Phenol Ethers and Terpenes of Six Heterotropa Species Distributed in the Ryukyu Islands and Formosa

A new intracellular peptidase, which we call “d-peptidase S,” was purified from Nocardia orientalis IFO 12806 (ISP 5040). The purified enzyme was homogeneous on disc gel electrophoresis. The molecular weight and the isoelectric point were estimated to be 52,000 and 4.9, respectively. The optimum pH for the hydrolysis of d-leucyl-d-leucine was 8.0 to 8.1, and the optimum temperature was 36°C. The purified enzyme usually hydrolyzed the peptide bonds preceding the hydrophobic D-amino acids of dipeptides. Tri- and tetra-peptides extending to the amino terminus of such peptides were also hydrolyzed. Therefore, the enzyme is a carboxylpeptidase-like peptidase specific to d-amino acid peptides. The Km values for d-leucyl-d-leucine and l-leucyl-d-leucine were 0.21 × 10^-3 and 0.44 × 10^-3 m respectively. The activity was inhibited by several sulfhydryl reagents and two chelators, 8-hydroxyquinoline and o-phenanthroline.     

Disruption of quinoprotein ethanol dehydrogenase gene and adjacent genes in Pseudomonas putida HK5

Pseudomonas putida HK5 produces three different quinoprotein alcohol dehydrogenases: ADH-I, ADH-IIB and ADH-IIG. Gene organization of qedA , the gene for ADH-I, and other 10 genes in the cluster was related to the genome sequences of five other Pseudomonas strains. Insertion mutations in either qedA , exaE or agmR eliminated ADH-I activity, although the mutants were still able to grow on ethanol but more slowly than the wild-type strain. Mutant analysis demonstrated the requirement of agmR and exaE in ADH-I expression, and the tentative involvement of agmR , but not exaE , in the induction of ADH-IIB and ADH-IIG activities.     

Further Characterization of Aminopeptidase of Rabbit Skeletal Muscle

Further investigation on characterization was conducted on purified neutral aminopeptidase of 160,000 daltons from rabbit skeletal muscle. The enzyme possesses arylamidase activity. The greater part of leucine-β-naphthylamide hydrolyzing activity of the muscle extract was attributed to the enzyme. The Km value for Ala-Gly-Phe-Ala, the most cleavable substrate tested, was 0.25 mm. Substrate inhibition was observed for Val-Val-Val-Ala and Val-Val-Val. The enzyme was inhibited by puromycin in a non-competitive manner, Ki being 4 × 10^?6 m. The enzyme was also inhibited by insulin and the oxidized B-chain of insulin. The tetrapeptide with N-terminal residue of d configuration, tRNA, pyruvate and α-ketoglutarate had no effect on the enzyme. On the basis of all properties determined so far, this muscle aminopeptidase is concluded to be identical to none of the known aminopeptidases from other tissues.     

Recent progress in studies on the health benefits of pyrroloquinoline quinone

Pyrroloquinoline quinone (PQQ), an aromatic tricyclic o-quinone, was identified initially as a redox cofactor for bacterial dehydrogenases. Although PQQ is not biosynthesized in mammals, trace amounts of PQQ have been found in human and rat tissues because of its wide distribution in dietary sources. Importantly, nutritional studies in rodents have revealed that PQQ deficiency exhibits diverse systemic responses, including growth impairment, immune dysfunction, and abnormal reproductive performance. Although PQQ is not currently classified as a vitamin, PQQ has been implicated as an important nutrient in mammals. In recent years, PQQ has been receiving much attention owing to its physiological importance and pharmacological effects. In this article, we review the potential health benefits of PQQ with a focus on its growth-promoting activity, anti-diabetic effect, anti-oxidative action, and neuroprotective function. Additionally, we provide an update of its basic pharmacokinetics and safety information in oral ingestion.     

