7YHE

Crystal structure of the triple mutant CmnC-L136Q,S138G,D249Y in complex with alpha-KG

Summary for 7YHE

• Entry DOI: 10.2210/pdb7yhe/pdb
• Descriptor: CmnC, 2-OXOGLUTARIC ACID, TRIETHYLENE GLYCOL, ... (5 entities in total)
• Functional Keywords: hydroxylase, oxygenase, oxidoreductase
• Biological source: Saccharothrix mutabilis subsp. capreolus
• Total number of polymer chains: 2
• Total formula weight: 78739.34

Authors

Huang, S.J.,Hsiao, Y.H.,Lin, E.C.,Hsiao, P.Y.,Chang, C.Y. (deposition date: 2022-07-13, release date: 2023-07-19, Last modification date: 2023-11-29)

Primary citation

Hsiao, Y.H.,Huang, S.J.,Lin, E.C.,Hsiao, P.Y.,Toh, S.I.,Chen, I.H.,Xu, Z.,Lin, Y.P.,Liu, H.J.,Chang, C.Y.
Crystal structure of the alpha-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.
Front Chem, 10:1001311-1001311, 2022

PubMed Abstract: CmnC is an α-ketoglutarate (α-KG)-dependent non-heme iron oxygenase involved in the formation of the l-capreomycidine (l-Cap) moiety in capreomycin (CMN) biosynthesis. CmnC and its homologues, VioC in viomycin (VIO) biosynthesis and OrfP in streptothricin (STT) biosynthesis, catalyze hydroxylation of l-Arg to form β-hydroxy l-Arg (CmnC and VioC) or β,γ-dihydroxy l-Arg (OrfP). In this study, a combination of biochemical characterization and structural determination was performed to understand the substrate binding environment and substrate specificity of CmnC. Interestingly, despite having a high conservation of the substrate binding environment among CmnC, VioC, and OrfP, only OrfP can hydroxylate the substrate enantiomer d-Arg. Superposition of the structures of CmnC, VioC, and OrfP revealed a similar folds and overall structures. The active site residues of CmnC, VioC, and OrfP are almost conserved; however Leu136, Ser138, and Asp249 around the substrate binding pocket in CmnC are replaced by Gln, Gly, and Tyr in OrfP, respectively. These residues may play important roles for the substrate binding. The mutagenesis analysis revealed that the triple mutant CmnC switches the substrate stereoselectivity from l-Arg to d-Arg with ∼6% relative activity. The crystal structure of CmnC in complex with d-Arg revealed that the substrate loses partial interactions and adopts a different orientation in the binding site. This study provides insights into the enzyme engineering to α-KG non-heme iron oxygenases for adjustment to the substrate stereoselectivity and development of biocatalysts.

PubMed: 36176888
DOI: 10.3389/fchem.2022.1001311

Experimental method

X-RAY DIFFRACTION (1.67 Å)