Kinetics of proton-linked flavin conformational changes in p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) is an FAD-dependent monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (pOHB) to 3,4-dihydroxybenzoate in an NADPH-dependent reaction. Two structural features are coupled to control the reactivity of PHBH with NADPH: a proton-transfer network that allows protons to be passed between the sequestered active site and solvent and a flavin that adopts two positions: "in", where the flavin is near pOHB, and "out", where the flavin is near NADPH. PHBH uses the proton-transfer network to test for the presence of a suitable aromatic substrate before allowing the flavin to adopt the NADPH-accessible conformation. In this work, kinetic analysis of the His72Asn mutant, with a disrupted proton-transfer network, showed that flavin movement could occur in the presence or absence of NADPH but that NADPH stimulated movement to the reactive conformation required for hydride transfer. Substrate and solvent isotope effects on the transient kinetics of reduction of the His72Asn mutant showed that proton transfer was linked to flavin movement and that the conformational change occurred in a step separate from that of hydride transfer. Proton transfers during the reductive half-reaction were observed directly in the wild-type enzyme by performing experiments in the presence of a fluorescent pH-indicator dye in unbuffered solutions. NADPH binding caused rapid proton release from the enzyme, followed by proton uptake after flavin reduction. Solvent and substrate kinetic isotope effects showed that proton-coupled flavin movement and reduction also occurred in different steps in wild-type PHBH. These results allow a detailed kinetic scheme to be proposed for the reductive half-reaction of the wild-type enzyme. Three kinetic models considered for substrate-induced isomerization are analyzed in the Appendix.     

Structure-function correlations of the reaction of reduced nicotinamide analogues with p-hydroxybenzoate hydroxylase substituted with a series of 8-substituted flavins

Structural and kinetic studies have revealed two flavin conformations in p-hydroxybenzoate hydroxylase (PHBH), the in-position and the out-position. Conversion between these two conformations is believed to be essential during catalysis. Although substrate hydroxylation occurs while the flavin in PHBH is in the in-conformation, the position of the flavin during reduction by NADPH is uncertain. To investigate the catalytic importance of the out-conformation of the flavin and to clarify the mechanism of flavin reduction in PHBH, we report quantitative structure-reactivity relationships (QSAR) using PHBH substituted separately with nine derivatives of FAD modified in the 8-position and four dihydronicotinamide analogues as reducing agents. The 8-position of the FAD isoalloxazine ring was chosen for modification because in PHBH it has minimal interactions with the protein and is accessible to solvent. The chemical sequence of events during catalysis by PHBH was not altered when using any of the modified flavins, and normal products were obtained. Although the rate of reduction of PHBH reconstituted with flavin derivatives is expected to be dependent on the redox potential of the flavin, no strict correlation was observed. Instead, the rate of reduction correlated with the kappa-substituent constant, which is based on size and hydrophobicity of the 8-substituent on the FAD. Substituents that sterically hinder attainment of the out-conformation decreased the rate of flavin reduction much more than expected on the basis of the redox potential of the flavin. The results of this QSAR analysis are consistent with the hypothesis that the flavin in PHBH must move to the out-conformation for proper formation of the charge-transfer complex between NADPH and FAD that is necessary for rapid flavin reduction.     

Real-time enzyme dynamics illustrated with fluorescence spectroscopy of p-hydroxybenzoate hydroxylase

We have used the flavoenzyme p-hydroxybenzoate hydroxylase (PHBH) to illustrate that a strongly fluorescent donor label can communicate with the flavin via single-pair F?rster resonance energy transfer (spFRET). The accessible Cys-116 of PHBH was labeled with two different fluorescent maleimides with full preservation of enzymatic activity. One of these labels shows overlap between its fluorescence spectrum and the absorption spectrum of the FAD prosthetic group in the oxidized state, while the other fluorescent probe does not have this spectral overlap. The spectral overlap strongly diminished when the flavin becomes reduced during catalysis. The donor fluorescence properties can then be used as a sensitive antenna for the flavin redox state. Time-resolved fluorescence experiments on ensembles of labeled PHBH molecules were carried out in the absence and presence of enzymatic turnover. Distinct changes in fluorescence decays of spFRET-active PHBH can be observed when the enzyme is performing catalysis using both substrates p-hydroxybenzoate and NADPH. Single-molecule fluorescence correlation spectroscopy on spFRET-active PHBH showed the presence of a relaxation process (relaxation time of 23 micros) that is related to catalysis. In addition, in both labeled PHBH preparations the number of enzyme molecules reversibly increased during enzymatic turnover indicating that the dimer-monomer equilibrium is affected.     