Different forms of quinoprotein aldose-(glucose-) dehydrogenase in Acinetobacter calcoaceticus

The ratios of the oxidation rates of aldose sugars, determined in cell-free extracts of Acinetobacter calcoaceticus, vary with the strain and growth conditions used. Three distinct forms of glucose dehydrogenase with different substrate specificities, occurring in variable proportions in these extracts, are responsible for this effect. One form is the already known soluble glucose dehydrogenase, the other two forms are complexes containing enzyme and components of the respiratory chain. The proportions in which the enzyme forms are found in the cell-free extract correlate with the oxidative behaviour of whole cells with respect to aldose sugars. It is concluded, therefore, that the enzyme forms are not an artefact of the isolation procedure but that they exist as such in vivo. Since the two complexes can be converted into the soluble enzyme form, aldose dehydrogenase can, probably, be integrated in three different ways into the respiratory chain.The presence of glucose during growth does not stimulate aldose dehydrogenase production. This is not surprising since the enzyme has no function in carbon metabolism, except perhaps in strains growing on pentoses at high pH. Therefore, the physiological role of quinoprotein aldose dehydrogenase in this organism may be primarily in energy generation.Non-standard abbreviations quinoproteins enzymes containing 2,7,9-tricarboxy-1 H-pyrrolo [2,3f] quinoline-4,5-dione (pyrrolo-quinoline quinone) as the coenzyme     

Substituent Effect on Photosynthesis of N2-α-Substituted Benzyl-N4-alkyl-2,4-diamino-6-chloro-s-triazines and Their Phytotoxic Property

The influence of substituents on the activities of a series of N²-α-substituted benzyl-N⁴-alkyl-2,4-diamino-6-chloro-s-triazines as inhibitors of photosystem II (PSII) was examined, and the phytotoxic differences between them and atrazine, as to the photosynthesis in leaf disks, mesophyll cells, intact chloroplasts and broken chloroplasts of spinach, and as to seedling-growth, were discussed. The inhibitory activity of the N²-α,α-dimethylbenzyl-N⁴-ethyl derivative (6), which was comparable on that of atrazine, was lower than those of the N²-α-alkylbenzyl analogues (1 ~5). The N⁴-?-alkyl-N²-α- methylbenzyl derivatives, in spite of the carbon length of the alkyl group, exhibited more potent activity than atrazine, but an a α β substitution of the N⁴-n-alkyl group caused a decrease in the activity with a few exceptions. These data may imply that the space of the binding site on PSII surrounding both the N² and N⁴ amino groups is relatively large. The binding between the receptor site and the N⁴ amino group, however, is easily influenced by a slight structural change in an inhibitor. The herbicidal compounds, N²-α-methylbenzyl-A^⁴-ethyl (1), A^²-α,α-dimethylbenzyl-N⁴-1-methylpropyl (30) and N²-α-methylbenzyl-N⁴,N⁴-diethyl (42) derivatives, exhibited potent inhibitory activity in the seedling growth test under dark/light conditions, whereas atrazine was very poor. The inhibitory activity of compound (1) toward photosynthesis was poor with leaf disks, compared to atrazine, whereas, the order of their activities was the reverse for plant preparations such as abaxial epidermis peeled leaf disks, mesophyll cells, intact chloroplasts and broken chloroplasts. It was indicated that a change in the phytotoxic symptom in the whole plant assay would be correlated to the permeability of the compound through the plant membrane(s).     

Construction and characterization of heterodimeric soluble quinoprotein glucose dehydrogenase

In order to study in greater detail the subunit interaction of the homodimeric soluble quinoprotein glucose dehydrogenase (PQQGDH-B), we developed an effective method of creating heterodimeric PQQGDH-B. Two different homodimers are combined, one of which has a polyarginine tail (Arg-tail), and subjected to a protein dissociation/redimerization procedure. Separation of the mixture by cation exchange chromatography results in three peaks showing GDH activity, eluting at 133, 231 and 273 mM NaCl concentration. These peaks were determined to correspond to the Arg-tailless homodimer, heterodimer, and Arg-tailed homodimer, respectively. To test this approach, we constructed and characterized heterodimeric PQQGDH-B composed of native (wild-type) and inactive mutant (His168Gln) subunits. The heterodimeric wild-type-His168Gln showed slightly decreased GDH activity and almost identical substrate specificity profile to the wild-type enzyme. Moreover, the Hill coefficient of the heterodimer was calculated as 1.13, indicating positive cooperativity.     

