인문학
사회과학
자연과학
공학
의약학
농수해양학
예술체육학
복합학
DBpia AI
AI 에이전트
AI 아이디어
AI 리더
AI 뷰어
Citeasy
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초록·키워드
Short chain flavin reductases (such as AbeF, BorF, and PheA2) use NADH to reduce FAD to FADH2 to provide reduced diffusible flavin for halogenases and monooxygenases in bacterial two-component systems. Despite sharing a common fold and significant sequence homology (AbeF/BorF identity = 30.57% while it is between AbeF / PheA2 identity = 26.14%), they have been shown to carry out the same reaction using three different kinetic mechanisms: ordered sequential bi bi (BorF), random sequential bi bi (AbeF), and ping-pong bi bi (PheA2). The underlying explanation for these different kinetic mechanisms is not obvious from structural data (crystal structures of BorF and PheA2 and AlphaFold2 predicted structure for AbeF). Site directed mutagenesis was applied to investigate the differences in kinetic mechanisms despite the high structural similarity in active sites. Structural analysis and multiple sequence alignments were used to identify positions that differ between BorF, AbeF, and PheA2 and were predicted to influence substrate binding. Point mutations were generated in AbeF and BorF and wild type and mutant proteins were expressed and purified from E. coli. Fluorescence quenching experiments were used to analyze flavin binding affinity and a spectrophotometric NADH oxidation assay was employed to analyze the flavin reductase activity. Mutants were compared with the wild types for the possible changes in their kinetic parameters and substrate binding. In the active site, PheA2 has Ser49 while AbeF and BorF have Cys49 and Cys79 respectively in the exact location. Kinetic assay results revealed that C49S and C79S mutants had higher Km values compared to the wild types. The findings of this study provide an initial understanding of the structure dependent mechanistic diversity in flavin reductases. This work was supported by National Institutes of Health and The University of Toledo
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