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Journal of Bacteriology, March 2005, p. 2084-2092, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.2084-2092.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Molecular Analysis of the Rebeccamycin L-Amino Acid Oxidase from Lechevalieria aerocolonigenes ATCC 39243

Tomoyasu Nishizawa,1 Courtney C. Aldrich,1 and David H. Sherman1,2*

Life Science Institute and Department of Medicinal Chemistry, College of Pharmacy,1 Departments of Chemistry and Microbiology and Immunology, Medical School, University of Michigan, Ann Arbor, Michigan2

Received 21 May 2004/ Accepted 14 December 2004

Rebeccamycin, a member of the tryptophan-derived indolocarbazole family, is produced by Lechevalieria aerocolonigenes ATCC 39243. The biosynthetic pathway that specifies biosynthesis of this important metabolite is comprised of 11 genes spanning 18 kb of DNA. A presumed early enzyme involved in elaboration of the rebeccamycin aglycone is encoded by rebO, located at the left-hand region of the reb gene cluster. The deduced protein product, RebO (51.9 kDa), is an L-amino acid oxidase (L-AAO) that has 27% identity to an L-AAO from Scomber japonicus (animal, mackerel) and is a member of the family of FAD-dependent oxidase enzymes. In order to study the biochemical properties of this key enzyme, the rebO gene was overexpressed and purified from Escherichia coli. Biochemical characterization showed that RebO is dimeric, with a molecular mass of approximately 101 kDa. Further analysis revealed that the enzyme contains a noncovalently bound FAD cofactor and is reoxidized at the expense of molecular oxygen by producing one molecule of hydrogen peroxide. Based on kinetic studies, RebO shows significant preference for 7-chloro-L-tryptophan, suggesting its likely role as the natural early pathway substrate. Furthermore, the native RebO enzyme has evident, albeit limited, flexibility as shown by bioconversion studies with unnatural substrates. This work provides the first analysis of a structural enzyme involved in construction of this important class of indolocarbazole natural products.

* Corresponding author. Mailing address: Life Science Institute, University of Michigan, 210 Washtenaw Ave., Ann Arbor, MI 48109-2216. Phone: (734) 615-9907. Fax: (734) 615-3641. E-mail: davidhs{at}umich.edu.

Journal of Bacteriology, March 2005, p. 2084-2092, Vol. 187, No. 6
0021-9193/05/$08.00+0     doi:10.1128/JB.187.6.2084-2092.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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