Proton Relay Network Research Articles

Depicting the proton relay network in human aromatase: New insights into the role of the alcohol-acid pair.

Human aromatase is the cytochrome P450 catalyzing the conversion of androgens into estrogens in a three steps reaction essential to maintain steroid hormones balance. Here we report the capture and spectroscopic characterization of its compound I (Cpd I), the main reactive species in cytochromes P450. The typical spectroscopic transitions indicating the formation of Cpd I are detected within 0.8 s when mixing aromatase with meta‐chloroperoxybenzoic acid. The estrogen product is obtained from the same reaction mixture, demonstrating the involvement of Cpd I in aromatization reaction. Site‐directed mutagenesis is applied to the acid‐alcohol pair D309 and T310 and to R192, predicted to be part of the proton relay network. Mutants D309N and R192Q do not lead to Cpd I with an associated loss of activity, confirming that these residues are involved in proton delivery for Cpd I generation. Cpd I is captured for T310A mutant and shows 2.9‐ and 4.4‐fold faster rates of formation and decay, respectively, compared to wild‐type (WT). However, its activity is lower than the WT and a larger amount of H2O2 is produced during catalysis, indicating that T310 has an essential role in proton gating for generation of Cpd 0 and Cpd I and for their stabilization. The data provide new evidences on the role of threonine belonging to the conserved “acid‐alcohol” pair and known to be crucial for oxygen activation in cytochromes P450.

Crystallographic snapshots of ternary complexes of thermophilic secondary alcohol dehydrogenase from Thermoanaerobacter pseudoethanolicus reveal the dynamics of ligand exchange and the proton relay network.

Three-dimensional structures of I86A and C295A mutant secondary alcohol dehydrogenase (SADH) from Thermoanaerobacter pseudoethanolicus were determined by x-ray crystallography. The tetrameric structure of C295A-SADH soaked with NADP+ and dimethyl sulfoxide (DMSO) was determined to 1.85 Å with an Rfree of 0.225. DMSO is bound to the tetrahedral zinc in each subunit, with ligands from SG of Cys-37, NE2 of His-59, and OD2 of Asp-150. The nicotinamide ring of NADP is hydrogen-bonded to the N of Ala-295 and the O of Val-265 and Gly-293. The O of DMSO is connected to a network of hydrogen bonds with OG of Ser-39, the 3'-OH of NADP, and ND1 of His-42. The structure of I86A-SADH soaked with 2-pentanol and NADP+ contains (R)-2-pentanol bound in each subunit, ligated to the tetrahedral zinc, and connected to the proton relay network. The structure of I86A-SADH soaked with 3-methylcyclohexanol and NADP+ has alcohol bound in three subunits. Two of the sites have the alcohol ligated to the zinc in an axial position, with OE2 of Glu-60 in the other axial position of a trigonal bipyramidal complex. One site has 3-methylcyclohexanol bound noncovalently, with the zinc in an inverted tetrahedral geometry with Glu-60. The fourth site also has the zinc in a trigonal bipyramidal complex with axial Glu-60 and water ligands. These structures demonstrate that ligand exchange of SADH involves pentacoordinate and inverted zinc complexes with Glu-60. Furthermore, we see a network of hydrogen bonds connecting the substrate oxygen to the external solvent that is likely to play a role in the mechanism of SADH.

Structural analysis of P450 AmphL from Streptomyces nodosus provides insights into substrate selectivity of polyene macrolide antibiotic biosynthetic P450s

