Consequences of bile salt biotransformations by intestinal bacteria.

Emerging evidence strongly suggest that the human "microbiome" plays an important role in both health and disease. Bile acids function both as detergents molecules promoting nutrient absorption in the intestines and as hormones regulating nutrient metabolism. Bile acids regulate metabolism via activation of specific nuclear receptors (NR) and G-protein coupled receptors (GPCRs). The circulating bile acid pool composition consists of primary bile acids produced from cholesterol in the liver, and secondary bile acids formed by specific gut bacteria. The various biotransformation of bile acids carried out by gut bacteria appear to regulate the structure of the gut microbiome and host physiology. Increased levels of secondary bile acids are associated with specific diseases of the GI system. Elucidating methods to control the gut microbiome and bile acid pool composition in humans may lead to a reduction in some of the major diseases of the liver, gall bladder and colon.

Structure and functional characterization of a bile acid 7α dehydratase BaiE in secondary bile acid synthesis.

Conversion of the primary bile acids cholic acid (CA) and chenodeoxycholic acid (CDCA) to the secondary bile acids deoxycholic acid (DCA) and lithocholic acid (LCA) is performed by a few species of intestinal bacteria in the genus Clostridium through a multistep biochemical pathway that removes a 7α-hydroxyl group. The rate-determining enzyme in this pathway is bile acid 7α-dehydratase (baiE). In this study, crystal structures of apo-BaiE and its putative product-bound [3-oxo-Δ(4,6) -lithocholyl-Coenzyme A (CoA)] complex are reported. BaiE is a trimer with a twisted α + ß barrel fold with similarity to the Nuclear Transport Factor 2 (NTF2) superfamily. Tyr30, Asp35, and His83 form a catalytic triad that is conserved across this family. Site-directed mutagenesis of BaiE from Clostridium scindens VPI 12708 confirm that these residues are essential for catalysis and also the importance of other conserved residues, Tyr54 and Arg146, which are involved in substrate binding and affect catalytic turnover. Steady-state kinetic studies reveal that the BaiE homologs are able to turn over 3-oxo-Δ(4) -bile acid and CoA-conjugated 3-oxo-Δ(4) -bile acid substrates with comparable efficiency questioning the role of CoA-conjugation in the bile acid metabolism pathway.

Structural and functional characterization of BaiA, an enzyme involved in secondary bile acid synthesis in human gut microbe.

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.

A substrate-assisted mechanism of nucleophile activation in a Ser-His-Asp containing C-C bond hydrolase.

The meta-cleavage product (MCP) hydrolases utilize a Ser-His-Asp triad to hydrolyze a carbon-carbon bond. Hydrolysis of the MCP substrate has been proposed to proceed via an enol-to-keto tautomerization followed by a nucleophilic mechanism of catalysis. Ketonization involves an intermediate, ES(red), which possesses a remarkable bathochromically shifted absorption spectrum. We investigated the catalytic mechanism of the MCP hydrolases using DxnB2 from Sphingomonas wittichii RW1. Pre-steady-state kinetic and LC ESI/MS evaluation of the DxnB2-mediated hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid to 2-hydroxy-2,4-pentadienoic acid and benzoate support a nucleophilic mechanism catalysis. In DxnB2, the rate of ES(red) decay and product formation showed a solvent kinetic isotope effect of 2.5, indicating that a proton transfer reaction, assigned here to substrate ketonization, limits the rate of acylation. For a series of substituted MCPs, this rate was linearly dependent on MCP pKa2 (ßnuc ∼ 1). Structural characterization of DxnB2 S105A:MCP complexes revealed that the catalytic histidine is displaced upon substrate-binding. The results provide evidence for enzyme-catalyzed ketonization in which the catalytic His-Asp pair does not play an essential role. The data further suggest that ES(red) represents a dianionic intermediate that acts as a general base to activate the serine nucleophile. This substrate-assisted mechanism of nucleophilic catalysis distinguishes MCP hydrolases from other serine hydrolases.

The lid domain of the MCP hydrolase DxnB2 contributes to the reactivity toward recalcitrant PCB metabolites.

