of p-nitrophenyl-phosphate was 0.034 mM. Although it has much in common with other alkaline phosphatases, the recombinant thermostable alkaline phosphatase possesses some unique features, such as high optimal pH and good thermostability.     

Extracellular 5-aminolevulinic acid production by Escherichia coli containing the Rhodopseudomonas palustris KUGB306 hemA gene

The Rhodopseudomonas palustris KUGB306 hemA gene codes for 5-aminolevulinic acid (ALA) synthase. This enzyme catalyzes the condensation of glycine and succinyl-CoA to yield ALA in the presence of the cofactor pyridoxal 5'- phosphate. The R. palustris KUGB306 hemA gene in the pGEX-KG vector system was transformed into Escherichia coli BL21. The effects of physiological factors on the extracellular production of ALA by the recombinant E. coli were studied. Terrific Broth (TB) medium resulted in significantly higher cell growth and ALA production than did Luria-Bertani (LB) medium. ALA production was significantly enhanced by the addition of succinate together with glycine in the medium. Maximal ALA production (2.5 g/l) was observed upon the addition of D-glucose as an ALA dehydratase inhibitor in the late-log culture phase. Based on the results obtained from the shake-flask cultures, fermentation was carried out using the recombinant E. coli in TB medium, with the initial addition of 90 mM glycine and 120 mM succinate, and the addition of 45 mM D-glucose in the late-log phase. The extracellular production of ALA was also influenced by the pH of the culture broth. We maintained a pH of 6.5 in the fermenter throughout the culture process, achieving the maximal levels of extracellular ALA production (5.15 g/l, 39.3 mM).     

Optimization of extracellular 5-aminolevulinic acid production from Escherichia coli transformed with ALA synthase gene of Bradyrhizobium japonicum

Extracellular accumulation of 5-aminolevulinic acid (ALA) by an E. coli overexpressing ALA synthase (ALAS) was achieved by inserting a hemA gene from Bradyrhizobium japonicum and expressed under the control of T7 promoter. At pH 7.0 extracellular ALA reached up to 15 mM in a jar fermenter with an addition of glycine (30 mM) and succinate (90 mM) in the medium. ALA accumulation was increased to 20 mM by adding levulinic acid (30 mM) to the cultures.     

Purification and characterization of an intracellular beta-glucosidase from the protoplast fusant of Aspergillus oryzae and Aspergillus niger

Protoplasts of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 were prepared using cellulose and snail enzyme with 0.6 M NaCl as osmotic stabilizer. Protoplast fusion has been performed using 35% polyethylene glycol 4.000 with 0.01 mM CaCl2. The fused protoplasts have been regenerated on regeneration medium and fusants were selected for further studies. An intracellular beta-glucosidase (EC 3.2.1.21) was purified from the protoplast fusant of Aspergillus oryzae 3.481 and Aspergillus niger 3.316 and characterized. The enzyme was purified 138.85-fold by ammonium sulphate precipitation, DE-22 ion exchange and Sephadex G-150 gel filtration chromatography with a specific activity of 297.14 U/mg of protein. The molecular mass of the purified enzyme was determined to be about 125 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme had an optimum pH of 5.4 and temperature of 65 degrees C, respectively. This enzyme showed relatively high stability against pH and temperature and was stable in the pH range of 3.0-6.6. Na+, K+, Ca2+, Mg2+ and EDTA completely inhibited the enzyme activity at a concentration of 10 mM. The enzyme activity was accelerated by Fe3+. The enzyme activity was strongly inhibited by glucose, the end product ofglucoside hydrolysis. The K(m) and V(max) values against salicin as substrate were 0.035 mM and 1.7215 micromol min(-1), respectively.     

Expression, enzymatic characterization, and high-level production of glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) in Escherichia coli

High-level production of recombinant glucose isomerase (rGI) is desirable for lactulose synthesis. In this study, the xylA gene encoding glucose isomerase from Actinoplanes missouriensis CICIM B0118(A) was cloned and expressed in E. coli BL21(DE3), and high-level production was performed by optimization of the medium composition. rGI was purified from a recombinant E. coli BL21(DE3) and characterized. The optimum pH value of the purified enzyme was 8.0 and it was relatively stable within the pH range of 7.0-9.0. Its optimum temperature was around 85 degrees C, and it exhibited good thermostability when the temperature was lower than 90 degrees C. The maximum enzyme activity required the presence of both Co2+ and Mg2+, at the concentrations of 200 microM and 8 mM, respectively. With high-level expression and the simple one-step chromatographic purification of the His-tagged recombinant enzyme, this GI could be used in industrial production of lactulose as a potential economic tool.     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Crystal structure of 5-aminolevulinate synthase, the first enzyme of heme biosynthesis, and its link to XLSA in humans