AmphL is a cytochrome P450 enzyme that catalyzes the C8 oxidation of 8-deoxyamphotericin B to the polyene macrolide antibiotic, amphotericin B. To understand this substrate selectivity, we solved the crystal structure of AmphL to a resolution of 2.0 Å in complex with amphotericin B and performed molecular dynamics (MD) simulations. A detailed comparison with the closely related P450, PimD, which catalyzes the epoxidation of 4,5-desepoxypimaricin to the macrolide antibiotic, pimaricin, reveals key catalytic structural features responsible for stereo- and regio-selective oxidation. Both P450s have a similar access channel that runs parallel to the active site I helix over the surface of the heme. Molecular dynamics simulations of substrate binding reveal PimD can “pull” substrates further into the P450 access channel owing to additional electrostatic interactions between the protein and the carboxyl group attached to the hemiketal ring of 4,5-desepoxypimaricin. This substrate interaction is absent in AmphL although the additional substrate -OH groups in 8-deoxyamphotericin B help to correctly position the substrate for C8 oxidation. Simulations of the oxy-complex indicates that these -OH groups may also participate in a proton relay network required for O2 activation as has been suggested for two other macrolide P450s, PimD and P450eryF. These findings provide experimentally testable models that can potentially contribute to a new generation of novel macrolide antibiotics with enhanced antifungal and/or antiprotozoal efficacy.

Updating the Paradigm: Redox Partner Binding and Conformational Dynamics in Cytochromes P450.

This Account summarizes recent findings centered on the role that redox partner binding, allostery, and conformational dynamics plays in cytochrome P450 proton coupled electron transfer. P450s are one of Nature's largest enzyme families and it is not uncommon to find a P450 wherever substrate oxidation is required in the formation of essential molecules critical to the life of the organism or in xenobiotic detoxification. P450s can operate on a remarkably large range of substrates from the very small to the very large, yet the overall P450 three-dimensional structure is conserved. Given this conservation of structure, it is generally assumed that the basic catalytic mechanism is conserved. In nearly all P450s, the O2 O-O bond must be cleaved heterolytically enabling one oxygen atom, the distal oxygen, to depart as water and leave behind a heme iron-linked O atom as the powerful oxidant that is used to activate the nearby substrate. For this process to proceed efficiently, externally supplied electrons and protons are required. Two protons must be added to the departing O atom while an electron is transferred from a redox partner that typically contains either a Fe2S2 or FMN redox center. The paradigm P450 used to unravel the details of these mechanisms has been the bacterial CYP101A1 or P450cam. P450cam is specific for its own Fe2S2 redox partner, putidaredoxin or Pdx, and it has long been postulated that Pdx plays an effector/allosteric role by possibly switching P450cam to an active conformation. Crystal structures, spectroscopic data, and direct binding experiments of the P450cam-Pdx complex provide some answers. Pdx shifts the conformation of P450cam to a more open state, a transition that is postulated to trigger the proton relay network required for O2 activation. An essential part of this proton relay network is a highly conserved Asp (sometimes Glu) that is known to be critical for activity in a number of P450s. How this Asp and proton delivery networks are connected to redox partner binding is quite simple. In the closed state, this Asp is tied down by salt bridges, but these salt bridges are ruptured when Pdx binds, leaving the Asp free to serve its role in proton transfer. An alternative hypothesis suggests that a specific proton relay network is not really necessary. In this scenario, the Asp plays a structural role in the open/close transition and merely opening the active site access channel is sufficient to enable solvent protons in for O2 protonation. Experiments designed to test these various hypotheses have revealed some surprises in both P450cam and other bacterial P450s. Molecular dynamics and crystallography show that P450cam can undergo rather significant conformational gymnastics that result in a large restructuring of the active site requiring multiple cis/trans proline isomerizations. It also has been found that X-ray driven substrate hydroxylation is a useful tool for better understanding the role that the essential Asp and surrounding residues play in catalysis. Here we summarize these recent results which provide a much more dynamic picture of P450 catalysis.

Coordination of Substrate Binding and Protonation in the N.gonorrhoeae MtrD Efflux Pump Controls the Functionally Rotating Transport Mechanism.

