Shy is a proteobacterial steroid hydratase which catalyzes steroid side chain degradation without requiring a catalytically inert partner domain.

Shy (side chain hydratase) and Sal (side chain aldolase), are involved in successive reactions in the pathway of bile acid side chain catabolism in Proteobacteria. Untagged Shy copurified with His-tagged Sal indicating that the two enzymes form a complex. Shy contains a MaoC and a DUF35 domain. When coexpressed with Sal, the DUF35 domain but not the MaoC domain of Shy was observed to copurify with Sal, indicating Sal interacts with Shy through its DUF35 domain. The MaoC domain of Shy (ShyMaoC) remained catalytically viable and could hydrate cholyl-enoyl-CoA with similar catalytic efficiency as in the Shy-Sal complex. Sal expressed with the DUF35 domain of Shy (Sal-ShyDUF35) was similarly competent for the retro-aldol cleavage of cholyl-3-OH-CoA. ShyMaoC showed a preference for C5 side chain bile acid substrates, exhibiting low activity toward C3 side chain substrates. The ShyMaoC structure was determined by X-ray crystallography, showing a hot dog fold with a short central helix surrounded by a twisted antiparallel ß-sheet. Modeling and mutagenesis studies suggest that the bile acid substrate occupies the large open cleft formed by the truncated central helix and repositioning of the active site housing. ShyMaoC therefore contains two substrate binding sites per homodimer, making it distinct from previously characterized MaoC steroid hydratases that are (pseudo) heterodimers with one substrate binding site per dimer. The characterization of Shy provides insight into how MaoC family hydratases have adapted to accommodate large polycyclic substrates that can facilitate future engineering of these enzymes to produce novel steroid pharmaceuticals.

Structure-function characterization of an aldo-keto reductase involved in detoxification of the mycotoxin, deoxynivalenol.

Deoxynivalenol (DON) is a mycotoxin, produced by filamentous fungi such as Fusarium graminearum, that causes significant yield losses of cereal grain crops worldwide. One of the most promising methods to detoxify this mycotoxin involves its enzymatic epimerization to 3-epi-DON. DepB plays a critical role in this process by reducing 3-keto-DON, an intermediate in the epimerization process, to 3-epi-DON. DepBRleg from Rhizobium leguminosarum is a member of the new aldo-keto reductase family, AKR18, and it has the unusual ability to utilize both NADH and NADPH as coenzymes, albeit with a 40-fold higher catalytic efficiency with NADPH compared to NADH. Structural analysis of DepBRleg revealed the putative roles of Lys-217, Arg-290, and Gln-294 in NADPH specificity. Replacement of these residues by site-specific mutagenesis to negatively charged amino acids compromised NADPH binding with minimal effects on NADH binding. The substrate-binding site of DepBRleg is larger than its closest structural homolog, AKR6A2, likely contributing to its ability to utilize a wide range of aldehydes and ketones, including the mycotoxin, patulin, as substrates. The structure of DepBRleg also suggests that 3-keto-DON can adopt two binding modes to facilitate 4-pro-R hydride transfer to either the re- or si-face of the C3 ketone providing a possible explanation for the enzyme's ability to convert 3-keto-DON to 3-epi-DON and DON in diastereomeric ratios of 67.2% and 32.8% respectively.

Bacterial Hydratases Involved in Steroid Side Chain Degradation Have Distinct Substrate Specificities.

Actinobacterial MaoC family enoyl coenzyme A (CoA) hydratases catalyze the addition of water across the double bond of CoA esters during steroid side chain catabolism. We determined that heteromeric MaoC type hydratases, exemplified by ChsH1-ChsH2Mtb of Mycobacterium tuberculosis and CasM-CasORjost from Rhodococcus jostii RHA1, are specific toward a 3-carbon side chain steroid metabolite, consistent with their roles in the last ß-oxidation cycle of steroid side chain degradation. Hydratases containing two fused MaoC domains are responsible for the degradation of longer steroid side chains. These hydratases, encoded in the cholesterol degradation gene clusters of M. tuberculosis and R. jostii RHA1, have broad specificity and were able to catalyze the hydration of the 5-carbon side chain of both cholesterol and bile acid metabolites. Surprisingly, the homologous hydratases from the bile acid degradation pathway have low catalytic efficiencies or no activity toward the 5-carbon side chain bile acid metabolites, cholyl-enoyl-CoA, lithocholyl-enoyl-CoA, and chenodeoxycholyl-enoyl-CoA. Instead, these hydratases preferred a cholate metabolite with oxidized steroid rings and a planar ring structure. Together, the results suggest that ring oxidation occurs prior to side chain degradation in the actinobacterial bile acid degradation pathway. IMPORTANCE Characterization of the substrate specificity of hydratases described here will facilitate the development of specific inhibitors that may be useful as novel therapeutics against M. tuberculosis and to metabolically engineer bacteria to produce steroid pharmaceuticals with desired steroid rings and side chain structures.

The steroid side-chain-cleaving aldolase Ltp2-ChsH2DUF35 is a thiolase superfamily member with a radically repurposed active site.

An aldolase from the bile acid-degrading actinobacterium Thermomonospora curvata catalyzes the C-C bond cleavage of an isopropyl-CoA side chain from the D-ring of the steroid metabolite 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). Like its homolog from Mycobacterium tuberculosis, the T. curvata aldolase is a protein complex of Ltp2 with a DUF35 domain derived from the C-terminal domain of a hydratase (ChsH2DUF35) that catalyzes the preceding step in the pathway. We determined the structure of the Ltp2-ChsH2DUF35 complex at 1.7 Å resolution using zinc-single anomalous diffraction. The enzyme adopts an αßßα organization, with the two Ltp2 protomers forming a central dimer, and the two ChsH2DUF35 protomers being at the periphery. Docking experiments suggested that Ltp2 forms a tight complex with the hydratase but that each enzyme retains an independent CoA-binding site. Ltp2 adopted a fold similar to those in thiolases; however, instead of forming a deep tunnel, the Ltp2 active site formed an elongated cleft large enough to accommodate 17-HOPC-CoA. The active site lacked the two cysteines that served as the nucleophile and general base in thiolases and replaced a pair of oxyanion-hole histidine residues with Tyr-246 and Tyr-344. Phenylalanine replacement of either of these residues decreased aldolase catalytic activity at least 400-fold. On the basis of a 17-HOPC-CoA -docked model, we propose a catalytic mechanism where Tyr-294 acts as the general base abstracting a proton from the D-ring hydroxyl of 17-HOPC-CoA and Tyr-344 as the general acid that protonates the propionyl-CoA anion following C-C bond cleavage.