The Catalytic Serine of <i>meta</i>-Cleavage Product Hydrolases Is Activated Differently for C–O Bond Cleavage Than for C–C Bond Cleavage

<i>meta</i>-Cleavage product (MCP) hydrolases catalyze C–C bond fission in the aerobic catabolism of aromatic compounds by bacteria. These enzymes utilize a Ser-His-Asp triad to catalyze hydrolysis via an acyl–enzyme intermediate. BphD, which catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) in biphenyl degradation, catalyzed the hydrolysis of an ester analogue, <i>p</i>-nitrophenyl benzoate (pNPB), with a <i>k</i>_cat value (6.3 ± 0.5 s^–1) similar to that of HOPDA (6.5 ± 0.5 s^–1). Consistent with the breakdown of a shared intermediate, product analyses revealed that BphD catalyzed the methanolysis of both HOPDA and pNPB, partitioning the products to benzoic acid and methyl benzoate in similar ratios. Turnover of HOPDA was accelerated up to 4-fold in the presence of short, primary alcohols (methanol > ethanol > <i>n</i>-propanol), suggesting that deacylation is rate-limiting during catalysis. In the steady-state hydrolysis of HOPDA, <i>k</i>_cat/<i>K</i>_m values were independent of methanol concentration, while both <i>k</i>_cat and <i>K</i>_m values increased with methanol concentration. This result was consistent with a simple model of nucleophilic catalysis. Although the enzyme could not be saturated with pNPB at methanol concentrations of >250 mM, <i>k</i>_obs values from the steady-state turnover of pNPB at low methanol concentrations were also consistent with a nucleophilic mechanism of catalysis. Finally, transient-state kinetic analysis of pNPB hydrolysis by BphD variants established that substitution of the catalytic His reduced the rate of acylation by more than 3 orders of magnitude. This suggests that for pNPB hydrolysis, the serine nucleophile is activated by the His-Asp dyad. In contrast, rapid acylation of the H265Q variant during C–C bond cleavage suggests that the serinate forms via a substrate-assisted mechanism. Overall, the data indicate that ester hydrolysis proceeds via the same acyl–enzyme intermediate as that of the physiological substrate but that the serine nucleophile is activated via a different mechanism

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Physiological Adaptation of the <i>Rhodococcus jostii</i> RHA1 Membrane Proteome to Steroids as Growth Substrates

<i>Rhodococcus jostii</i> RHA1 is a catabolically versatile soil actinomycete that can utilize a wide range of organic compounds as growth substrates including steroids. To globally assess the adaptation of the protein composition in the membrane fraction to steroids, the membrane proteomes of RHA1 grown on each of cholesterol and cholate were compared to pyruvate-grown cells using gel-free SIMPLE-MudPIT technology. Label-free quantification by spectral counting revealed 59 significantly regulated proteins, many of them present only during growth on steroids. Cholesterol and cholate induced distinct sets of steroid-degrading enzymes encoded by paralogous gene clusters, consistent with transcriptomic studies. CamM and CamABCD, two systems that take up cholate metabolites, were found exclusively in cholate-grown cells. Similarly, 9 of the 10 Mce4 proteins of the cholesterol uptake system were found uniquely in cholesterol-grown cells. Bioinformatic tools were used to construct a model of Mce4 transporter within the RHA1 cell envelope. Finally, comparison of the membrane and cytoplasm proteomes indicated that several steroid-degrading enzymes are membrane-associated. The implications for the degradation of steroids by actinomycetes, including cholesterol by the pathogen <i>Mycobacterium tuberculosis</i>, are discussed

Hemoglobin Binding and Catalytic Heme Extraction by IsdB Near Iron Transporter Domains