5-Aminolevulinate synthase (ALAS) is the first and rate-limiting enzyme of heme biosynthesis in humans, animals, other non-plant eukaryotes, and alpha-proteobacteria. It catalyzes the synthesis of 5-aminolevulinic acid, the first common precursor of all tetrapyrroles, from glycine and succinyl-coenzyme A (sCoA) in a pyridoxal 5'-phosphate (PLP)-dependent manner. X-linked sideroblastic anemias (XLSAs), a group of severe disorders in humans characterized by inadequate formation of heme in erythroblast mitochondria, are caused by mutations in the gene for erythroid eALAS, one of two human genes for ALAS. We present the first crystal structure of homodimeric ALAS from Rhodobacter capsulatus (ALAS(Rc)) binding its cofactor PLP. We, furthermore, present structures of ALAS(Rc) in complex with the substrates glycine or sCoA. The sequence identity of ALAS from R. capsulatus and human eALAS is 49%. XLSA-causing mutations may thus be mapped, revealing the molecular basis of XLSA in humans. Mutations are found to obstruct substrate binding, disrupt the dimer interface, or hamper the correct folding. The structure of ALAS completes the structural analysis of enzymes in heme biosynthesis.     

Characterization of a thermophilic DNA ligase from the archaeon Thermococcus fumicolans

A PCR protocol was used to identify and sequence a gene encoding a DNA ligase from Thermococcus fumicolans (Tfu). The recombinant enzyme, expressed in Escherichia coli BL21(DE3) pLysS, was purified to homogeneity and characterized. The optimum temperature and pH of Tfu DNA ligase were 65 degrees C and 7.0, respectively. The optimum concentration of MgCl2, which is indispensable for the enzyme activity, was 2 mM. We showed that Tfu DNA ligase displayed nick joining and blunt-end ligation activity using either ATP or NAD+, as a cofactor. In addition, our results would suggest that Tfu DNA ligase is likely to use the same catalytic residues with the two cofactors. The ability for DNA ligases, to use either ATP or NAD+, as a cofactor, appears to be specific of DNA ligases from Thermococcales, an order of hyperthermophilic microorganisms that belongs to the euryarchaeotal branch of the archaea domain.     

Expression and purification of pheophorbidase, an enzyme catalyzing the formation of pyropheophorbide during chlorophyll degradation: comparison with the native enzyme

Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.) were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg L(-1) of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs.     

蜡样芽胞杆菌磷脂酶C在乳酸克鲁维酵母中重组表达、纯化及酶学性质分析

【目的】构建蜡样芽胞杆菌(Bacillus cereus)磷脂酶C(Phospholipase C,PLC)的重组乳酸克鲁维酵母(Kluyveromyces lactis)菌株、纯化重组蛋白并对其进行酶学性质分析。【方法】以B.cereus基因组DNA为模板,PCR扩增得到磷脂酶C基因(bcplc),构建重组乳酸克鲁维酵母表达质粒并转化到乳酸克鲁维酵母中,实现bcplc基因的表达。利用镍柱亲和层析纯化和脱盐柱得到电泳纯的重组磷脂酶C(rbcPLC)。【结果】成功构建产磷脂酶C的重组乳酸克鲁维酵母并纯化了重组磷脂酶C,纯化后rbcPLC经SDS-PAGE分析在40 kDa附近出现显性条带。NPPC法测得rbcPLC酶活为19251 U/mg,最适反应温度为80°C,最适pH为9.0。在低于40°C时,pH 7.0-8.0时,rbcPLC重组酶较稳定。Cu~(2+)和Co~(2+)对其有明显的抑制作用;Zn~(2+)、Mn~(2+)、Ca~(2+)、Mg~(2+)对其有明显的促进作用。【结论】首次实现了对蜡样芽胞杆菌来源的磷脂酶C在乳酸克鲁维酵母中的重组表达、纯化及其酶学性质分析,为其它食品安全性微生物来源的磷脂酶C的研究提供了借鉴意义。     

Cloning and characterization of a gene encoding phosphoketolase in a Lactobacillus paraplantarum isolated from Kimchi

A gene coding for phosphoketolase, a key enzyme of carbohydrate catabolism in heterofermentative lactic acid bacteria (LAB), was cloned from a Lactobacillus paraplantarum C7 and expressed in Escherichia coli. The gene is 2502 bp long and codes for a 788-amino-acids polypeptide with a molecular mass of 88.7 kDa. A Shine-Dalgamo sequence (aaggag) and an inverted-repeat terminator sequence are located upstream and downstream of the phosphoketolase gene, respectively. The gene exhibits an identity of >52% with phosphoketolases of other LAB. The phosphoketolase of Lb. paraplantarum C7 (LBPK) contains several highly conserved phosphoketolase signature regions and typical thiamine pyrophosphate (TPP) binding sites, as reported for other TPP-dependent enzymes. The phosphoketolase gene was fused to a glutathione S-transferase (GST::LBPK) gene for purification. The GST::LBPK fusion protein was detected in the soluble fraction of a recombinant Escherichia coli BL21. The GST::LBPK fusion protein was purified with a yield of 4.32 mg/400 ml by GSTrap HP affinity column chromatography and analyzed by N-terminal sequencing. LBPK was obtained by factor Xa treatment of fusion protein and the final yield was 3.78 mg/400 ml. LBPK was examined for its N-terminal sequence and phosphoketolase activity. The K(M) and Vmax values for fructose-6-phosphate were 5.08 +/- 0.057 mM (mean +/- SD) and 499.21 +/- 4.33 micromol/min/mg, respectively, and the optimum temperature and pH for the production of acetyl phosphate were 45 degrees C and 7.0, respectively.     

