Motohiro Nishimura

Molecular Cloning of Streptomyces Genes Encoding Vanillate Demethylase

The vanillate demethylase genes from Streptomyces sp. NL15-2K were cloned and sequenced. The vanA and vanB gene homologs, which encode the terminal oxygenase subunit (VanA) and the ferredoxin-type reductase subunit (VanB) of the enzyme respectively, were found in the sequenced 7.5-kb DNA region. Expression of the vanAB genes in Streptomyces lividans 1326 resulted in in vivo demethylation of veratric acid to vanillic acid.

Inhibitory Effects of Carbohydrates on Cholesterol Esterase Biosynthesis in Streptomyces lavendulae H646-SY2.

The inhibitory effects of various carbohydrates on cholesterol esterase (CHE) biosynthesis were investigated in a CHE-producing Streptomyces lavendulae H646-SY2. A marked decrease in CHE production and preferential utilization of glucose were observed when glucose was added to a medium containing palmitic acid as the sole carbon source and an inducer of CHE production. This catabolite repression was also caused by other carbohydrates such as sucrose, glycerol, galactose and arabinose. Sucrose, however, did not inhibit the utilization of palmitic acid by the strain, suggesting that the mechanism of the catabolite repression by sucrose is different from that by glucose.

Structural evidence that puromycin hydrolase is a new type of aminopeptidase with a prolyl oligopeptidase family fold

Puromycin [PM, Supporting Information Fig. S1(A)]1 and blasticidin S [BS, Supporting Information Fig. S1(B)]2 are nucleoside antibiotics. We have previously found that BS-producing Streptomyces morookaensis JCM4673 inactivates the own product in the presence of acetyl CoA.3 We have also found that the bacterium has an enzyme to inactivate PM without the cofactor.4 The enzyme catalyzes the hydrolysis of an amide linkage between the aminonucleoside and O-methyl-L-tyrosine moieties in the PM molecule. We have revealed that the S. morookaensis PM hydrolase (PMH) has some aminopeptidase-like properties closely related to those of proline iminopeptidases [EC 3.4.11.5].5 Proline iminopeptidases (PIPs) are among the serine peptidases catalyzing the removal of the N-terminal proline from a substrate with strict specificity.6 However, PMH exhibits broad substrate specificity toward b-naphthylamide derivatives of amino acids, although it prefers L-prolyl-bnaphthylamide in such substrates. We have cloned and sequenced a gene encoding PMH from S. morookaensis.7 The deduced amino acid sequence was similar to those of several peptidases classified into the prolyl oligopeptidase (POP) family.8 This family includes POP (EC 3.4.21.26),9 which cleaves the internal peptide bond on the C-terminal side of the prolyl residues, oligopeptidase B (EC 3.4.21.83),10 which cleaves the internal peptide bond on the C-terminal side of the lysyl or arginyl residues, dipeptidyl peptidase IV (DPP IV; EC 3.4.14.5),11 which removes N-terminal dipeptides from the polypeptides having unsubstituted N-termini and the penultimate proline residue, prolyl tripeptidyl aminopeptidase (PTP; EC 3.4.14.12),12 which removes N-terminal tripeptides from polypeptides having a proline residue at the third position, and acyl-peptide hydrolase (ACPH; EC 3.4.19.1),13 which catalyzes the hydrolysis of the amino-terminal peptide bond of an N-acetylated peptide to generate an N-acetylated amino acid and a peptide with a free amino terminus. After we have found PMH, it has been shown that PMH homologues, which had been annotated as putative ACPH, from S. griseus and S. coelicolor display aminopeptidase activities [EC 3.4.11].14 However, the tertiary structure of aminopeptidases, classified into the POP family, is not yet determined. To understand the manner of recognition of the substrate and the catalytic

Characterization of two isozymes of coniferyl alcohol dehydrogenase from Streptomyces sp. NL15-2K.

We purified two isozymes of coniferyl alcohol dehydrogenase (CADH I and II) to homogeneity from cell-free extracts of Streptomyces sp. NL15-2K. The apparent molecular masses of CADH I and II were determined to be 143 kDa and 151 kDa respectively by gel filtration, whereas their subunit molecular masses were determined to be 35,782.2 Da and 37,597.7 Da respectively by matrix-assisted laser-desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Thus, it is probable that both isozymes are tetramers. The optimum pH and temperature for coniferyl alcohol dehydrogenase activity were pH 9.5 and 45 °C for CADH I and pH 8.5 and 40 °C for CADH II. CADH I oxidized various aromatic alcohols and allyl alcohol, and was most efficient on cinnamyl alcohol, whereas CADH II exhibited high substrate specificity for coniferyl alcohol, and showed no activity as to the other alcohols, except for cinnamyl alcohol and 3-(4-hydroxy-3-methoxyphenyl)-1-propanol. In the presence of NADH, CADH I and II reduced cinnamaldehyde and coniferyl aldehyde respectively to the corresponding alcohols.

Molecular cloning and expression of the Streptomyces coniferyl alcohol dehydrogenase gene in Escherichia coli.

Coniferyl alcohol dehydrogenase (CADH) is a key enzyme in catabolism of lignin-related aromatic compounds in bacteria. In Streptomyces sp. NL15-2K, CADH is a tetramer of identical subunits with an individual molecular mass of 39 kDa. This work describes the cloning and sequencing of the CADH gene from Streptomyces sp. NL15-2K, optimization of a protocol for high-level active CADH expression, and purification of recombinant CADH. A BLAST search and motif analyses of the predicted CADH amino acid sequence indicated the enzyme belongs to the medium-chain zinc-dependent alcohol dehydrogenase group. Cell density at heat-shock treatment, temperatures for heat shock and culture, duration of heat shock, concentration of isopropyl-β-d-thiogalactopyranoside (IPTG) as an inducer, and culture time after induction were adjusted for optimal CADH expression. Expression of active CADH under optimized conditions was approximately 4-fold higher than in the absence of heat shock. CADH purified from recombinant Escherichia coli was in the tetrameric form, as was natural CADH from NL15-2K.