Oxidative Catalysis in the Non-heme Iron Oxygenases “VioC” and “OrfP”

By: Lamia Tabassum Badhon

Advisor: Dr. Nicholas R. Silvaggi

Abstract

The non-heme Fe(II)-dependent oxygenases catalyze a wide range of enzymatic reactions in important cellular process ranging from DNA repair and cell signaling to biodegradation of xenobiotics and synthesis of secondary metabolites. They are particularly well represented in natural product biosynthetic clusters. For example, the biosynthetic pathways for two bioactive natural product compounds, viomycin and streptothricin, both involve interesting non-heme Fe(II)-dependent oxygenases. Viomycin is an antibiotic with potent activity against pathogenic bacteria including MRSA, and contains the non-proteinogenic amino acid capreomycidine. The synthesis of capreomycidine by the producing organism, Streptomyces vinaceus involves the action of two enzymes, VioC and VioD on the amino acid L-arginine. VioC is an arginine beta hydroxylase that stereo specifically hydroxylates arginine at the beta-carbon. The second enzyme, VioD, is a pyridoxal 5’-phosphate (PLP)-dependent enzyme that catalyzes an intramolecular transamination reaction to form a new C-N bond between the beta-carbon and one of the terminal nitrogens of the guanidinium group to form the Pyrimidine ring of the capreomycidine side chain. The aminoglycoside antibiotic streptothricin contains an unusual bicyclic component called streptolidine. Interestingly, streptolidine, like capreomycidine, is produced from L-arginine by two enzymes, OrfP and OrfR in Streptomyces lavendulae. OrfP is an ortholog of VioC (43% sequence identity) and OrfR is an ortholog of VioD (40% sequence identity). Despite having a very similar structure to VioC, and working on the same substrate, OrfP catalyzes a dihydroxylation reaction, installing hydroxyl groups at both the beta and gamma positions. The dihydroxylated arginine product is then cyclized by VioD to give hydroxy-capreomycidine, which is subsequently rearranged to streptolidine. Interestingly, VioC is flexible to accept a variety of L-arginine analogs as substrate. However, OrfP is limited to catalyze double hydroxylation of arginine only. The active site contains a crucial aspartate residue (D157) that is required the dihydroxylation reaction. Stereospecific hydroxylations are still challenging synthesis chemistry tasks. Enzymes like VioC and orfP may be useful tools for a possible semi-synthetic approach to these antibiotics.