A rate-limiting conformational change of the flavin in p-hydroxybenzoate hydroxylase is necessary for ligand exchange and catalysis: studies with 8-mercapto- and 8-hydroxy-flavins

The FAD of p-hydroxybenzoate hydroxylase (PHBH) is known to exist in two conformations. The FAD must be in the in-position for hydroxylation of p-hydroxybenzoate (pOHB), whereas the out-position is essential for reduction of the flavin by NADPH. In these investigations, we have used 8-mercapto-FAD and 8-hydroxy-FAD to probe the movement of the flavin in catalysis. Under the conditions employed, 8-mercapto-FAD (pK(a) = 3.8) and 8-hydroxy-FAD (pK(a) = 4.8) are mainly anionic. The spectral characteristics of the anionic forms of these flavins are very sensitive to their environment, making them sensitive probes for detecting movement of the flavin during catalysis. With these flavin analogues, the enzyme hydroxylates pOHB efficiently, but at a rate much slower than that of enzyme with FAD. Reaction of oxygen with reduced forms of these modified enzymes in the absence of substrate appears to proceed through the formation of the flavin-C4a-hydroperoxide intermediate, as with normal enzyme, but the decay of this intermediate is so fast compared to its formation that very little accumulates during the reaction. However, after elimination of H2O2 from the flavin-C4a-hydroperoxide, a perturbed oxidized enzyme spectrum is observed (Eox*), and this converts slowly to the spectrum of the resting oxidized form of the enzyme (Eox). In the presence of pOHB, PHBH reconstituted with 8-mercapto-FAD also shows the additional oxidized intermediate (Eox*) after the usual oxygenated C4a-intermediates have formed and decayed in the course of the hydroxylation reaction. This Eox* to Eox step is postulated to be due to flavin movement. Furthermore, binding of pOHB to resting (Eox) follows a three-step equilibrium mechanism that is also consistent with flavin movement being the rate-limiting step. The rate for the slowest step during pOHB binding is similar to that observed for the conversion of Eox* to Eox during the oxygen reaction in the absence or presence of substrate. Steady-state kinetic analysis of PHBH substituted with 8-mercapto-FAD demonstrated that the apparent k(cat) is also similar to the rate of Eox* conversion to Eox. Presumably, the protein environment surrounding the flavin in Eox* differs slightly from that of the final resting form of the enzyme (Eox).     

Role of protein flexibility in the catalytic cycle of p-hydroxybenzoate hydroxylase elucidated by the Pro293Ser mutant

Proline 293 of p-hydroxybenzoate hydroxylase from Pseudomonas aeruginosa is in a highly conserved region of the flavoprotein aromatic hydroxylases. It is thought to impart rigidity to the backbone, as it partially cradles the FAD in these hydroxylases. Thus, this residue has been substituted with serine by site-directed mutagenesis to investigate the importance of flexibility of the peptide segment in catalysis. Differential scanning calorimetry demonstrated that the mutation has decreased the stability of the folded mutant protein compared to the wild-type PHBH. The increased flexibility in the protein backbone enhanced the accessibility of the flavin hydroperoxide intermediate to the solvent, causing an increase in the elimination of H(2)O(2) from this labile intermediate and, consequently, a decrease in the efficiency of substrate hydroxylation. Additionally, the increased accessibility of this mutant form of the enzyme makes it more susceptible than the wild-type enzyme to being trapped in the hydroxyflavin intermediate form in the presence of high levels of p-hydroxybenzoate. The mutation also lowers the pK(a) of the phenolic oxygen of bound p-hydroxybenzoate, and eliminates the pH dependence of the rate constant for flavin reduction by NADPH. These experimental observations lead to a model that explains how the wild-type protein can sense the charge of the 4-substituent of the aromatic ligand and link this charge to a flavin conformational change that is required for reaction with NADPH: (i) The peptide oxygen of Pro 293 is repelled by the negative charge of the phenolic oxygen of p-hydroxybenzoate. (ii) This repulsion is transmitted through the peptide backbone, causing the movement of Asn 300. (iii) The change in the position of Asn 300 triggers the movement of the flavin from the largely buried "in" conformation to the exposed, reactive "out" conformation.     

