It is proposed to investigate the biosynthesis of three novel microbial metabolites. These are sparsomycin, a sulfur containing antitumor agent from Streptomyces sparsogenes, furanomycin, a dihydrofuran antibiotic from Streptomyces threomyceticus, and valanimycin, an azoxy compound and antitumor agent from Streptomyces viridifaciens. Investigations in the previous grant period have established the primary metabolic precursors of sparsomycin, and future work will focus on elucidation of the unusual biochemistry that is likely to be involved in the formation of the uracil and monooxodithioacetal moieties present in the antibiotic. The mechanism of formation of the dithioacetal moiety may prove to be related to the biosynthesis of the enzyme cofactors biotin and lipoic acid. Investigations of furanomycin carried out several years ago revealed that the antibiotic is a polyketide derived from a propionate starter unit and two acetate units. Recent investigations of furanomycin have shown that the ether moiety of the antibiotic is derived from molecular oxygen and that oxygen is introduced at C-2 of propionate with inversion of configuration and loss of the 2 pro-R hydrogen atom. These results suggest that the biosynthesis of furanomycin may be related to the biosynthetic pathway proposed for-the commercially important polyether antibiotics and proceed via the intermediacy of trans, cis - 2,3,5,6-heptadienoic acid. Future investigations of furanomycin biosynthesis will evaluate this possibility. Preliminary investigations of the biosynthesis of the valanimycin by Yamato et al. indicate that the antibiotic is probably derived from valine and alanine. These findings will be verified by more rigorous experiments, the origin of the two nitrogen atoms in the azoxy linkage will be determined, and investigations will then be carried out to elucidate the mechanism of formation of the N-N bond of the azoxy group. The information gained from these latter studies should provide insight into the mechanism of N-N bond formation in a wide variety of biologically active natural products. The methods utilized to study each of the three microbial metabolites will be: 1) the administration of specifically labeled precursors in singly or doubly-labeled form to the appropriate organisms followed by location of the labels in the natural products by NMR spectrometry, and 2) the use of cell-free extracts.

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National Institute of General Medical Sciences (NIGMS)
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Bio-Organic and Natural Products Chemistry Study Section (BNP)
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Rice University
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Garg, Ram P; Parry, Ronald J (2010) Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP). Microbiology 156:472-83
Garg, Ram P; Alemany, Lawrence B; Moran, Sean et al. (2009) Identification, characterization, and bioconversion of a new intermediate in valanimycin biosynthesis. J Am Chem Soc 131:9608-9
Garg, Ram P; Qian, Xuelei L; Alemany, Lawrence B et al. (2008) Investigations of valanimycin biosynthesis: elucidation of the role of seryl-tRNA. Proc Natl Acad Sci U S A 105:6543-7
Zhang, Xiujun; Alemany, Lawrence B; Fiedler, Hans-Peter et al. (2008) Biosynthetic investigations of lactonamycin and lactonamycin z: cloning of the biosynthetic gene clusters and discovery of an unusual starter unit. Antimicrob Agents Chemother 52:574-85
Garg, Ram P; Gonzalez, Jose M; Parry, Ronald J (2006) Biochemical characterization of VlmL, a Seryl-tRNA synthetase encoded by the valanimycin biosynthetic gene cluster. J Biol Chem 281:26785-91
Tao, Tao; Alemany, Lawrence B; Parry, Ronald J (2003) Valanimycin biosynthesis: investigations of the mechanism of isobutylhydroxylamine incorporation. Org Lett 5:1213-5
Garg, Ram P; Ma, Yunqing; Hoyt, Jeffrey C et al. (2002) Molecular characterization and analysis of the biosynthetic gene cluster for the azoxy antibiotic valanimycin. Mol Microbiol 46:505-17
Skae, P; Parry, R J (2001) Determination of the stereochemistry of hydride transfer from NADPH to FAD catalyzed by VlmR, a flavin reductase from the valanimycin biosynthetic pathway. Org Lett 3:1117-9
Ma, Y; Patel, J; Parry, R J (2000) A novel valanimycin-resistance determinant (vlmF) from Streptomyces viridifaciens MG456-hF10. Microbiology 146 ( Pt 2):345-52
Parry, R J; Li, W (1997) Purification and characterization of isobutylamine N-hydroxylase from the valanimycin producer Streptomyces viridifaciens MG456-hF10. Arch Biochem Biophys 339:47-54

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