DxnB2 and BphD are meta-cleavage product (MCP) hydrolases that catalyze C-C bond hydrolysis of the biphenyl metabolite 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA). BphD is a bottleneck in the bacterial degradation of polychlorinated biphenyls (PCBs) by the Bph catabolic pathway due in part to inhibition by 3-Cl HOPDAs. By contrast, DxnB2 from Sphingomonas wittichii RW1 catalyzes the hydrolysis of 3-Cl HOPDAs more efficiently. X-ray crystallographic studies of the catalytically inactive S105A variant of DxnB2 complexed with 3-Cl HOPDA revealed a binding mode in which C1 through C6 of the dienoate are coplanar. The chlorine substituent is accommodated by a hydrophobic pocket that is larger than the homologous site in BphDLB400 from Burkholderia xenovorans LB400. The planar binding mode observed in the crystalline complex was consistent with the hyper- and hypsochromically shifted absorption spectra of 3-Cl and 3,9,11-triCl HOPDA, respectively, bound to S105A in solution. Moreover, ES(red), an intermediate possessing a bathochromically shifted spectrum observed in the turnover of HOPDA, was not detected, suggesting that substrate destabilization was rate-limiting in the turnover of these PCB metabolites. Interestingly, electron density for the first α-helix of the lid domain was poorly defined in the dimeric DxnB2 structures, unlike in the tetrameric BphDLB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/ß-hydrolase core domain, as a determinant in the oligomeric state and suggests its involvement in catalysis. Finally, an increased mobility of the DxnB2 lid may contribute to the enzyme's ability to hydrolyze PCB metabolites, highlighting how lid architecture contributes to substrate specificity in α/ß-hydrolases.

The molecular basis for inhibition of BphD, a C-C bond hydrolase involved in polychlorinated biphenyls degradation: large 3-substituents prevent tautomerization.

The microbial degradation of polychlorinated biphenyls (PCBs) by the biphenyl catabolic (Bph) pathway is limited in part by the pathway's fourth enzyme, BphD. BphD catalyzes an unusual carbon-carbon bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA), in which the substrate is subject to histidine-mediated enol-keto tautomerization prior to hydrolysis. Chlorinated HOPDAs such as 3-Cl HOPDA inhibit BphD. Here we report that BphD preferentially hydrolyzed a series of 3-substituted HOPDAs in the order H>F>Cl>Me, suggesting that catalysis is affected by steric, not electronic, determinants. Transient state kinetic studies performed using wild-type BphD and the hydrolysis-defective S112A variant indicated that large 3-substituents inhibited His-265-catalyzed tautomerization by 5 orders of magnitude. Structural analyses of S112A.3-Cl HOPDA and S112A.3,10-diF HOPDA complexes revealed a non-productive binding mode in which the plane defined by the carbon atoms of the dienoate moiety of HOPDA is nearly orthogonal to that of the proposed keto tautomer observed in the S112A.HOPDA complex. Moreover, in the 3-Cl HOPDA complex, the 2-hydroxo group is moved by 3.6 A from its position near the catalytic His-265 to hydrogen bond with Arg-190 and access of His-265 is blocked by the 3-Cl substituent. Nonproductive binding may be stabilized by interactions involving the 3-substituent with non-polar side chains. Solvent molecules have poor access to C6 in the S112A.3-Cl HOPDA structure, more consistent with hydrolysis occurring via an acyl-enzyme than a gem-diol intermediate. These results provide insight into engineering BphD for PCB degradation.

The tautomeric half-reaction of BphD, a C-C bond hydrolase. Kinetic and structural evidence supporting a key role for histidine 265 of the catalytic triad.

BphD of Burkholderia xenovorans LB400 catalyzes an unusual C-C bond hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to afford benzoic acid and 2-hydroxy-2,4-pentadienoic acid (HPD). An enol-keto tautomerization has been proposed to precede hydrolysis via a gem-diol intermediate. The role of the canonical catalytic triad (Ser-112, His-265, Asp-237) in mediating these two half-reactions remains unclear. We previously reported that the BphD-catalyzed hydrolysis of HOPDA (lambda(max) is 434 nm for the free enolate) proceeds via an unidentified intermediate with a red-shifted absorption spectrum (lambda(max) is 492 nm) (Horsman, G. P., Ke, J., Dai, S., Seah, S. Y. K., Bolin, J. T., and Eltis, L. D. (2006) Biochemistry 45, 11071-11086). Here we demonstrate that the S112A variant generates and traps a similar intermediate (lambda(max) is 506 nm) with a similar rate, 1/tau approximately 500 s(-1). The crystal structure of the S112A:HOPDA complex at 1.8-A resolution identified this intermediate as the keto tautomer, (E)-2,6-dioxo-6-phenyl-hex-3-enoate. This keto tautomer did not accumulate in either the H265A or the S112A/H265A double variants, indicating that His-265 catalyzes tautomerization. Consistent with this role, the wild type and S112A enzymes catalyzed tautomerization of the product HPD, whereas H265A variants did not. This study thus identifies a keto intermediate, and demonstrates that the catalytic triad histidine catalyzes the tautomerization half-reaction, expanding the role of this residue from its purely hydrolytic function in other serine hydrolases. Finally, the S112A:HOPDA crystal structure is more consistent with hydrolysis occurring via an acyl-enzyme intermediate than a gem-diol intermediate as solvent molecules have poor access to C6, and the closest ordered water is 7 A away.