The Isd (iron-regulated surface determinant) system is a multiprotein transporter that allows bacterium <i>Staphylococcus aureus</i> to take up iron from hemoglobin (Hb) during human infection. In this system, IsdB is a cell wall-anchored surface protein that contains two near iron transporter (NEAT) domains, one of which binds heme. IsdB rapidly extracts heme from Hb and transfers it to IsdA for relay into the bacterial cell. Using a series of recombinant IsdB constructs that included at least one NEAT domain, we demonstrated that both domains are required to bind Hb with high affinity (<i>K</i>_D = 0.42 ± 0.05 μM) and to extract heme from Hb. Moreover, IsdB extracted heme only from oxidized metHb, although it also bound oxyHb and the Hb–CO complex. In a reconstituted model of the biological heme relay pathway, IsdB catalyzed the transfer of heme from metHb to IsdA with a <i>K</i>_m for metHb of 0.75 ± 0.07 μN and a <i>k</i>_cat of 0.22 ± 0.01 s^–1. The latter is consistent with the transfer of heme from metHb to IsdB being the rate-limiting step. With both NEAT domains and the linker region present in a single contiguous polypeptide, high-affinity Hb binding was achieved, rapid heme uptake was observed, and multiple turnovers of heme extraction from metHb and transfer to IsdA were conducted, representing all known Hb–heme uptake functions of the full-length IsdB protein

Snapshots of the Catalytic Cycle of an O₂, Pyridoxal Phosphate-Dependent Hydroxylase

Enzymes that catalyze hydroxylation of unactivated carbons normally contain heme and nonheme iron cofactors. By contrast, how a pyridoxal phosphate (PLP)-dependent enzyme could catalyze such a hydroxylation was unknown. Here, we investigate RohP, a PLP-dependent enzyme that converts l-arginine to (<i>S</i>)-4-hydroxy-2-ketoarginine. We determine that the RohP reaction consumes oxygen with stoichiometric release of H₂O₂. To understand this unusual chemistry, we obtain ∼1.5 Å resolution structures that capture intermediates along the catalytic cycle. Our data suggest that RohP carries out a four-electron oxidation and a stereospecific alkene hydration to give the (<i>S</i>)-configured product. Together with our earlier studies on an O₂, PLP-dependent l-arginine oxidase, our work suggests that there is a shared pathway leading to both oxidized and hydroxylated products from l-arginine

A Substrate-Assisted Mechanism of Nucleophile Activation in a Ser–His–Asp Containing C–C Bond Hydrolase

The <i>meta</i>-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 p<i>K</i>_a2 (β_nuc ∼ 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 <i>meta</i>-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 <i>Sphingomonas wittichii</i> 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 BphD_LB400 from <i>Burkholderia xenovorans</i> 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 BphD_LB400. Structural comparison of MCP hydrolases identified the NC-loop, connecting the lid to the α/β-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/β-hydrolases

Identification of an Acyl-Enzyme Intermediate in a <i>meta</i>-Cleavage Product Hydrolase Reveals the Versatility of the Catalytic Triad

<i>Meta</i>-cleavage product (MCP) hydrolases are members of the α/β-hydrolase superfamily that utilize a Ser-His-Asp triad to catalyze the hydrolysis of a C–C bond. BphD, the MCP hydrolase from the biphenyl degradation pathway, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. A 1.6 Å resolution crystal structure of BphD H265Q incubated with HOPDA revealed that the enzyme’s catalytic serine was benzoylated. The acyl-enzyme is stabilized by hydrogen bonding from the amide backbone of ‘oxyanion hole’ residues, consistent with formation of a tetrahedral oxyanion during nucleophilic attack by Ser112. Chemical quench and mass spectrometry studies substantiated the formation and decay of a Ser112-benzoyl species in wild-type BphD on a time scale consistent with turnover and incorporation of a single equivalent of ¹⁸O into the benzoate produced during hydrolysis in H₂¹⁸O. Rapid-scanning kinetic studies indicated that the catalytic histidine contributes to the rate of acylation by only an order of magnitude, but affects the rate of deacylation by over 5 orders of magnitude. The orange-colored catalytic intermediate, ES^red, previously detected in the wild-type enzyme and proposed herein to be a carbanion, was not observed during hydrolysis by H265Q. In the newly proposed mechanism, the carbanion abstracts a proton from Ser112, thereby completing tautomerization and generating a serinate for nucleophilic attack on the C6-carbonyl. Finally, quantification of an observed pre-steady-state kinetic burst suggests that BphD is a half-site reactive enzyme. While the updated catalytic mechanism shares features with the serine proteases, MCP hydrolase-specific chemistry highlights the versatility of the Ser-His-Asp triad