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Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11), 15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).

Research has been conducted on the biosynthesis of the isopropenoid antibiotics produced by actinomycins. The cloning and sequencing analysis of the cyclase genes which encode proteins demonstrating similarities to the eukaryotic diterpene cyclases and eubacterial pentalenene synthase has been carried out and the results are described.

Site-directed mutagenesis was used to investigate the cloning, sequencing and overexpression in Escherichia coli cells of the low-specificity L-threonine aldolase (L-TA) gene from the Pseudomonas strain NCIMB 10558, purification and characterization of the recombinant enzyme and the identification of the active-site lysine residue of the enzyme. The amino acid sequence displayed less than 20% identity with those of low-specificity L-TA from Saccharomyces cerevisiae, L-allo-threonine aldolase from Aeromonas jandaei and other hypothetical proteins from other microorganisms.

The Corynebacterium strain St-10 capable of gaseous styrene assimilation was used to purify a novel phenylacetaldehyde reductase 50-fold to homogeneity. The enzyme, which was observed to be vital in in vivo styrene metabolism, featured four identical subunits and had a 155,000 molecular weight. The decrease of several ketones, aldehydes and phenylacetaldehyde was achieved through the enzyme. However, the enzyme failed to promote the dehydrogenation of 2-phenylethanol.

Abstract Background Biogenic emissions of methyl halides (CH3Cl, CH3Br and CH3I) are the major source of these compounds in the atmosphere; however, there are few reports about the halide profiles and strengths of these emissions. Halide ion methyltransferase (HMT) and halide/thiol methyltransferase (HTMT) enzymes concerning these emissions have been purified and characterized from several organisms including marine algae, fungi, and higher plants; however, the correlation between emission profiles of methyl halides and the enzymatic properties of HMT/HTMT, and their role in vivo remains unclear. Results Thirty-five higher plant species were screened, and high CH3I emissions and HMT/HTMT activities were found in higher plants belonging to the Poaceae family, including wheat (Triticum aestivum L.) and paddy rice (Oryza sativa L.), as well as the Brassicaceae family, including daikon radish (Raphanus sativus). The in vivo emission of CH3I clearly correlated with HMT/HTMT activity. The emission of CH3I from the sprouting leaves of R. sativus, T. aestivum and O. sativa grown hydroponically increased with increasing concentrations of supplied iodide. A gene encoding an S-adenosylmethionine halide/thiol methyltransferase (HTMT) was cloned from R. sativus and expressed in Escherichia coli as a soluble protein. The recombinant R. sativus HTMT (RsHTMT) was revealed to possess high specificity for iodide (I-), bisulfide ([SH]-), and thiocyanate ([SCN]-) ions. Conclusion The present findings suggest that HMT/HTMT activity is present in several families of higher plants including Poaceae and Brassicaceae, and is involved in the formation of methyl halides. Moreover, it was found that the emission of methyl iodide from plants was affected by the iodide concentration in the cultures. The recombinant RsHTMT demonstrated enzymatic properties similar to those of Brassica oleracea HTMT, especially in terms of its high specificity for iodide, bisulfide, and thiocyanate ions. A survey of biogenic emissions of methyl halides strongly suggests that the HTM/HTMT reaction is the key to understanding the biogenesis of methyl halides and methylated sulfur compounds in nature.

The preparation and purification of [super 13]C-labeled menaquinone (MK), a lipid soluble molecule, is described. The studies helped to identify a new pathway for the biosynthesis of MK in Streptomyces species and other pathogenic bacteria.

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanal acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrate catalyzed by PAR.

Penicillium citrinum was found to catalyze the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity. From the strain, a cDNA clone encoding a novel NADPH-dependent alkyl 4-halo-3-oxobutyrate reductase (KER) was isolated.