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\begin{document}

\title{Identification two key residues at the intersection of subdomains of a
thioether monooxygenase for improving its sulfoxidation performance}



\author[1]{Shi Miao Ren}%
\author[1]{Feng Liu}%
\author[1]{Yin-Qi Wu}%
\author[1]{Qi Chen}%
\author[1]{Zhi-Jun Zhang}%
\author[1]{Hui-Lei Yu}%
\author[2]{Jian-He Xu}%
\affil[1]{East China University of Science and Technology}%
\affil[2]{East China University of Science \& Technology}%


\vspace{-1em}



  
  \date{June 14, 2024}


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\begin{abstract}
ABSTRACT: AcCHMO, a cyclohexanone monooxygenase from Acinetobacter
calcoaceticus, is a typical Type I Baeyer-Villiger monooxygenase.
AcCHMOM6 is a mutant of AcCHMO we obtained previously that could oxidase
the omeprazole sulfide to chiral sulfoxide drug esomeprazole. Based on
the structural characteristics of AcCHMO, focused mutagenesis strategy
was adopted at the intersections of FAD binding domain, NADPH binding
domain and \selectlanguage{greek}α-\selectlanguage{english}helical domain. By the focused mutagenesis and subsequent
global evolution, two key residues (55-Leu and 497-Pro) at the
intersection of subdomains were identified, of which the L55Y
mutagenesis accelerated the H- transfer from NADPH to FAD, while the
P497S mutagenesis widened the bottleneck radius of the substrate tunnel
and alleviated the substrate inhibition remarkably. By combination of
the two mutagenesis, AcCHMOM7 (L55Y/P497S) increased its specific
activity from 18.5 U/g to 108 U/g, and its Ki of the substrate sulfide
was increased from 34 \selectlanguage{greek}μ\selectlanguage{english}M to 265 \selectlanguage{greek}μ\selectlanguage{english}M. These results indicated that the
catalytic performance can be elevated by modification of the sensitive
sites in the intersection of subdomains of AcCHMO, which also provided
some insights for the engineering of other type I BVMOs or other
multi-subdomain proteins.%
\end{abstract}%



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\textbf{Hosted file}

\verb`Manuscript-Biotechnol. Bioeng.-Shi Miao Ren.doc`  available at \url{https://authorea.com/users/344765/articles/471166-identification-two-key-residues-at-the-intersection-of-subdomains-of-a-thioether-monooxygenase-for-improving-its-sulfoxidation-performance}

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