Coenzyme recognition in para-hydroxybenzoate hydroxylase

The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [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; Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. Kinetic studies detected the movement of the flavin from the buried conformation to the exposed conformation caused by the binding of NADPH prior to its reaction with the flavin. The pH dependence of the rate constant for flavin reduction in wild-type PHBH and the His72Asn mutant indicates that the deprotonation of bound p-hydroxybenzoate is also required for flavin movement, and is accomplished by the same internal proton transport network previously found to be involved in substrate oxidation. The linkage of substrate deprotonation to flavin movement constitutes a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.

1H, 13C, 15N and 31P NMR was applied to a study of free and protein-bound flavins in order to obtain a better insight into the mechanism by which the function of the flavin coenzyme is "tuned" upon binding to apoflavoproteins. A thorough 13C and 15N investigation of free flavins provided a detailed view on the effects of sterical hindrance and polarization on the structure of oxidized and reduced flavins. Moreover, this study resulted in a semi-empirical basis for the interpretation of NMR data obtained on protein-bound flavins. Remarkable results of the study of free flavins are the fact, that oxidized isoalloxazine is not fully planar in an apolar medium, and that fully reduced isoalloxazine is more planar than believed up till now. Unsubstituted reduced isoalloxazine in water has an almost fully sp 2hybridized N(10) and an N(5) atom which is approximately 70% sp 2hybridized. Upon modification the hybridization of N(5) and N(10) can be modulated rather independently due to steric hindrance. The so-called "butterfly" motion has a low activation barrier, which is probably an important feature for the mentioned tuning mechanism.Flavodoxin from Megasphaeraelsdenii is the protein which is most extensively studied in this thesis. By a 13C relaxation study it was shown that the isoalloxazine ring is strongly immobilized upon binding to apoflavodoxin. Also the phosphate group of FMN is strongly immobilized in the interior of the protein. Moreover, the phosphate group is dianionic in the complex, regardless of the redox state of bound FMN. Based on the NMR data, it was shown that the redox potential for the transition semiquinone/hydroquinone is mainly governed by charge interactions. For the first time a quantification of the redox potential modification by the apoprotein could be established, thereby showing that, contrary to literature reports, the redox potential modulation is not governed by steric effects exerted on the isoalloxazine moiety. Both by 31P NMR and 1H NMR it was shown that M.elsdenii flavodoxin acts as a one-electron transferring protein shuttling between the semiquinone and hydroquinone state, because the oxidized state is ruled out as a biologically relevant redox state in this protein due to the introduction of a high activation barrier between the oxidized and semiquinone state. Moreover, the electron transfer mechanism is of an outer sphere type. Based on the NMR data some arguments are presented that a specific complex formation between M.elsdenii flavodoxin and the electron donor or acceptor is not needed for an effective electron transfer. An important part of the active center of M.elsdenii flavodoxin was elucidated using time resolved photochemically induced dynamic nuclear polarization (CIDNP) and modern two-dimensional NMR techniques. The use of the paramagnetic semiquinone state in combination with 2D NMR techniques allowed the generation of NMR spectra of only the active center. Complete assignments were given in both the oxidized and the hydroquinone state for Trp-91, Ala-56 and Tyr-89, which are all very close to the isoalloxazine ring. It was shown that the relative position of Trp-91 with respect to the prosthetic group is slightly different in the two redox states. The active center of flavodoxin from M.elsdenii and Clostridium MP appear to be similar. The only small difference between results of the crystallographic study on Clostridium MP flavodoxin and 1H NMR results on M.elsdenii flavodoxin was observed for the relative position of Ala-56 and Tyr-89.A detailed 13C and 15N NMR study was performed on the complex of riboflavin and Riboflavin Binding Protein from the egg yolk and egg white. Subtle information on hydrogen bonding, conformation of the isoalloxazine ring and solvent accessibility were obtained in oxidized and reduced state. As far as the results could be compared with reported binding studies of riboflavin analogues, the results appear to be in excellent agreement. The pyrimidine ring is exposed to solvent, except for 0(4α) in both redox states. N(10) is forced into the molecular plane, which in turn probably causes N(1) to be somewhat out of the plane. The NMR results are consistent with a partial opening of the protein at pH 6 and a more stable conformation at pH 9. Based on the NMR results a possible function for the complex in the embryonic development is suggested.As an example of the class of dehydrogenases, lipoamide dehydrogenase from Azotobacter vinelandii was studied by 13C NMR. In the oxidized state hydrogen bonds exist to 0(2α) and 0(4α), but the polarization of the isoalloxazine ring probably does not extend to the N(10) atom. The 4 electron reduced protein (both the essential disulfide and the flavin are reduced) contains an essentially planar N(10) atom, and again hydrogen bonds to 0(2α) and 0(4α). The 2 electron reduced protein (EH 2 ) appeared to be particularly interesting. It consists of an equal mixture of protein in which the disulfide is reduced and the flavin oxidized, and protein in which the disulfide is oxidized and the flavin reduced. The results allowed a detailed description of the electronic structure of the two-electron reduced protein. From the NMR results it is concluded that the redox potential of both centers is roughly the same. The results show that if the disulfide is reduced one thiol group is present as an anionic thiol group and extremely close to the C(4a) atom of the flavin. The exchange of reduction equivalents between the two redox centers in EH 2 is slow (<5 s -1). In combination with reported kinetic data, it became evident that NAD +accelerates considerably the exchange of reduction equivalents between the disulfide and the flavin. The results show that this transfer must be implemented in the reaction cycle and offer a nice explanation of some "anomalous" kinetic data.As an example of the class of hydroxylases, p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens was studied by 1C NMR. Without substrate the isoalloxazine ring is probably exposed to solvent. Upon substrate binding a drastic change of the polarization of C(2) occurs, which is in accord with the published enzyme-substrate complex as revealed by crystallographic methods. It is suggested, that the active center without substrate is rather mobile in order to facilitate the binding of substrate. A conformational change accompanies the binding of substrate. The active center, as present in the oxidized enzyme-substrate complex, is probably already essentially formed upon reduction of the enzyme in the absence of substrate, which explains the drastically decreased binding rate of substrate after reduction. Upon complex formation' with substrate in the reduced state the N(5) is forced into the molecular plane of flavin. Some arguments are presented suggesting that the sp 2hybridization degree of N(5) and N(10) are the main factors which govern the reactivity (or "activation") of oxygen, but not the πelectron density at the C(4a) center of flavin. An electron transfer from the hydroquinone state towards oxygen probably precedes the formation of a C(4a) peroxyflavin by radical pair combination.