Unusual folded conformation of nicotinamide adenine dinucleotide bound to flavin reductase P.

The 2.1 A resolution crystal structure of flavin reductase P with the inhibitor nicotinamide adenine dinucleotide (NAD) bound in the active site has been determined. NAD adopts a novel, folded conformation in which the nicotinamide and adenine rings stack in parallel with an inter-ring distance of 3.6 A. The pyrophosphate binds next to the flavin cofactor isoalloxazine, while the stacked nicotinamide/adenine moiety faces away from the flavin. The observed NAD conformation is quite different from the extended conformations observed in other enzyme/NAD(P) structures; however, it resembles the conformation proposed for NAD in solution. The flavin reductase P/NAD structure provides new information about the conformational diversity of NAD, which is important for understanding catalysis. This structure offers the first crystallographic evidence of a folded NAD with ring stacking, and it is the first enzyme structure containing an FMN cofactor interacting with NAD(P). Analysis of the structure suggests a possible dynamic mechanism underlying NADPH substrate specificity and product release that involves unfolding and folding of NADP(H).     

Protein dynamics control proton transfers to the substrate on the His72Asn mutant of p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase (PHBH) hydroxylates activated benzoates using NADPH as a reductant and O(2) as an oxygenating substrate. Because the flavin, when reduced, will quickly react with oxygen in either the presence or absence of a phenolic substrate, it is important to regulate flavin reduction to prevent the uncontrolled reaction of NADPH and oxygen to form H(2)O(2). Reduction is controlled by the protonation state of the aromatic substrate p-hydroxybenzoate (pOHB), which when ionized to the phenolate facilitates the movement of flavin between two conformations, termed "in" and "out". When the hydrogen bond network that provides communication between the substrate and solvent is disrupted by changing its terminal residue, His72, to Asn, protons from solution no longer equilibrate rapidly with pOHB bound to the active site [Palfey, B. A., Moran, G. R., Entsch, B., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 1153-1158]. Thus, one population of the His72Asn enzyme reduces rapidly and has the phenolate form of pOHB bound at the active site and the flavin in the out conformation. The remaining population of the His72Asn enzyme reduces slowly and has the phenolic form of pOHB bound and the flavin in the in conformation. We have investigated the mechanisms of proton transfer between solvent and pOHB bound to the His72Asn form of the enzyme by double-mixing and single-mixing stopped-flow experiments. We find that, depending on the initial ionization state of bound pOHB and the new pH of the solution, the ionization/protonation of pOHB proceeds through the direct reaction of hydronium or hydroxide with the enzyme-ligand complex and leads to the conversion of one flavin conformation to the other. Our kinetic data indicate that the enzyme with the flavin in the in conformation reacts in two steps. Inspection of crystal structures suggests that the hydroxide ion would react at the re-face of the flavin, and its reaction with pOHB is limited by the movement of Pro293, a conserved residue in similar flavoprotein hydroxylases. We hypothesize that this type of breathing mode by the protein may have been used to compensate for the lack of an efficient proton-transfer network in ancestral hydroxylases, permitting useful catalysis prior to the emergence of specialized proton-transfer mechanisms.     

Constraining enzyme conformational change by an antibody leads to hyperbolic inhibition

Although it has been known for many years that antibodies display properties characteristic of allosteric effectors, the molecular mechanisms responsible for these effects remain poorly understood. Here, we describe a single-domain antibody fragment (nanobody) that modulates protein function by constraining conformational change in the enzyme dihydrofolate reductase (DHFR). Nanobody 216 (Nb216) behaves as a potent allosteric inhibitor of DHFR, giving rise to mixed hyperbolic inhibition kinetics. The crystal structure of Nb216 in complex with DHFR reveals that the nanobody binds adjacent to the active site. Half of the epitope consists of residues from the flexible Met20 loop. This loop, which ordinarily oscillates between occluded and closed conformations during catalysis, assumes the occluded conformation in the Nb216-bound state. Using stopped flow, we show that Nb216 inhibits DHFR by stabilising the occluded Met20 loop conformation. Surprisingly, kinetic data indicate that the Met20 loop retains sufficient conformational flexibility in the Nb216-bound state to allow slow substrate turnover to occur.     

