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Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.

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^{L136Q,S138G,D249Y} switches the substrate stereoselectivity from l-Arg to d-Arg with ∼6% relative activity. The crystal structure of CmnC^{L136Q,S138G,D249Y} 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.

SUBMITTER: Hsiao YH

PROVIDER: S-EPMC9513391 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.

Hsiao Yu-Hsuan YH Huang Szu-Jo SJ Lin En-Chi EC Hsiao Po-Yun PY Toh Shu-Ing SI Chen I-Hsuan IH Xu Zhengren Z Lin Yu-Pei YP Liu Hsueh-Ju HJ Chang Chin-Yuan CY

Frontiers in chemistry 20220913

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 ...[more]

PMID: 36176888

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Project description:The ubiquitous ATP synthase uses an electrochemical gradient to synthesize cellular energy in the form of ATP. The production of this electrochemical gradient relies on liposoluble proton carriers like ubiquinone (UQ), which is used in the respiratory chains of eukaryotes and proteobacteria. The biosynthesis of UQ requires three hydroxylation reactions on contiguous positions of an aromatic ring. In Escherichia coli, each of three UQ flavin monooxygenases (FMOs), called UbiF, UbiH, and UbiI, modifies a single position of the aromatic ring. This pattern of three hydroxylation reactions/three proteins has been accepted as a paradigm in UQ biology. Using a phylogenetic analysis, we found that UbiF, UbiH, and UbiI are detected only in a small fraction of proteobacteria, and we identified two new types of UQ FMOs: UbiM, which is distributed in members of the alpha, beta, and gamma classes of proteobacteria, and UbiL, which is restricted to members of the alphaproteobacteria. Remarkably, the ubiL and ubiM genes were found in genomes with fewer than three UQ hydroxylase-encoding genes. We demonstrated, using biochemical approaches, that UbiL from Rhodospirillum rubrum and UbiM from Neisseria meningitidis hydroxylate, respectively, two and three positions of the aromatic ring during UQ biosynthesis. We conclude that bacteria have evolved a large repertoire of hydroxylase combinations for UQ biosynthesis, including pathways with either three specialist enzymes or pathways with one or two generalist enzymes of broader regioselectivity. The emergence of the latter is potentially related to genome reduction events. IMPORTANCE UQ, a key molecule for cellular bioenergetics that is conserved from proteobacteria to humans, appeared in an ancestral proteobacterium more than 2 billion years ago. UQ biosynthesis has been studied only in a few model organisms, and thus, the diversity of UQ biosynthesis pathways is largely unknown. In the work reported here, we conducted a phylogenomic analysis of hydroxylases involved in UQ biosynthesis. Our results support the existence of at least two UQ hydroxylases in the proteobacterial ancestor, and yet, we show that their number varies from one to four in extant proteobacterial species. Our biochemical experiments demonstrated that bacteria containing only one or two UQ hydroxylases have developed generalist enzymes that are able to catalyze several steps of UQ biosynthesis. Our study documents a rare case where evolution favored the broadening of an enzyme's regioselectivity, which resulted in gene loss in several proteobacterial species with small genomes.

Dataset Information

Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.

Publications

Crystal structure of the α-ketoglutarate-dependent non-heme iron oxygenase CmnC in capreomycin biosynthesis and its engineering to catalyze hydroxylation of the substrate enantiomer.

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