Multidrug resistance is a serious problem that threatens the effective treatment of the widespread sexually transmitted disease gonorrhea, caused by the Gram-negative bacterium Neisseria gonorrhoeae. The drug efflux pump primarily implicated in N.gonorrhoeae antimicrobial resistance is the inner membrane transporter MtrD, which forms part of the tripartite multiple transferable resistance (Mtr) CDE efflux system. A structure of MtrD was first solved in 2014 as a symmetrical homotrimer, and then, recently, as an asymmetrical homotrimer. Through a series of molecular dynamics simulations and mutagenesis experiments, we identify the combination of substrate binding and protonation states of the proton relay network that drives the transition from the symmetric to the asymmetric conformation of MtrD. We characterize the allosteric coupling between the functionally important local regions that control conformational changes between the access, binding, and extrusion states and allow for transition to the asymmetric MtrD conformation. We also highlight a significant rotation of the transmembrane helices caused by protonation of the proton relay network, which widens the intermonomeric gap that is a hallmark of the rotational transporter mechanism. This is the first analysis and description of the transport mechanism for the N.gonorrhoeae MtrD transporter and provides evidence that antimicrobial efflux in MtrD follows the functionally rotating transport mechanism seen in protein homologues from the same transport protein superfamily.

A requirement for an active proton delivery network supports a compound I-mediated C–C bond cleavage in CYP51 catalysis

CYP51 enzymes (sterol 14α-demethylases) are cytochromes P450 that catalyze multistep reactions. The CYP51 reaction occurs in all biological kingdoms and is essential in sterol biosynthesis. It removes the 14α-methyl group from cyclized sterol precursors by first forming an alcohol, then an aldehyde, and finally eliminating formic acid with the introduction of a Δ14-15 double bond in the sterol core. The first two steps are typical hydroxylations, mediated by an electrophilic compound I mechanism. The third step, C-C bond cleavage, has been proposed to involve either compound I (i.e. FeO3+) or, alternatively, a proton transfer-independent nucleophilic ferric peroxo anion (compound 0, i.e. Fe3+O2-). Here, using comparative crystallographic and biochemical analyses of WT human CYP51 (CYP51A1) and its D231A/H314A mutant, whose proton delivery network is destroyed (as evidenced in a 1.98-Å X-ray structure in complex with lanosterol), we demonstrate that deformylation of the 14α-carboxaldehyde intermediate requires an active proton relay network to drive the catalysis. These results indicate a unified, compound I-based mechanism for all three steps of the CYP51 reaction, as previously established for CYP11A1 and CYP19A1. We anticipate that our approach can be applied to mechanistic studies of other P450s that catalyze multistep reactions, such as C-C bond cleavage.

Proton Relay Network in the Bacterial P450s: CYP101A1 and CYP101D1.

Cytochrome P450s are among nature's most powerful catalysts. Their ability to activate molecular dioxygen to form high-valent ferryl intermediates (Compounds I and II) enables a wide array of chemistries ranging from simple epoxidations to more complicated C-H bond oxidations. Oxygen activation is achieved by reduction of the ferrous dioxygen complex, which requires the transfer of an electron from a redox partner and subsequent double protonation to yield a water molecule and a ferryl porphyrin π-cation radical (Compound I). Previous studies of the CYP101 family of cytochrome P450s demonstrated the importance of the conserved active site Asp25X residue in this protonation event, although its precise role is yet to be unraveled. To further explore the origin of protons in oxygen activation, we analyzed the effects of an Asp to Glu mutation at the 25X position in P450cam and in CYP101D1. This mutation inactivates P450cam but not CYP101D1. A series of mutagenic, crystallographic, kinetic, and molecular dynamics studies indicate that this mutation locks P450cam into a closed, inactive conformation. In CYP101D1, the D259E mutant changes the rate-limiting step to reduction of the P450-oxy complex, thus opening a window into the critical proton-coupled electron transfer step in P450 catalysis.

Outer-Sphere Tyrosine 159 within the 3-Mercaptopropionic Acid Dioxygenase S-H-Y Motif Gates Substrate-Coordination Denticity at the Non-Heme Iron Active Site.