Identification of the active conformation and the importance of length of the flexible loop 72-83 in regulating the conformational change of undecaprenyl pyrophosphate synthase

Increasing evidence has shown that intrinsic disorder of proteins plays a key role in their biological functions. In the case of undecaprenyl pyrophosphate synthase (UPPs), which catalyzes the chain elongation of farnesyl pyrophosphate (FPP) to undecaprenyl pyrophosphate via eight consecutive condensation reactions with isopentenyl pyrophosphate, a highly flexible loop 72-83 was previously linked to protein conformational change required for catalysis [Chen, Y. H., Chen, A. P.-C., Chen, C. T., Wang, A. H.-J., and Liang, P. H., (2002) J. Biol. Chem. 277, 7369-7376]. The crystal structure and fluorescence studies suggested that the alpha3 helix connected to the loop moves toward the active site when the substrate is bound. To identify the active conformation and study the role of the loop for conformational change, the UPPs mutants with amino acids inserted into or deleted from the loop were examined. The inserted mutant with extra Ala residues fails to display the intrinsic fluorescence quenching upon FPP binding, and its crystal structure reveals only the open form. These phenomena appear to be different from the wild-type enzyme in which open and closed conformers were observed and suggest that the extended loop fails to pull the alpha3 helix and/or the extra amino acids in the loop cause steric hindrance on the alpha3 helix movement. The loop-shortening mutants with deletion of V82 and S83 or S72 also adopt an open conformation with the loop stretched, although they show decreased intrinsic fluorescence with FPP bound, similar to that seen in the wild-type enzyme. We conclude that the closed conformation is apparently the active conformation. Change of the length of the loop 72-83 impairs the ability of conformational change and causes remarkably lower activity of UPPs.     

Charge neutralization in the active site of the catalytic trimer of aspartate transcarbamoylase promotes diverse structural changes

A classical model for allosteric regulation of enzyme activity posits an equilibrium between inactive and active conformations. An alternative view is that allosteric activation is achieved by increasing the potential for conformational changes that are essential for catalysis. In the present study, substitution of a basic residue in the active site of the catalytic (C) trimer of aspartate transcarbamoylase with a non‐polar residue results in large interdomain hinge changes in the three chains of the trimer. One conformation is more open than the chains in both the wild‐type C trimer and the catalytic chains in the holoenzyme, the second is closed similar to the bisubstrate‐analog bound conformation and the third hinge angle is intermediate to the other two. The active‐site 240s loop conformation is very different between the most open and closed chains, and is disordered in the third chain, as in the holoenzyme. We hypothesize that binding of anionic substrates may promote similar structural changes. Further, the ability of the three catalytic chains in the trimer to access the open and closed active‐site conformations simultaneously suggests a cyclic catalytic mechanism, in which at least one of the chains is in an open conformation suitable for substrate binding whereas another chain is closed for catalytic turnover. Based on the many conformations observed for the chains in the isolated catalytic trimer to date, we propose that allosteric activation of the holoenzyme occurs by release of quaternary constraint into an ensemble of active‐site conformations.     

Properties of p-hydroxybenzoate hydroxylase when stabilized in its open conformation