The conserved residues His-162 and Arg-269 of the flavoprotein p-hydroxybenzoate hydroxylase (EC 1.14.13.2) are located at the entrance of the interdomain cleft that leads toward the active site. To study their putative role in NADPH binding, His-162 and Arg-269 were selectively changed by site-specific mutagenesis. The catalytic properties of H162R, H162Y, and R269K were similar to the wild-type enzyme. However, less conservative His-162 and Arg-269 replacements strongly impaired NADPH binding without affecting the conformation of the flavin ring and the efficiency of substrate hydroxylation. The crystal structures of H162R and R269T in complex with 4-hydroxybenzoate were solved at 3.0 and 2.0 A resolution, respectively. Both structures are virtually indistinguishable from the wild-type enzyme-substrate complex except for the substituted side chains. In contrast to wild-type p-hydroxybenzoate hydroxylase, H162R is not inactivated by diethyl pyrocarbonate. NADPH protects wild-type p-hydroxybenzoate hydroxylase from diethylpyrocarbonate inactivation, suggesting that His-162 is involved in NADPH binding. Based on these results and GRID calculations we propose that the side chains of His-162 and Arg-269 interact with the pyrophosphate moiety of NADPH. An interdomain binding mode for NADPH is proposed which takes a novel sequence motif (Eppink, M. H. M., Schreuder, H. A., and van Berkel, W. J. H. (1997) Protein Sci. 6, 2454-2458) into account.