Thiol dioxygenases are non-heme mononuclear iron enzymes that catalyze the O2-dependent oxidation of free thiols (-SH) to produce the corresponding sulfinic acid (-SO2-). Regardless of the phylogenic domain, the active site for this enzyme class is typically comprised of two major features: (1) a mononuclear ferrous iron coordinated by three protein-derived histidines and (2) a conserved sequence of outer Fe-coordination-sphere amino acids (Ser-His-Tyr) spatially adjacent to the iron site (∼3 Å). Here, we utilize a promiscuous 3-mercaptopropionic acid dioxygenase cloned from Azotobacter vinelandii (Av MDO) to explore the function of the conserved S-H-Y motif. This enzyme exhibits activity with 3-mercaptopropionic acid (3mpa), l-cysteine (cys), as well as several other thiol-bearing substrates, thus making it an ideal system to study the influence of residues within the highly conserved S-H-Y motif (H157 and Y159) on substrate specificity and reactivity. The pKa values for these residues were determined by pH-dependent steady-state kinetics, and their assignments verified by comparison to H157N and Y159F variants. Complementary electron paramagnetic resonance and Mössbauer studies demonstrate a network of hydrogen bonds connecting H157-Y159 and Fe-bound ligands within the enzymatic Fe site. Crucially, these experiments suggest that the hydroxyl group of Y159 hydrogen bonds to Fe-bound NO and, by extension, Fe-bound oxygen during native catalysis. This interaction alters both the NO binding affinity and rhombicity of the 3mpa-bound iron-nitrosyl site. In addition, Fe coordination of cys is switched from thiolate only to bidentate (thiolate/amine) for the Y159F variant, indicating that perturbations within the S-H-Y proton relay network also influence cys Fe binding denticity.

Proton relay network in P450cam formed upon docking of putidaredoxin.

Cytochromes P450 are versatile heme-based enzymes responsible for vital life processes. Of these, P450cam (substrate camphor) has been most studied. Despite this, precise mechanisms of the key O─O cleavage step remain partly elusive to date; effects observed in various enzyme mutants remain partly unexplained. We have carried out extended (to 1000 ns) MM-MD and follow-on quantum mechanics/molecular mechanics computations, both on the well-studied FeOO state and on Cpd(0) (compound 0). Our simulations include (all camphor-bound): (a) WT (wild type), FeOO state. (b) WT, Cpd(0). (c) Pdx (Putidaredoxin, redox partner of P450)-docked-WT, FeOO state. (d) Pdx-docked WT, Cpd(0). (e) Pdx-docked T252A mutant, Cpd(0). Among our key findings: (a) Effect of Pdx docking appears to go far beyond that indicated in prior studies: it leads to specific alterations in secondary structure that create the crucial proton relay network. (b) Specific proton relay networks we identify are: FeOO(H)⋯T252⋯nH 2 O⋯D251 in WT; FeOO(H)⋯nH 2 O⋯D251 in T252A mutant; both occur with Pdx docking. (c) Direct interaction of D251 with -FeOOH is, respectively, rare/frequent in WT/T252A mutant. (d) In WT, T252 is in the proton relay network. (e) Positioning of camphor appears significant: when camphor is part of H-bonding network, second protonation appears to be facilitated.

Structural Insight into Eukaryotic Sterol Transport through Niemann-Pick Type C Proteins

Niemann-Pick type C (NPC) proteins are essential for sterol homeostasis, believed to drive sterol integration into the lysosomal membrane before redistribution to other cellular membranes. Here, using a combination of crystallography, cryo-electron microscopy, and biochemical and invivo studies on the Saccharomyces cerevisiae NPC system (NCR1 and NPC2), we present a framework for sterol membrane integration. Sterols are transferred between hydrophobic pockets of vacuolar NPC2 and membrane-protein NCR1. NCR1 has its N-terminal domain (NTD) positioned to deliver a sterol to a tunnel connecting NTD to the luminal membrane leaflet 50Å away. A sterol is caught inside this tunnel during transport, and a proton-relay network of charged residues in the transmembrane region is linked to this tunnel supporting a proton-driven transport mechanism. We propose a model for sterol integration that clarifies the role of NPC proteins in this essential eukaryotic pathway and that rationalizes mutations in patients with Niemann-Pick disease type C.

Ligand and Redox Partner Binding Generates a New Conformational State in Cytochrome P450cam (CYP101A1).