p-Hydroxybenzoate hydroxylase is extensively studied as a model for single-component flavoprotein monooxygenases. It catalyzes a reaction in two parts: (1) reduction of the FAD in the enzyme by NADPH in response to binding of p-hydroxybenzoate to the enzyme and (2) oxidation of reduced FAD with oxygen in an environment free from solvent to form a hydroperoxide, which then reacts with p-hydroxybenzoate to form an oxygenated product. These different reactions are coordinated through conformational rearrangements of the protein and the isoalloxazine ring during catalysis. Until recently, it has not been clear how p-hydroxybenzoate gains access to the buried active site. In 2002, a structure of a mutant form of the enzyme without substrate was published that showed an open conformation with solvent access to the active site [Wang, J., Ortiz-Maldonado, M., Entsch, B., Massey, V., Ballou, D., and Gatti, D. L. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 608-613]. The wild-type enzyme does not form high-resolution crystals without substrate. We hypothesized that the wild-type enzyme without substrate also forms an open conformation for binding p-hydroxybenzoate, but only transiently. To test this idea, we have studied the properties of two different mutant forms of the enzyme that are stabilized in the open conformation. These mutant enzymes bind p-hydroxybenzoate very fast, but with very low affinity, as expected from the open structure. The mutant enzymes are extremely inactive, but are capable of slowly forming small amounts of product by the normal catalytic pathway. The lack of activity results from the failure of the mutants to readily form the out conformation required for flavin reduction by NADPH. The mutants form a large fraction of an abnormal conformation of the reduced enzyme with p-hydroxybenzoate bound. This conformation of the enzyme is unreactive with oxygen. We conclude that transient formation of this open conformation is the mechanism for sequestering p-hydroxybenzoate to initiate catalysis. This overall study emphasizes the role that protein dynamics can play in enzymatic catalysis.     

Using Raman spectroscopy to monitor the solvent-exposed and "buried" forms of flavin in p-hydroxybenzoate hydroxylase

X-ray crystallographic studies of several complexes involving FAD bound to p-hydroxybenzoate hydroxylase (PHBH) have revealed that the isoalloxazine ring system of FAD is capable of adopting in two positions on the protein. In one, the "in" form, the ring is surrounded by protein groups and has little contact with solvent; in the second, "out" form, the ring is largely solvent exposed. Using Raman difference spectroscopy, it has been possible to obtain Raman spectra for the flavin ring in both conformational states for different complexes in solution. The spectra consist of a rich assortment of isoalloxazine ring modes whose normal mode origin can be assigned by using density functional theory and ab initio calculations. Further insight into the sensitivity of these modes to changes in environment is provided by the Raman spectra of lumiflavin in the solid state, in DMSO and in aqueous solution. For the protein complexes, the Raman difference spectra of flavin bound to wt PHBH and wt PHBH plus substrate, p-hydroxybenzoate, provided examples of the "in" conformation. These data are compared to those for flavin bound to wt PHBH plus 2,4-dihydroxybenzoate, where X-ray analysis show that the flavin is "out". There are several spectral regions where characteristic differences exist for flavin in the "in" or "out" conformation, these occur near 1700, 1500, 1410, 1350, 1235, and 1145 cm(-)(1). These spectral features can be used as empirical marker bands to determine the populations of "in" and "out" for any complex of PHBH and to monitor changes in those populations with perturbations to the system, e.g., by changing temperature or pH. Thus, it will now be possible to determine the conformational state of the flavin in PHBH for those complexes that have resisted X-ray crystallographic analysis. Raman difference data are also presented for the Tyr222Phe mutant. The Raman data show that the isoalloxazine ring is predominantly "out" for Tyr222Phe. However, in the presence of the substrate p-hydroxybenzoate there is clear evidence from the Raman marker bands that a mixed population of "in" and "out" exists with the majority being in the "out" state. This is consistent with the conclusions drawn from crystallographic studies on this complex (Gatti, D. L., Palfey, B. A., Lah, M. S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science, 266, 110-114).     

Biochemical characterization of a flavin adenine dinucleotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism

Pyoverdin is the hydroxamate siderophore produced by the opportunistic pathogen Pseudomonas aeruginosa under the iron-limiting conditions of the human host. This siderophore includes derivatives of ornithine in the peptide backbone that serve as iron chelators. PvdA is the ornithine hydroxylase, which performs the first enzymatic step in preparation of these derivatives. PvdA requires both flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate (NADPH) for activity; it was found to be a soluble monomer most active at pH 8.0. The enzyme demonstrated Michaelis-Menten kinetics in an NADPH oxidation assay, but a hydroxylation assay indicated substrate inhibition at high ornithine concentration. PvdA is highly specific for both substrate and coenzyme, and lysine was shown to be a nonsubstrate effector and mixed inhibitor of the enzyme with respect to ornithine. Chloride is a mixed inhibitor of PvdA with respect to ornithine but a competitive inhibitor with respect to NADPH, and a bulky mercurial compound (p-chloromercuribenzoate) is a mixed inhibitor with respect to ornithine. Steady-state experiments indicate that PvdA/FAD forms a ternary complex with NADPH and ornithine for catalysis. PvdA in the absence of ornithine shows slow substrate-independent flavin reduction by NADPH. Biochemical comparison of PvdA to p-hydroxybenzoate hydroxylase (PHBH, from Pseudomonas fluorescens) and flavin-containing monooxygenases (FMOs, from Schizosaccharomyces pombe and hog liver microsomes) leads to the hypothesis that PvdA catalysis proceeds by a novel reaction mechanism.     