Cystalysin is a C(beta)-S(gamma) lyase from the oral pathogen Treponema denticola catabolyzing L-cysteine to produce pyruvate, ammonia and H(2)S. With its ability to induce cell lysis, cystalysin represents a new class of pyridoxal 5'-phosphate (PLP)-dependent virulence factors. The crystal structure of cystalysin was solved at 1.9 A resolution and revealed a folding and quaternary arrangement similar to aminotransferases. Based on the active site architecture, a detailed catalytic mechanism is proposed for the catabolism of S-containing amino acid substrates yielding H(2)S and cysteine persulfide. Since no homologies were observed with known haemolysins the cytotoxicity of cystalysin is attributed to this chemical reaction. Analysis of the cystalysin-L-aminoethoxyvinylglycine (AVG) complex revealed a 'dead end' ketimine PLP derivative, resulting in a total loss of enzyme activity. Cystalysin represents an essential factor of adult periodontitis, therefore the structure of the cystalysin-AVG complex may provide the chemical basis for rational drug design.

The catalytic center of nitric-oxide synthase (NOS) consists of a thiolate-coordinated heme macrocycle, a tetrahydrobiopterin (H4B) cofactor, and an l-arginine (l-Arg)/N-hydroxyarginine substrate binding site. To determine how the interplay between the cofactor, the substrates, and the protein matrix housing the heme regulates the enzymatic activity of NOS, the CO-, NO-, and CN(-)-bound adducts of the oxygenase domain of the inducible isoform of NOS (iNOS(oxy)) were examined with resonance Raman spectroscopy. The Raman data of the CO-bound ferrous protein demonstrated that the presence of l-Arg causes the Fe-C-O moiety to adopt a bent structure because of an H-bonding interaction whereas H4B binding exerts no effect. Similar behavior was found in the CN(-)-bound ferric protein and in the nitric oxide (NO)-bound ferrous protein. In contrast, in the NO-bound ferric complexes, the addition of l-Arg alone does not affect the structural properties of the Fe-N-O moiety, but H4B binding forces it to adopt a bent structure, which is further enhanced by the subsequent addition of l-Arg. The differential interactions between the various heme ligands and the protein matrix in response to l-Arg and/or H4B binding is coupled to heme distortions, as reflected by the development of a variety of out-of-plane heme modes in the low frequency Raman spectra. The extent and symmetry of heme deformation modulated by ligand, substrate, and cofactor binding may provide important control over the catalytic and autoinhibitory properties of the enzyme.

The crystal structure of the enzyme-substrate complex of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4-hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr-->Phe mutant enzymes. The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201-->Phe and Tyr385-->Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p-hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349]. Enzyme Tyr201-->Phe has a high Km for NADPH and produces only 5% of 3,4-dihydroxybenzoate/catalytic cycle. Unlike the wild-type enzyme, the Tyr201-->Phe mutant does not stabilize the phenolate form of 4-hydroxybenzoate. With enzyme Tyr385-->Phe, flavin reduction is rate-limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385-->Phe prefers the binding of the phenolic form of 4-hydroxybenzoate. Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4-fluorobenzoate differentially stimulates the stabilization of the 4 alpha-hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5-trihydroxybenzoate from 3,4-dihydroxybenzoate. The affinity of enzyme Tyr201-->Phe for the dianionic substrate 2,3,5,6-tetrafluoro-4-hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment. In contrast to data reported for p-hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4-hydroxycinnamate to wild-type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201-->Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385-->Phe, the phenolic pKa of bound 4-hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385-->Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.