Characterization of a group of pyrroloquinoline quinone‐dependent dehydrogenases that are involved in the conversion of L‐sorbose to 2‐Keto‐L‐gulonic acid in Ketogulonicigenium vulgare WSH‐001

Ketogulonicigenium vulgare WSH‐001 is an industrial strain used for vitamin C production. Based on genome sequencing and pathway analysis of the bacterium, some of its potential pyrroloquinoline quinone (PQQ)‐dependent dehydrogenases were predicted, including KVU_pmdA_0245, KVU_2142, KVU_2159, KVU_1366, KVU_0203, KVU_0095, and KVU_pmdB_0115. BLAST and function domain searches showed that enzymes encoded by these genes may act as putative PQQ‐dependent L ‐sorbose dehydrogenases (SDH) or L ‐sorbosone dehydrogenases (SNDH). To validate whether these dehydrogenases are PQQ‐dependent or not, these seven putative dehyrogenases were overexpressed in Escherichia coli BL21 (DE3) and purified for characterization. Biochemical and kinetic characterization of the purified proteins have led to the identification of seven enzymes that possess the ability to oxidize L ‐sorbose or L ‐sorbosone to varying degrees. In addition, the dehydrogenation of sorbose in K. vulgare is validated to be PQQ dependent, identification of these PQQ‐dependent dehydrogenases expanded the PQQ‐dependent dehydrogenase family. Besides, the optimal combination of enzymes that could more efficiently catalyze the conversion of sorbose to gulonic acid was proposed. These are important in supporting the development of metabolic engineering strategies and engineering of efficient strains for one‐step production of vitamin C in the future. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29:1398–1404, 2013     

甲基营养菌MP688萄糖脱氢酶基因分离鉴定及性质研究

目的：鉴定甲基营养菌MP688中的葡萄糖脱氢酶基因。方法：对甲基营养菌MP688基因组序列进行比对和分析,找到与已知细菌葡萄糖脱氢酶同源性最高的基因序列mpq_2164,且该基因所编码蛋白经分析具有跨膜结构域。设计51物扩增mpq_2164和缺失跨膜区域序列的s-mpq_2164,将PCR产物克隆到表达载雄pET-15b上,在大肠杆菌BL21中完成异源重组表达,然后通过组氨酸标签镍柱亲和层析纯化,采用DCIP法测定葡萄糖脱氢酶的活力。结果：分离了甲基营养菌MP688中的葡糖糖脱氢酶基因,并实现了s-mpq_2164的高效异源重组表达;MPQ2164的氯基酸序列与已知的葡萄糖脱氢酶相似性很低,但酶活测定结果表明S-MPQ-2164具有很高的葡糖糖脱氢酶活性。结论：MPQ_2164是-个依赖于吡咯喹啉醌的葡萄糖脱氢酶,去掉跨膜结构域有利于该蛋白的异源嘉{大,     

Methylation Analysis of Fractions from the Leuconostoc mesenteroides NRRL B-1299 Dextran

Methylation analysis of five fractions of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 has shown that each fraction was a highly branched dextran with the branches being joined mainly through C-2. Detection of a small amount of 4-O-mono-methyl-d-glucose has suggested that parts of the d-glucose residues were doubly branched at both C-2 and C-3. Detection of a larger amount of 2,4,6-tri-O-methyl-d-glucose in the hydrolyzates of the methylated products of the borate insoluble fractions has shown a greater percentage of linear α-1,3-linked d-glucose residues in these fractions. It is suggested that the solubility of the dextran is closely related to the content of linear α-1,3-linked d-glucose residues.     

Enantioselectivity of Enzymatic Cleavage Reaction of 13-Hydroperoxylinolenic Acid to C6-Aldehyde and C12-Oxo Acid in Tea Chloroplasts

A protease with strict specificity to lysyl peptide bonds like that of Achromobacter protease I was purified from a crude enzyme powder obtained from a culture filtrate of Achromobacter lyticus M497-1 and characterized. The purified enzyme had the following differences from protease I. The enzyme had an isoelectric point of 5.3, lower than the value of 6.9 for protease I. The amino acid composition of the enzyme had higher proportions of His, Glu, and Gly and lower proportions of Arg and Thr than protease I. The enzyme was unstable (30% residual activity) in the presence of 7 m urea (pH 8.0, 30°C, 20 min); protease I was resistant to the same conditions (80% residual activity). The k_cat/Km values for the hydrolysis of Tos-Lys-OMe and Lys-pNA by the enzyme were lower than those of protease I.     

The Composition of Saccharogenic Amylase from Rhizopus javanicus and the Isolation of Glycopeptides

Saccharogenic amylase from Rhizopus javanicus sp. 3–46 was known to be a glycoprotein which contained 27 residues of mannose and 4 residues of N-acetylglucosamine per mole of the saccharogenic amylase. Attempts have been made to obtain glycopeptides from the saccharogenic amylase. Three glycopeptides, GP-I-a, GP-I-b and GP-II, were separated from a Pronase digest of heat-denatured saccharogenic amylase by gel filtration on Sephadex G-50 and chromatography on DEAE-Sephadex A-25. GP-I-a contained asparagine, glycine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 6: 2. GP-I-b contained asparagine, threonine, mannose and N-acetylglucosamine in a molar ratio of 1: 1: 9:2. GP-II consisted of threonine, serine, proline, alanine and mannose in a molar ratio of 6: 2: 2: 2: 12.     

