Structural basis for the erythro‐stereospecificity of the l‐arginine oxygenase VioC in viomycin biosynthesis

Abstract

The nonheme iron oxygenase VioC from Streptomyces vinaceus catalyzes Fe(II)‐dependent and α‐ketoglutarate‐dependent Cβ‐hydroxylation of l‐arginine during the biosynthesis of the tuberactinomycin antibiotic viomycin. Crystal structures of VioC were determined in complexes with the cofactor Fe(II), the substrate l‐arginine, the product (2S,3S)‐hydroxyarginine and the coproduct succinate at 1.1–1.3 Å resolution. The overall structure reveals a β‐helix core fold with two additional helical subdomains that are common to nonheme iron oxygenases of the clavaminic acid synthase‐like superfamily. In contrast to other clavaminic acid synthase‐like oxygenases, which catalyze the formation of threo diastereomers, VioC produces the erythro diastereomer of Cβ‐hydroxylated l‐arginine. This unexpected stereospecificity is caused by conformational control of the bound substrate, which enforces a gauche(–) conformer for χ1 instead of the trans conformers observed for the asparagine oxygenase AsnO and other members of the clavaminic acid synthase‐like superfamily. Additionally, the substrate specificity of VioC was investigated. The side chain of the l‐arginine substrate projects outwards from the active site by undergoing interactions mainly with the C‐terminal helical subdomain. Accordingly, VioC exerts broadened substrate specificity by accepting the analogs l‐homoarginine and l‐canavanine for Cβ‐hydroxylation.

Visualizing the Reaction Cycle in an Iron(II)- and 2-(Oxo)-glutarate-Dependent Hydroxylase

“…21 In the study elucidating its role in the viomycin pathway, multiple structures of VioC complexes were solved to near-atomic resolution by x-ray crystallography. 22 However, the x-ray datasets provide only limited insight into the reaction mechanism because the complexes characterized are not on the catalytic pathway. In one, 2OG was replaced by tartrate; in a second, the 3-hydroxy L-Arg product was bound along with succinate; and in a third, the iron cofactor was absent (fig.…”

Crystal Structure of the Ectoine Hydroxylase, a Snapshot of the Active Site

“…3), this loop corresponds to residues 195-211 in the VsEctD ectoine hydroxylase, and it is also disordered in both crystal structures of this protein (22,44). Notably, in the ligand-free crystal structures of the phytanoyl-CoA hydroxylases PhyH (55), the asparagine hydroxylase AsnO from S. coelicolor (60), the L-arginine oxygenase VioC from Streptomyces vinaceus (61), and the taurine dioxygenases from E. coli and Pseudomonas putida (62), a similar loop is disordered as well and became only visible in crystal structures with bound substrates. It is thought that this mobile loop functions as a lid that shields the enzyme reaction chamber during catalysis from the solvent.…”

Crystal Structure of a Novel N-Substituted L-Amino Acid Dioxygenase from Burkholderia ambifaria AMMD

“…Although a few Fe(II)/α-KG-dependent dioxygenases are known to hydroxylate free amino acids [13], [14], their substrate specificities are restricted to hydrophilic amino acids such as L-arginine and L-asparagine. Therefore, the mechanism for the substrate specificity of SadA remains poorly understood.…”