Structural features in the KshA terminal oxygenase protein that determine substrate preference of 3-ketosteroid 9α-hydroxylase enzymes.
Rieske nonheme monooxygenase 3-ketosteroid 9α-hydroxylase (KSH) enzymes play a central role in bacterial steroid catabolism. KSH is a two-component iron-sulfur-containing enzyme, with KshA representing the terminal oxygenase component and KshB the reductase component. We previously reported that the KshA1 and KshA5 homologues of Rhodococcus rhodochrous DSM43269 have clearly different substrate preferences. KshA protein sequence alignments and three-dimensional crystal structure information for KshA(H37Rv) of Mycobacterium tuberculosis H37Rv served to identify a variable region of 58 amino acids organized in a β sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the β sheets between KshA1 and KshA5 resulted in active chimeric enzymes with substrate preferences clearly resembling those of the donor enzymes. Exchange of smaller parts of the KshA1 and KshA5 β-sheet regions revealed that a highly variable loop region located at the entrance of the active site strongly contributes to KSH substrate preference. This loop region may be subject to conformational changes, thereby affecting binding of different substrates in the active site. This study provides novel insights into KshA structure-function relationships and shows that KSH monooxygenase enzymes are amenable to protein engineering for the development of biocatalysts with improved substrate specificities.
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.
Mirjan
M
Petrusma
Lubbert
L
Dijkhuizen
Robert
R
van der Geize
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D011487:Protein Conformation; D011994:Recombinant Proteins; D012240:Rhodococcus / Q000201:enzymology; D013379:Substrate Specificity
D001426:Bacterial Proteins; D011994:Recombinant Proteins; C540657:3-ketosteroid 9alpha-hydroxylase, Mycobacterium tuberculosis; D006899:Mixed Function Oxygenases