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

Abstract

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.Over the past 50 years, many antibacterial, antifungal, and anticancer agents have been identified from secondary metabolites isolated from soil microorganisms. In particular, actinomycetes produce a wide variety of bioactive natural products. Based on phylogenetic analysis from 16S rRNA gene sequences (18), Lechevalieria aerocolonigenes ATCC 39243 is a member of the family Pseudonocardiaceae and is the producer of the indolocarbazole antitumor antibiotic rebeccamycin (Fig.

Enzymatic Generation of the Chromopyrrolic Acid Scaffold of Rebeccamycin by the Tandem Action of RebO and RebD

“…It has recently been established that RebO is a flavin-dependent L-tryptophan oxidase, akin to the well-known L-amino acid oxidases, generating the imine form of indole 3-pyruvate, with concomitant two-electron reduction of O 2 to H 2 O 2 (14). 7Cl-Trp is the preferred substrate for RebO (14), which generates the imine form of 7Cl-IPA as its immediate oxidation product.…”

“…We were interested in determining the activity of RebD in forming CPA (4), and in ascertaining the substrate requirements of this enzyme. Because of the limited availability of 7Cl-Trp, the natural substrate for the RebO/RebD system (14), much of this study on the tandem action of RebO and RebD was performed using L-Trp as the substrate.…”

SO-LAAO, a Novell-Amino Acid Oxidase That EnablesStreptococcus oligofermentansTo OutcompeteStreptococcus mutansby Generating H2O2from Peptone

“…By an integrated approach using physiology, biochemistry, and genetics, an ORF protein assigned as SO-LAAO, which is the homologue of flavoprotein SSA-0323 in S. sanguinis, was verified to possess LAAO activity and to be responsible for H 2 O 2 production from L-amino acids in S. oligofermentans. LAAOs, which catalyze the oxidative deamination of amino acids to yield ammonia, hydrogen peroxide, and ketoacids with oxygen consumed (9), have been widely detected in snake and insect venoms (1,30) and in some fungi, algae, and bacteria (17,25,38). According to the substrate spectra, LAAOs can be divided into two categories, one with a broad spectrum of substrates, like the LAAO of Rhodococcus opacus DSM 43250, which catalyzes not only almost all the 20 L-amino acids but also some derivatives (9), and another with a restricted substrate spectrum, like lysine oxidase of Marinomonas mediterranea, which uses lysine exclusively (21).…”

Crystal structures and catalytic mechanism of cytochrome P450 StaP that produces the indolocarbazole skeleton

“…Biochemical characterizations of the biosyntheses of staurosporine or rebeccamycin have been reported for several enzymes (7)(8)(9)(10). However, until now, the only crystal structure available was that of RebH which provides 7-chlorotryptophan, a precursor of rebeccamycin (11).…”