It has become increasingly clear that cytochromes P450 can cycle back and forth between two extreme conformational states termed the closed and open states. In the well-studied cytochrome P450cam, the binding of its redox partner, putidaredoxin (Pdx), shifts P450cam toward the open state. Shifting to the open state is thought to be important in the formation of a proton relay network essential for O-O bond cleavage and formation of the active Fe(IV)═O intermediate. Another important intermediate is the oxy-P450cam complex when bound to Pdx. Trapping this intermediate in crystallo is challenging owing to its instability, but the CN- complex is both stable and an excellent mimic of the O2 complex. Here we present the P450cam-Pdx structure complexed with CN-. CN- results in large conformational changes including cis/trans isomerization of proline residues. Changes include large rearrangements of active-site residues and the formation of new active-site access channel that we have termed channel 2. The formation of channel 2 has also been observed in our previous molecular dynamics simulations wherein substrate binding to an allosteric site remote from the active site opens up channel 2. This new structure supports an extensive amount of previous work showing that distant regions of the structure are dynamically coupled and underscores the potentially important role that large conformational changes and dynamics play in P450 catalysis.

1,2-Diol Dehydration by the Radical SAM Enzyme AprD4: A Matter of Proton Circulation and Substrate Flexibility.

Regiospecific dehydration of vicinal diols by enzymes is a difficult reaction that usually requires activation by dedicated organic cofactors. The enzymatic use of radical-based chemistry is an effective but challenging alternative as radical intermediates are difficult to control. Here we report the X-ray structure of the radical S-adenosyl-l-methionine (SAM) dehydratase AprD4 involved in the biosynthesis of the aminoglycoside (AG) antibiotic apramycin. Using in vitro characterizations and theoretical calculations based on our crystal structure, we have been able to propose a detailed mechanism of AprD4 catalysis, which involves a complex partially substrate-induced proton relay network in the enzyme active site and highlights the key role of the protein matrix in driving high-energy intermediates.

Mechanisms of proton relay and product release by Class A β-lactamase at ultrahigh resolution.

Structural data are available in the PDB database under the accession numbers 5TOP, 5TOY, and 5VLE.

Effect of Redox Partner Binding on Cytochrome P450 Conformational Dynamics.

Previous crystal structures of cytochrome P450cam complexed with its redox partner, putidaredoxin (Pdx), shows that P450cam adopts the open conformation. It has been hypothesized that the Pdx-induced shift toward the open state frees the essential Asp251 from salt bridges with Arg186 and Lys178 so that Asp251 can participate in a proton relay network required for O2 activation. This in part explains why P450cam has such a strict requirement for Pdx. One problem with this view is that looser substrate-protein interactions in the open state may not be compatible with the observed regio- and stereoselective hydroxylation. In the present study, molecular dynamics simulations show that Pdx binding favors a conformation that stabilizes the active site and decreases camphor mobility yet retains a partially open conformation compatible with the required proton relay network. The R186A mutant which frees Asp251 in the absence of Pdx retains good enzyme activity, and the crystal structure shows that product, 5-exo-hydroxycamphor, is bound. This indicates that rupture of the Asp251-Arg186 relaxes selectivity with respect to source of electrons and enables X-ray generated reducing equivalents to support substrate hydroxylation. These combined computational and experimental results are consistent with the proposed role of Pdx in assisting the release of Asp251 from ion pairs so that it can participate in proton-coupled electron transfer.

