1. Biochemical Characterization of the Two-Component Flavin-Dependent Monooxygenase Involved in Valanimycin Biosynthesis
Hao Li, Benedicta Forson, Meital Eckshtain-Levi, Hannah Valentino, Julia S Martín Del Campo, John J Tanner, Pablo Sobrado Biochemistry. 2021 Jan 12;60(1):31-40. doi: 10.1021/acs.biochem.0c00679. Epub 2020 Dec 22.
The flavin reductase (FRED) and isobutylamine N-hydroxylase (IBAH) from Streptomyces viridifaciens constitute a two-component, flavin-dependent monooxygenase system that catalyzes the first step in valanimycin biosynthesis. FRED is an oxidoreductase that provides the reduced flavin to IBAH, which then catalyzes the hydroxylation of isobutylamine (IBA) to isobutylhydroxylamine (IBHA). In this work, we used several complementary methods to investigate FAD binding, steady-state and rapid reaction kinetics, and enzyme-enzyme interactions in the FRED:IBAH system. The affinity of FRED for FADox is higher than its affinity for FADred, consistent with its function as a flavin reductase. Conversely, IBAH binds FADred more tightly than FADox, consistent with its role as a monooxygenase. FRED exhibits a strong preference (28-fold) for NADPH over NADH as the electron source for FAD reduction. Isothermal titration calorimetry was used to study the association of FRED and IBAH. In the presence of FAD, either oxidized or reduced, FRED and IBAH associate with a dissociation constant of 7-8 μM. No interaction was observed in the absence of FAD. These results are consistent with the formation of a protein-protein complex for direct transfer of reduced flavin from the reductase to the monooxygenase in this two-component system.
2. Identification, characterization, and bioconversion of a new intermediate in valanimycin biosynthesis
Ram P Garg, Lawrence B Alemany, Sean Moran, Ronald J Parry J Am Chem Soc. 2009 Jul 22;131(28):9608-9. doi: 10.1021/ja901243p.
The antibiotic valanimycin is a naturally occurring azoxy compound isolated from Streptomyces viridifaciens. Detailed investigations have shown that valanimycin is derived from L-valine and L-serine via the intermediacy of O-(L-seryl)isobutylhydroxylamine. Sequence analysis of the valanimycin biosynthetic genes provides relatively few clues concerning the nature of the later stages of the pathway. Two exceptions are provided by the vlmJ and vlmK genes. The translation product of vlmJ exhibits similarity to diacylglycerol kinases, while the translation product of vlmK exhibits a weak similarity to the MmgE/PrpD superfamily of proteins. This superfamily includes 2-methylcitrate dehydratase. This communication describes the isolation and structure elucidation of valanimycin hydrate from vlmJ and vlmK mutants of S. viridifaciens. Additional studies have shown that the conversion of valanimycin hydrate into valanimycin by S. viridifaciens requires both the vlmJ and vlmK genes and that VlmJ catalyzes the ATP-dependent phosphorylation of the hydroxyl group of valanimycin hydrate prior to VlmK-catalyzed dehydration.
3. Regulation of valanimycin biosynthesis in Streptomyces viridifaciens: characterization of VlmI as a Streptomyces antibiotic regulatory protein (SARP)
Ram P Garg, Ronald J Parry Microbiology (Reading). 2010 Feb;156(Pt 2):472-483. doi: 10.1099/mic.0.033167-0. Epub 2009 Nov 5.
Streptomyces antibiotic regulatory proteins (SARPs) have been shown to activate transcription by binding to a tandemly arrayed set of heptameric direct repeats located around the -35 region of their cognate promoters. Experimental evidence is presented here showing that vlmI is a regulatory gene in the valanimycin biosynthetic gene cluster of Streptomyces viridifaciens and encodes a protein belonging to the SARP family. The organization of the valanimycin biosynthetic gene cluster suggests that the valanimycin biosynthetic genes are located on three potential transcripts, vlmHORBCD, vlmJKL and vlmA. Disruption of vlmI abolished valanimycin biosynthesis. Western blot analyses showed that VlmR and VlmA are absent from the vlmI mutant and that the production of VlmK is severely diminished. These results demonstrate that the expression of these genes from the three potential transcripts is under the positive control of VlmI. The vlmA-vlmH and vlmI-vlmJ intergenic regions both exhibit a pattern of heptameric direct repeats. Gel shift assays with VlmI overproduced in Escherichia coli as a C-terminal FLAG-tagged protein clearly demonstrated that VlmI binds to DNA fragments from both regions that contain these heptameric repeats. When a high-copy-number vlmI expression plasmid was introduced into Streptomyces coelicolor M512, which contains mutations in the undecylprodigiosin and actinorhodin activators redD and actII-orf4, undecylprodigiosin production was restored, showing that vlmI can complement a redD mutation. Introduction of the same vlmI expression plasmid into an S. viridifaciens vlmI mutant restored valanimycin production to wild-type levels.