Protein dynamics and electrostatics in the function of p-hydroxybenzoate hydroxylase

para-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor, FAD, by NADPH in response to binding p-hydroxybenzoate to the enzyme, then oxidation of reduced FAD by oxygen to form a hydroperoxide, which oxygenates p-hydroxybenzoate to form 3,4-dihydroxybenzoate. These diverse reactions all occur within a single polypeptide and are achieved through conformational rearrangements of the isoalloxazine ring and protein residues within the protein structure. In this review, we examine the complex dynamic behavior of the protein that enables regulated fast and specific catalysis to occur. Original research papers (principally from the past 15 years) provide the information that is used to develop a comprehensive overview of the catalytic process. Much of this information has come from detailed analysis of many specific mutants of the enzyme using rapid reaction technology, biophysical measurements, and high-resolution structures obtained by X-ray crystallography. We describe how three conformations of the enzyme provide a foundation for the catalytic cycle. One conformation has a closed active site for the conduct of the oxygen reactions, which must occur in the absence of solvent. The second conformation has a partly open active site for exchange of substrate and product, and the third conformation has a closed protein structure with the isoalloxazine ring rotated out to the surface for reaction with NADPH, which binds in a surface cleft. A fundamental feature of the enzyme is a H-bond network that connects the phenolic group of the substrate in the buried active site to the surface of the protein. This network serves to protonate and deprotonate the substrate and product in the active site to promote catalysis and regulate the coordination of conformational states for efficient catalysis.     

Synergistic interactions of multiple mutations on catalysis during the hydroxylation reaction of p-hydroxybenzoate hydroxylase: studies of the Lys297Met, Asn300Asp, and Tyr385Phe mutants reconstituted with 8-Cl-flavin

The oxygen transfer to p-hydroxybenzoate catalyzed by p-hydroxybenzoate hydroxylase (PHBH) has been shown to occur via a C4a-hydroperoxide of the flavin. Two factors are likely to be important in facilitating the transfer of oxygen from the C4a-hydroperoxide to the substrate. (a) The positive electrostatic potential of the active site partially stabilizes the negative charge centered on the oxygen of the flavin-C4a-alkoxide leaving group during the transition state [Ortiz-Maldonado, M., Ballou, D. P., and Massey, V. (1999) Biochemistry 38, 8124-8137]. (b) The hydrogen-bonding network ionizes the substrate to promote its nucleophilic attack on the electrophilic C4a-hydroperoxide intermediate [Entsch, B., Palfey, B. A., Ballou, D. P., and Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. This ionization is also aided by the positive electrostatic potential of the active site [Moran, G. R., Entsch, B., Palfey, B. A., and Ballou, D. P. (1997) Biochemistry 36, 7548-7556]. Substituents on the flavin can specifically affect the stability of the alkoxide leaving-group, whereas changes to specific enzyme residues can affect the charge in the active site and the hydrogen-bonding network. We have used wild-type (WT) PHBH and several mutant forms, all with normal FAD and with 8-Cl-FAD substituted for FAD, to assess the relative contributions of the two effects. Lys297Met and Asn300Asp have decreased positive charge in the active site, and these variants engender approximately 35-fold slower hydroxylation rates than the WT enzyme. Substitution of 8-Cl-FAD in these mutant forms gives approximately 1.8-fold increases in hydroxylation rates, compared with a > or =4.8-fold increase for WT with this flavin. The hydroxylation catalyzed by Tyr385Phe, a mutant enzyme form with a disrupted hydrogen-bonding network that compromises the ionization of the substrate without changing the positive charge of the active site, is stimulated 1.5-fold by substituting the enzyme with 8-Cl-FAD. The substrate, tetrafluoro-p-hydroxybenzoate, is fully ionized in WT PHBH, but this phenolate is a poor nucleophile because of the electron-withdrawing effects of the fluorine substituents. With tetrafluoro-p-hydroxybenzoate as the substrate, substitution of FAD with 8-Cl-FAD in the WT enzyme stabilizes the leaving alkoxide and leads to a 2.3-fold increase in the hydroxylation rate compared to that with FAD. Either the use of substrates that do not communicate with the proton network or the mutation of amino acid residues that perturb this interaction may prevent a necessary conformational change that allows proper orientation between reactants during the hydroxylation reaction or permits the essential protonation of the initially formed nascent flavin-C4a-peroxide anion. Thus, both activation of substrate by the proton network and stabilization of the leaving alkoxide appear to be important for oxygen transfer catalyzed by PHBH. The full effect of the substituents on the flavin (4.8-fold) can only be realized when the optimal transition state can be achieved, and this optimal state is not fully realized with the mutant forms.     