Active Site and Laminarin Binding in Glycoside Hydrolase Family 55

The Carbohydrate Active Enzyme (CAZy) database indicates that glycoside hydrolase family 55 (GH55) contains both endo- and exo-β-1,3-glucanases. The founding structure in the GH55 is PcLam55A from the white rot fungus Phanerochaete chrysosporium (Ishida, T., Fushinobu, S., Kawai, R., Kitaoka, M., Igarashi, K., and Samejima, M. (2009) Crystal structure of glycoside hydrolase family 55 β-1,3-glucanase from the basidiomycete Phanerochaete chrysosporium. J. Biol. Chem. 284, 10100-10109). Here, we present high resolution crystal structures of bacterial SacteLam55A from the highly cellulolytic Streptomyces sp. SirexAA-E with bound substrates and product. These structures, along with mutagenesis and kinetic studies, implicate Glu-502 as the catalytic acid (as proposed earlier for Glu-663 in PcLam55A) and a proton relay network of four residues in activating water as the nucleophile. Further, a set of conserved aromatic residues that define the active site apparently enforce an exo-glucanase reactivity as demonstrated by exhaustive hydrolysis reactions with purified laminarioligosaccharides. Two additional aromatic residues that line the substrate-binding channel show substrate-dependent conformational flexibility that may promote processive reactivity of the bound oligosaccharide in the bacterial enzymes. Gene synthesis carried out on ∼30% of the GH55 family gave 34 active enzymes (19% functional coverage of the nonredundant members of GH55). These active enzymes reacted with only laminarin from a panel of 10 different soluble and insoluble polysaccharides and displayed a broad range of specific activities and optima for pH and temperature. Application of this experimental method provides a new, systematic way to annotate glycoside hydrolase phylogenetic space for functional properties.

Crystal Structure of Cindoxin, the P450cin Redox Partner

The crystal structure of the flavin mononucleotide (FMN)-containing redox partner to P450cin, cindoxin (Cdx), has been determined to 1.3 Å resolution. The overall structure is similar to that of the FMN domain of human cytochrome P450 reductase. A Brownian dynamics–molecular dynamics docking method was used to produce a model of Cdx with its redox partner, P450cin. This Cdx–P450cin model highlights the potential importance of Cdx Tyr96 in bridging the FMN and heme cofactors as well P450cin Arg102 and Arg346. Each of the single-site Ala mutants exhibits ∼10% of the wild-type activity, thus demonstrating the importance of these residues for binding and/or electron transfer. In the well-studied P450cam system, redox partner binding stabilizes the open low-spin conformation of P450cam and greatly decreases the stability of the oxy complex. In sharp contrast, Cdx does not shift P450cin to a low-spin state, although the stability of oxy-P450cin is decreased 10-fold in the presence of Cdx. This indicates that Cdx may have a modest effect on the open–closed equilibrium in P450cin compared to that in P450cam. It has been postulated that part of the effector role of Pdx on P450cam is to promote a significant structural change that makes available a proton relay network involving Asp251 required for O2 activation. The structure around the corresponding Asp in P450cin, Asp241, provides a possible structural reason for why P450cin is less dependent on its redox partner for functionally important structural changes.

Structural Basis for the Functional Roles of Critical Residues in Human Cytochrome P450 Aromatase

Cytochrome P450 aromatase (CYP19A1) is the only enzyme known to catalyze the biosynthesis of estrogens from androgens. The crystal structure of human placental aromatase (pArom) has paved the way toward understanding the structure-function relationships of this remarkable enzyme. Using an amino terminus-truncated recombinant human aromatase (rArom) construct, we investigate the roles of key amino acids in the active site, at the intermolecular interface, inside the access channel, and at the lipid-protein boundary for their roles in enzyme function and higher-order organization. Replacing the active site residue D309 with an N yields an inactive enzyme, consistent with its proposed involvement in aromatization. Mutation of R192 at the lipid interface, pivotal to the proton relay network in the access channel, results in the loss of enzyme activity. In addition to the distal catalytic residues, we show that mutation of K440 and Y361 of the heme-proximal region critically interferes with substrate binding, enzyme activity, and heme stability. The D-E loop deletion mutant Del7 that disrupts the intermolecular interaction significantly reduces enzyme activity. However, the less drastic Del4 and point mutants E181A and E181K do not. Furthermore, native gel electrophoresis, size-exclusion chromatography, and analytical ultracentrifugation are used to show that mutations in the intermolecular interface alter the quaternary organization of the enzyme in solution. As a validation for interpretation of the mutational results in the context of the innate molecule, we determine the crystal structure of rArom to show that the active site, tertiary, and quaternary structures are identical to those of pArom.

Synergistic effects of mutations in cytochrome P450cam designed to mimic CYP101D1.

