Structural Features in the KshA Terminal Oxygenase Protein That Determine Substrate Preference of 3-Ketosteroid 9 -Hydroxylase Enzymes

Abstract

Rieske nonheme monooxygenase 3-ketosteroid 9␣-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 ␤ sheet that is part of the so-called helix-grip fold of the predicted KshA substrate binding pocket. Exchange of the ␤ 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 ␤-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.

Substrate Specificities and Conformational Flexibility of 3-Ketosteroid 9α-Hydroxylases

“…The steady-state kinetic studies corroborate and extend previous studies of KshA1 that has been implicated in bile acid catabolism based on substrate preference (24), gene deletion studies, and phylogenetic analysis (39). These studies further establish KshA5's broad substrate specificity for short side chain steroids and, unexpectedly, its strong substrate inhibition.…”

“…The role of the mouth loop had been described in studies of chimeras of KshA1 and KshA5 (39). Thus, the broad substrate preference of KshA5 was eliminated by substituting its mouth loop with that of KshA1.…”

“…Moreover, displacement of these helices was not observed in KshA1 or KshA Mtb , and it remains to be seen whether this conformational change is unique to KshA5. Nevertheless, the high functionality of the KshA1/KshA5 chimeras (39) suggests that KshAs undergo similar conformational changes. More generally, our data suggest that conformational changes in the RO active site during the catalytic cycle may be larger than has been recognized.…”

“…By identifying the substrate-binding residues of KshAs and identifying determinants of specificity in KshA, this study provides a framework for engineering an important class of monooxygenases for biocatalytic applications (2,39). Further studies should also establish the occurrence and significance of helix displacement in ROs.…”

Systems-level modeling of mycobacterial metabolism for the identification of new (multi-)drug targets

“…For example, the cholesterol degradation pathway in Mtb [8] contains a large number of enzymes, many of them essential for cholesterol degradation and thus possible drug targets. However, stable toxic intermediates such as cholest-4-en-3-one and catechol derivatives accumulate if the enzymes HsaC, KshA, Cyp125 and Cyp142 are non-functional [59,60]. The accumulation of such intermediates can be fatal to Mtb, increasing the potential of these enzymes as drug targets.…”