Potato tuber succinate semialdehyde dehydrogenase: purification and characterization

Succinate semialdehyde dehydrogenase (SSADH) has been purified from potato tubers with 39% yield, 832-fold purification, and a specific activity of 6.5 units/mg protein. The final preparation was homogeneous as judged from native and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel filtration on Sepharose 6B gave a relative molecular mass (Mr) of 145,000 for the native enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single polypeptide band of Mr 35,000. Thus the enzyme appears to be a tetramer of identical subunits. Chromatofocusing of the enzyme gave a pI of 8.7. The enzyme was maximally active at pH 9.0 in 100 mM sodium pyrophosphate buffer. In 100 mM Tris-HCl buffer, pH 9.0, the enzyme gave only 20% of the activity found in pyrophosphate buffer and had a shorter linear rate. The enzyme was specific for succinate semialdehyde (SSA) as substrate and could not utilize acetaldehyde, glyceraldehyde 3-phosphate, malonaldehyde, lactate, or ethanol as substrates. The enzyme was also specific for NAD+ as cofactor and NADP+ and 3-acetylpyridine adenine dinucleotide could not serve as cofactors. Potato SSADH had a Km of 4.6 microM for SSA when assayed in pyrophosphate buffer and was inhibited by that substrate at concentrations greater than 120 microM. The Km for NAD+ was found to be 31 microM. The enzyme required exogenous addition of a thiol compound for maximal activity and was inhibited by the thiol-directed reagents p-hydroxymercuribenzoate, dithionitrobenzoate, and N-ethyl-maleimide, by heavy metal ions Hg2+, Cu2+, Cd2+, and Zn2+, and by arsenite. These results indicate a requirement of a SH group for catalytic activity.     

Enzyme system involved in the synthesis of thiamin triphosphate. I. Purification and characterization of protein-bound thiamin diphosphate: ATP phosphoryltransferase

An enzyme system catalyzing the synthesis of thiamin triphosphate consists of an enzyme (protein-bound thiamin diphosphate:ATP phosphoryltransferase), thiamin diphosphate bound to a macromolecule as substrate, ATP, Mg2+, and a low molecular weight cofactor. This system was established by combining a purified enzyme and an essentially pure, macromolecule-bound substrate prepared from rat livers. This macromolecule was found to be a protein, and the transphosphorylation of thiamin diphosphate to thiamin triphosphate with ATP and enzyme was shown to occur on this macromolecule which binds thiamin diphosphate. Free thiamin, thiamin monophosphate, thiamin diphosphate, and thiamin triphosphate have no effect on this reaction. Thus, the overall reaction is: thiamin diphosphate-protein + ATP in equilibrium thiamin triphosphate-protein + ADP. So-called thiamin diphosphate:ATP phosphoryltransferase (EC 2.7.4.15) activity was not detected in rat brain or liver. The enzyme was extracted from acetone powder of a crude mitochondrial fraction of bovine brain cortex and purified to homogeneity with a 0.6% yield after DEAE-cellulose chromatography, a first gel filtration, hydroxylapatite chromatography, chromatofocusing, and a second gel filtration. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Its molecular weight was estimated to be 103,000. The pH optimum was 7.5, and the Km was determined to be 6 X 10(-4) M for ATP. ATP was found to be the most effective phosphate donor among the nucleoside triphosphates. Amino acid analysis of the purified enzyme revealed an abundance of glutaminyl, glutamyl, and aspartyl residues. Sulfhydryl reagents inhibited the enzyme reaction. Metals such as Fe2+, Zn2+, Pb2+, and Cu2+ strongly inhibited the activity. The enzyme was unstable, and glycerol (20%) and dithiothreitol (1.0 mM) were found to preserve the enzyme activity.     

K30, H150, and H168 Are Essential Residues for Coordinating Pyridoxal 5′-Phosphate of O-Acetylserine Sulfhydrylase from Acidithiobacillus ferrooxidans

O-acetylserine sulfhydrylase (OASS) is a key enzyme involved in the pathway of the cysteine biosynthesis. The gene of OASS from Acidithiobacillus ferrooxidans ATCC 23270 was cloned and expressed in E. coli, the soluble protein was purified by one-step affinity chromatography to apparent homogeneity. Colors and UV–vis scanning results of the recombinant protein confirmed that it was a pyridoxal 5′-phosphate (PLP)-containing protein. Sequence alignment and site-directed mutation of the enzyme revealed that the cofactor PLP is covalently bound in Schiff base linkage with K30, as well as the two residues H150 and H168 were the crucial residues for PLP binding and stabilization.