The in vivo and in vitro substrate specificity of quinoprotein glucose dehydrogenase of Acinetobacter calcoaceticus LMD79.41

Quinoprotein glucose dehydrogenase (GDH; EC 1.1.99.17) was partially purified from cell-free extracts of Acinetobacter calcoaceticus LMD79.41. The enzyme oxidized monosaccharides (d-glucose, d-allose, 2-deoxy-d-glucose, d-galactose, d-mannose, d-xylose, d-ribose and l-arabinose) as well as disaccharides (d-lactose, d-maltose and d-cellobiose).Intact cells of A. calcoaceticus LMD79.41 also oxidized these monosaccharides, but not the disaccharides.The difference in substrate specificity can not be explained by impermeability of the outer membrane for disaccharides, since right-side-out membrane vesicles did not oxidize disaccharides either. Destruction of the cytoplasmic membrane strongly affected the catalytic properties of GDH. Not only did the affinity towards some monosaccharides change substantially, but disaccharides also became good substrates upon solubilization of the enzyme. Thus, at least in A. calcoaceticus LMD79.41, the oxidation of disaccharides by GDH can be considered as an in vitro ‘artefact’ caused by the removal of the enzyme from its natural environment.     

Surface charge engineering of PQQ glucose dehydrogenase for downstream processing

The ion-exchange chromatography behavior of recombinant glucose dehydrogenase harboring pyrroloquinoline quinone (PQQGDH) was modified to greatly simplify its purification. The surface charge of PQQGDH was engineered by either fusing a three-arginine tail to the C-terminus of PQQGDH (PQQGDH+Arg3) or by substituting three residues exposed on the surface of the enzyme to Arg by site-directed mutagenesis (3RPQQGDH). During cation exchange chromatography, both surface charge-engineered enzymes eluted at much higher salt concentrations than the wild-type enzyme. After the chromatography purification step, both PQQGDH+Arg3 and 3RPQQGDH appeared as single bands on SDS-PAGE, while extra bands appeared with the wild-type protein sample. Although all tested kinetic parameters of both engineered enzymes are similar to those of wild type, both modifications resulted in enzymes with increased thermal stability. Our achievements have resulted in the greater production of an improved quality PQQGDH by a simplified process.     

Stabilization of quaternary structure of water-soluble quinoprotein glucose dehydrogenase

Water-soluble quinoprotein glucose dehydrogease (PQQGDH-B) is a dimeric enzyme whose application for glucose sensing is the focus of much attention. We attempted to increase the thermal stability of PQQGDH-B by introducing a disulfide bond at the dimer interface. The Ser residue at position 415 was selected for substitution with Cys, as structural information revealed that its side chains face each other at the dimer interface of PQQGDH-B. PQQGDH-B with Ser415Cys showed 30-fold greater thermal stability at 55°C than did the wild-type enzyme without any decrease in catalytic activity. After incubation at 70°C for 10 min, Ser415Cys retained 90% of the GDH activity of the wild-type enzyme. Disulfide bond formation between the mutant subunits was confirmed by analyses with sodium dodecylsulfate-polyacrylamide gel electrophoresis in the presence and absence of reductants. Our results indicate that the introduction of one Cys residue in each monomer of PQQGDH-B resulted in formation of a disulfide bond at the dimer interface and thus achieved a large increase in the thermal stability of the enzyme.     

Distribution of Tyrosine Phenol Lyase in Microorganisms

The distribution of tyrosine phenol lyase activity in microorganisms was studied with intact cells in a synthetic reaction mixture containing l-serine and phenol or pyrocatechol. This activity was found in various bacteria, most of which belonged to the Enterobacteriaceae; especially to the genera Escherichia, Proteus and Erwinia. Cells of Erwinia herbicola ATCC 21434 were selected as a promising source of enzyme.

Intact cells of Erwinia herbicola ATCC 21434 prepared from a broth cultured for 24 hr contained markedly high enzymic activity and catalyzed the synthetic reaction of l-tyrosine or 3,4-dihydroxyphenyl-l-alanine (l-dopa) from l-serine and phenol or pyrocatechol in significantly high yields.