Structural Analysis of a Novel Cyclohexylamine Oxidase from Brevibacterium oxydans IH-35A

Cyclohexylamine oxidase (CHAO) is a flavoprotein first described in Brevibacterium oxydans strain IH-35A that carries out the initial step of the degradation of the industrial chemical cyclohexylamine to cyclohexanone. We have cloned and expressed in Escherichia coli the CHAO-encoding gene (chaA) from B. oxydans, purified CHAO and determined the structures of both the holoenzyme form of the enzyme and a product complex with cyclohexanone. CHAO is a 50 kDa monomer with a PHBH fold topology. It belongs to the flavin monooxygenase family of enzymes and exhibits high substrate specificity for alicyclic amines and sec-alkylamines. The overall structure is similar to that of other members of the flavin monooxygenase family, but lacks either of the C- or N-terminal extensions observed in these enzymes. Active site features of the flavin monooxygenase family are conserved in CHAO, including the characteristic aromatic cage. Differences in the orientations of residues of the CHAO aromatic cage result in a substrate-binding site that is more open than those of its structural relatives. Since CHAO has a buried hydrophobic active site with no obvious route for substrates and products, a random acceleration molecular dynamics simulation has been used to identify a potential egress route. The path identified includes an intermediate cavity and requires transient conformation changes in a shielding loop and a residue at the border of the substrate-binding cavity. These results provide a foundation for further studies with CHAO aimed at identifying features determining substrate specificity and for developing the biocatalytic potential of this enzyme.     

The Cys319 loop modulates the transition between dehydrogenase and hydrolase conformations in inosine 5'-monophosphate dehydrogenase

X-ray crystal structures of enzyme-ligand complexes are widely believed to mimic states in the catalytic cycle, but this presumption has seldom been carefully scrutinized. In the case of Tritrichomonas foetus inosine 5'-monophosphate dehydrogenase (IMPDH), 10 structures of various enzyme-substrate-inhibitor complexes have been determined. The Cys319 loop is found in at least three different conformations, suggesting that its conformation changes as the catalytic cycle progresses from the dehydrogenase step to the hydrolase reaction. Alternatively, only one conformation of the Cys319 loop may be catalytically relevant while the others are off-pathway. Here we differentiate between these two hypotheses by analyzing the effects of Ala substitutions at three residues of the Cys319 loop, Arg322, Glu323, and Gln324. These mutations have minimal effects on the value of k(cat) (≤5-fold) that obscure large effects (>10-fold) on the microscopic rate constants for individual steps. These substitutions increase the equilibrium constant for the dehydrogenase step but decrease the equilibrium between open and closed conformations of a mobile flap. More dramatic effects are observed when Arg322 is substituted with Glu, which decreases the rates of hydride transfer and hydrolysis by factors of 2000 and 130, respectively. These experiments suggest that the Cys319 loop does indeed have different conformations during the dehydrogenase and hydrolase reactions as suggested by the crystal structures. Importantly, these experiments reveal that the structure of the Cys319 loop modulates the closure of the mobile flap. This conformational change converts the enzyme from a dehydrogenase into hydrolase, suggesting that the conformation of the Cys319 loop may gate the catalytic cycle.     

Substitutions in a flexible loop of horse liver alcohol dehydrogenase hinder the conformational change and unmask hydrogen transfer.