A close orthologue to cytochrome P450cam (CYP101A1) that catalyzes the same hydroxylation of camphor to 5-exo-hydroxycamphor is CYP101D1. There are potentially important differences in and around the active site that could contribute to subtle functional differences. Adjacent to the heme iron ligand, Cys357, is Leu358 in P450cam, whereas this residue is Ala in CYP101D1. Leu358 plays a role in binding of the P450cam redox partner, putidaredoxin (Pdx). On the opposite side of the heme, about 15-20 Å away, Asp251 in P450cam plays a critical role in a proton relay network required for O2 activation but forms strong ion pairs with Arg186 and Lys178. In CYP101D1 Gly replaces Lys178. Thus, the local electrostatic environment and ion pairing are substantially different in CYP101D1. These sites have been systematically mutated in P450cam to the corresponding residues in CYP101D1 and the mutants analyzed by crystallography, kinetics, and UV-vis spectroscopy. Individually, the mutants have little effect on activity or structure, but in combination there is a major drop in enzyme activity. This loss in activity is due to the mutants being locked in the low-spin state, which prevents electron transfer from the P450cam redox partner, Pdx. These studies illustrate the strong synergistic effects on well-separated parts of the structure in controlling the equilibrium between the open (low-spin) and closed (high-spin) conformational states.

Elucidating the reaction mechanism of the benzoate oxidation pathway encoded aldehyde dehydrogenase from Burkholderia xenovorans LB400

Oxidation of cis-3,4-dehydroadipyl-CoA semialdehyde to cis-3,4-dehydroadipyl-CoA by the aldehyde dehydrogenase, ALDH(C) (EC.1.2.1.77), is an essential step in the metabolism of benzoate in Burkholderia xenovorans LB400. In a previous study, we established a structural blueprint for this novel group of ALDH enzymes. Here, we build significantly on this initial work and propose a detailed reaction mechanism for ALDH(C) based on comprehensive structural and functional investigations of active site residues. Kinetic analyses reveal essential roles for C296 as the nucleophile and E257 as the associated general base. Structural analyses of E257Q and C296A variants suggest a dynamic charge repulsion relationship between E257 and C296 that contributes to the inherent flexibility of E257 in the native enzyme, which is further regulated by E496 and E167. A proton relay network anchored by E496 and supported by E167 and K168 serves to reset E257 for the second catalytic step. We also propose that E167, which is unique to ALDH(C) and its homologs, serves a critical role in presenting the catalytic water to the newly reset E257 such that the enzyme can proceed with deacylation and product release. Collectively, the reaction mechanism proposed for ALDH(C) promotes a greater understanding of these novel ALDH enzymes, the ALDH super-family in general, and benzoate degradation in B. xenovorans LB400.

Disruption of the Proton Relay Network in the Class 2 Dihydroorotate Dehydrogenase fromEscherichia coli

Dihydroorotate dehydrogenases (DHODs) are FMN-containing enzymes that catalyze the conversion of dihydroorotate (DHO) to orotate in the de novo synthesis of pyrimidines. During the reaction, a proton is transferred from C5 of DHO to an active site base and the hydrogen at C6 of DHO is transferred to N5 of the isoalloxazine ring of the flavin as a hydride. In class 2 DHODs, a hydrogen bond network observed in crystal structures has been proposed to deprotonate the C5 atom of DHO. The active site base (Ser175 in the Escherichia coli enzyme) hydrogen bonds to a crystallographic water molecule that sits on a phenylalanine (Phe115 in the E. coli enzyme) and hydrogen bonds to a threonine (Thr178 in the E. coli enzyme), residues that are conserved in class 2 enzymes. The importance of these residues in the oxidation of DHO was investigated using site-directed mutagenesis. Mutating Ser175 to alanine had severe effects on the rate of flavin reduction, slowing it by more than 3 orders of magnitude. Changing the size and/or hydrophobicity of the residues of the hydrogen bond network, Thr178 and Phe115, slowed flavin reduction as much as 2 orders of magnitude, indicating that the active site base and the hydrogen bond network work together for efficient deprotonation of DHO.