Results of the isolation and identification of the products showed that the amino acid synthesized by this enzymatic method was identical with l-tyrosine or l-dopa.     

Participation and Properties of Lipoxygenase and Hydroperoxide Lyase in Volatile C6-Aldehyde Formation from C18-Unsaturated. Fatty Acids in Isolated Tea Chloroplasts

Isolated tea chloroplasts utilized linoleic acid, linolenicacid and their 13-hydroperoxides as substrates for volatileC₆-aldehyde formation. Optimal pH values for oxygen uptake,hydroperoxide lyase and the overall reaction from C₁₈-fattyacids to C₆-aldehydes were 6.3, 7.0 and 6.3, respectively. Methyllinoleate, linoleyl alcohol and -linolenic acid were poor substratesfor the overall reaction, but linoleic and linolenic acids weregood substrates. The 13-hydroperoxides of the above fatty acidsand alcohol also showed substrate specificity similar to thatof fatty acids. Oxygen uptakes (relative V_max) with methyl linoleate,linoleyl alcohol, linolenic acid, -linolenic acid and arachidonicacid were comparable to or higher than that with linoleic acid.In winter leaves, the activity for C₆-aldehyde formation fromC₁₈-fatty acids was raduced to almost zero. This was due tothe reduction in oxygenation. The findings presented here provideevidence for the involvement of lipoxygenase and hydroperoxidelyase in C₆-aldehyde formation in isolated chloroplasts. (Received July 11, 1981; Accepted November 5, 1981)     

Glu742 substitution to Lys enhances the EDTA tolerance ofEscherichia coli PQQ glucose dehydrogenase

Summary Based on homology analysis of the PQQ (pyrroloquinoline quinone) glucose dehydrogenase (PQQGDH) gene fromEscherichia coli andAcinetobacter calcoaceticus, Glu742 was substituted to Lys by site directed mutagenesis of theE. coli PQQGDH gene (gcd). The mutant enzyme, E742K showed higher tolerance towards EDTA inactivation than wild type PQQGDH. This is the first mutagenesis study of putative a PQQ binding site in PQQ enzyme.     

Purification and Properties of Two Different Dihydroxyacetone Reductases in Gluconobacter suboxydans Grown on Glycerol

It is well known that in oxidative fermentation microbial growth is improved by the addition of glycerol. In a wild strain, glycerol was converted rapidly to dihydroxyacetone (DHA) quantitatively in the early growth phase by the action of quinoprotein glycerol dehydrogenase (GLDH), and then DHA was incorporated into the cells by the early stationary phase. Two DHA reductases (DHARs), NADH-dependent (NADH-DHAR) (EC 1.1.1.6) and NADPH-dependent (NADPH-DHAR) (EC 1.1.1.156), were detected in the same cytoplasm of Gluconobacter suboxydans IFO 3255. The former appeared to be inducible and labile in nature while the latter was constitutive and stable. The two DHARs were separated each other and were finally purified to crystalline enzymes. This report might be the first one dealing with NADPH-DHAR that has been crystallized. The two DHARs were specific only to DHA reduction to glycerol and thus contributed to cytoplasmic DHA metabolism, resulting in an improved biomass yield with the addition of glycerol.     

The role of copper in bovine serum amine oxidase

Summary The role of copper in bovine serum amine oxidase was investigated by studying the effect of copper-binding inhibitors on the reactions of the pyrroloquinoline quinone carbonyl and on the reaction with oxygen. Hydrazines and hydrazides were used as carbonyl reagents and one of the hydrazines, benzylhydrazine, which was found to behave as a pseudo-substrate, was used to probe the reaction with oxygen. The presence ofN,N-diethyldithiocarbamate, a chelator that binds copper irreversibly, did not prevent the reactions at the carbonyl, but slowed down their rate and modified the conformation of the adducts. The same happened to the reaction with oxygen, which was slowed down but not abolished. Copper, which was never seen in the reduced state, thus appears to control all reactions without being directly involved in the binding of either hydrazines or oxygen. The enzyme functionality was in fact preserved upon substitution of copper with cobalt. The specific activity of the cobalt-substituted enzyme was only reduced to about 40% the native amine oxidase value. This is the first case so far in which the role of copper can be performed by a different metal ion.Abbreviations BSAO bovine serum amine oxidase - DDC N,N-diethyldithiocarbamate - PQQ pyrroloquinoline quinone