Abstract
Despite significant influence of secondary bile acids on human health and disease, limited structural and biochemical information is available for the key gut microbial enzymes catalyzing its synthesis. Herein, we report apo- and cofactor bound crystal structures of BaiA2, a short chain dehydrogenase/reductase from Clostridium scindens VPI 12708 that represent the first protein structure of this pathway. The structures elucidated the basis of cofactor specificity and mechanism of proton relay. A conformational restriction involving Glu42 located in the cofactor binding site seems crucial in determining cofactor specificity. Limited flexibility of Glu42 results in imminent steric and electrostatic hindrance with 2'-phosphate group of NADP(H). Consistent with crystal structures, steady state kinetic characterization performed with both BaiA2 and BaiA1, a close homolog with 92% sequence identity, revealed specificity constant (kcat /KM ) of NADP(+) at least an order of magnitude lower than NAD(+) . Substitution of Glu42 with Ala improved specificity toward NADP(+) by 10-fold compared to wild type. The cofactor bound structure uncovered a novel nicotinamide-hydroxyl ion (NAD(+) -OH(-) ) adduct contraposing previously reported adducts. The OH(-) of the adduct in BaiA2 is distal to C4 atom of nicotinamide and proximal to 2'-hydroxyl group of the ribose moiety. Moreover, it is located at intermediary distances between terminal functional groups of active site residues Tyr157 (2.7 Å) and Lys161 (4.5 Å). Based on these observations, we propose an involvement of NAD(+) -OH(-) adduct in proton relay instead of hydride transfer as noted for previous adducts.
Highlights
Deoxycholic acid (DCA) and lithocholic acid (LCA) are secondary bile acids that are generated in the gut by bacterial transformation of primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), respectively 1
All bile acid substrates are obtained from Steraloids (Newport, RI) except glycocholic acid (GCA) and taurocholic acid (TCA) that are obtained from Sigma Aldrich
Our work has uncovered a novel nicotinamide-OH− adduct, which we propose to be involved in proton relay instead of hydride transfer as noted earlier
Summary
Deoxycholic acid (DCA) and lithocholic acid (LCA) are secondary bile acids that are generated in the gut by bacterial transformation of primary bile acids, cholic acid (CA) and chenodeoxycholic acid (CDCA), respectively 1. The proposed pathway for this transformation by Clostridium scindens (C. scindens) VPI 12708 involves a network of enzymes that catalyze the removal of C7-hydroxyl group from primary bile acids (Fig. 1). Qin et al have found it to be one of the lowest abundant member of the “core microbiome”, present at approximately two orders of magnitude lower than the most copious microbes in human gut 3. Studies implicated secondary bile acids as effector molecules involved in human health influencing various signaling pathways associated with cancer and metabolism 4–6. It can be speculated that variation in population of a less abundant microbial species may influence the level of key metabolites in the human body and thereby human health. The microbial enzymes involved in this synthesis could represent potential drug targets for manipulating secondary bile acid level
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