When horse liver alcohol dehydrogenase binds coenzyme, a rotation of about 10 degrees brings the catalytic domain closer to the coenzyme binding domain and closes the active site cleft. The conformational change requires that a flexible loop containing residues 293-298 in the coenzyme binding domain rearranges so that the coenzyme and some amino acid residues from the catalytic domain can be accommodated. The change appears to control the rate of dissociation of the coenzyme and to be necessary for installation of the proton relay system. In this study, directed mutagenesis produced the activated Gly293Ala/Pro295Thr enzyme. X-ray crystallography shows that the conformations of both free and complexed forms of the mutated enzyme and wild-type apoenzyme are very similar. Binding of NAD(+) and 2,2, 2-trifluoroethanol do not cause the conformational change, but the nicotinamide ribose moiety and alcohol are not in a fixed position. Although the Gly293Ala and Pro295Thr substitutions do not disturb the apoenzyme structure, molecular modeling shows that the new side chains cannot be accommodated in the closed native holoenzyme complex without steric alterations. The mutated enzyme may be active in the "open" conformation. The turnover numbers with ethanol and acetaldehyde increase 1.5- and 5.5-fold, respectively, and dissociation constants for coenzymes and other kinetic constants increase 40-2,000-fold compared to those of the native enzyme. Substrate deuterium isotope effects on the steady state V or V/K(m) parameters of 4-6 with ethanol or benzyl alcohol indicate that hydrogen transfer is a major rate-limiting step in catalysis. Steady state oxidation of benzyl alcohol is most rapid above a pK of about 9 for V and V/K(m) and is 2-fold faster in D(2)O than in H(2)O. The results are consistent with hydride transfer from a ground state zinc alkoxide that forms a low-barrier hydrogen bond with the hydroxyl group of Ser48.     

Closed conformation of the active site loop of rabbit muscle triosephosphate isomerase in the absence of substrate: evidence of conformational heterogeneity

The active site loop of triosephosphate isomerase (TIM) exhibits a hinged-lid motion, alternating between the two well defined "open" and "closed" conformations. Until now the closed conformation had only been observed in protein complexes with substrate analogues. Here, we present the first rabbit muscle apo TIM structure, refined to 1.5A resolution, in which the active site loop is either in the open or in the closed conformation in different subunits of the enzyme. In the closed conformation described here, the lid loop residues participate in stabilizing hydrogen bonds characteristic of holo TIM structures, whereas chemical interactions observed in the open loop conformation are similar to those found in the apo structures of TIM. In the closed conformation, a number of water molecules are observed at the projected ligand atom positions that are hydrogen bonded to the active site residues. Additives used during crystallization (DMSO and Tris molecules and magnesium atoms) were modeled in the electron density maps. However, no specific binding of these molecules is observed at, or close to, the active site and the lid loop. To further investigate this unusual closed conformation of the apo enzyme, two more rabbit muscle TIM structures, one in the same and another in a different crystal form, were determined. These structures present the open lid conformation only, indicating that the closed conformation cannot be explained by crystal contact effects. To rationalize why the active site loop is closed in the absence of ligand in one of the subunits, extensive comparison with previously solved TIM structures was carried out, supported by the bulk of available experimental information about enzyme kinetics and reaction mechanism of TIM. The observation of both open and closed lid conformations in TIM crystals might be related to a persistent conformational heterogeneity of this protein in solution.     

NikD, an unusual amino acid oxidase essential for nikkomycin biosynthesis: structures of closed and open forms at 1.15 and 1.90 A resolution

NikD is an unusual amino-acid-oxidizing enzyme that contains covalently bound FAD, catalyzes a 4-electron oxidation of piperideine-2-carboxylic acid to picolinate, and plays a critical role in the biosynthesis of nikkomycin antibiotics. Crystal structures of closed and open forms of nikD, a two-domain enzyme, have been determined to resolutions of 1.15 and 1.9 A, respectively. The two forms differ by an 11 degrees rotation of the catalytic domain with respect to the FAD-binding domain. The active site is inaccessible to solvent in the closed form; an endogenous ligand, believed to be picolinate, is bound close to and parallel with the flavin ring, an orientation compatible with redox catalysis. The active site is solvent accessible in the open form, but the picolinate ligand is approximately perpendicular to the flavin ring and a tryptophan is stacked above the flavin ring. NikD also contains a mobile cation binding loop.