Biliverdin reductase is unique among all enzymes described to date in having two pH optima, 6.75 and 8.7, at which NADH or NADPH, respectively, are required for activity. The enzyme converts biliverdin to bilirubin in mammals. The mature enzyme, which is 293 amino acids long, has 3 cysteine residues, and is sulfhydryl dependent. To understand the role of the cysteine residues in enzyme activity, we examined the effects of the neutral substitution with alanine of each of three residues, individually and in combination, by site-directed mutagenesis. These residues in the predicted amino acid sequence of rat biliverdin reductase correspond to amino acids 73, 280 and 291. The modification of the amino-proximal cysteine (Cys73), which is flanked by a tyrosine residue, completely inactivated the enzyme with NADH at pH 6.75 and NADPH at pH 8.7. The loss of reductase activity was not due to changes in three-dimensional characteristics of the protein as suggested by its mobility in a non-denaturing gel. Although modification of either of the two cysteines located near the C-terminus (Cys280 and Cys291) significantly reduced activity with both cofactors, these mutations did not inactivate the enzyme. Comparison of Km values for the Cys280-->Ala and Cys291-->Ala mutants with the wild type protein, at pH 8.7, suggests that Cys280 principally functions in substrate binding while Cys291 is predominantly involved in cofactor binding. This assignment probably also applies at pH 6.75. Comparison of kcat of the mutants with wild type shows that mutation of Cys280 decreases Vmax of the enzyme. Mutation of both C-terminal cysteines caused inactivation of the enzyme, comparable to that produced by mutation of Cys73. Analysis by circular dichroism at far-ultraviolet wavelengths suggests that the alterations in activity are not the result of changes in the secondary structure of these mutants. These results are consistent with Cys73 having a central role in substrate/cofactor binding while biliverdin reductase can function, albeit at a reduced rate, with only one of the near C-terminus cysteines. The results are further consistent with the suggestion that although the two C-terminal cysteines have preferential affinities, they can serve similar functions in the interaction with substrate/cofactor.

Pyridoxal 5'-phosphate-dependent enzymes may be grouped into five structural superfamilies of proteins, corresponding to as many fold types. The fold type I is by far the largest and most investigated group. An important feature of this fold, which is characterized by the presence of two domains, appears to be the existence of three clusters of evolutionarily conserved hydrophobic contacts. Although two of these clusters are located in the central cores of the domains and presumably stabilize their scaffold, allowing the correct alignment of the residues involved in cofactor and substrate binding, the role of the third cluster is much less evident. A site-directed mutagenesis approach was used to carry out a model study on the importance of the third cluster in the structure of a well characterized member of the fold type I group, serine hydroxymethyltransferase from Escherichia coli. The experimental results obtained indicated that the cluster plays a crucial role in the stabilization of the quaternary, native assembly of the enzyme, although it is not located at the subunit interface. The analysis of the crystal structure of serine hydroxymethyltransferase suggested that this stabilizing effect may be due to the strict structural relation between the cluster and two polypeptide loops, which, in fold type I enzymes, mediate the interactions between the subunits and are involved in cofactor binding, substrate binding and catalysis.

The conformational models of the active site of adenosine deaminase (ADA) and its complexes in the basic state with adenosine and 13 isosteric analogues of the aza, deaza, and azadeaza series were constructed. The optimization of the conformational energy of the active site and the nucleoside bound with it in the complex was achieved in the force field of the whole enzyme (the 1ADD structure was used) within the molecular mechanics model using the AMBER 99 potentials. The stable conformational states of each of the complexes, as well as the optimal conformation of the ADA in the absence of ligand, were determined. It was proved that the conformational state that is close to the structure of the ADA complex with 1-deazaadenosine (1ADD) known from the X-ray study corresponds to one of the local minima of the potential surface. Another, a significantly deeper minimum was determined; it differs from the first minimum by the mutual orientation of side chains of amino acid residues. A similar conformational state is optimal for the ADA active site in the absence of the bound ligand. A qualitative correlation exists between the values of potential energies of the complexes in this conformation and the enzymatic activity of ADA toward the corresponding nucleosides. The dynamics of conformational conversions of the active site after the binding of substrate or its analogues, as well as the possibility of the estimation of the inhibitory properties of nucleosides on the basis of calculations, are discussed.

Structural and kinetic investigations of the carboxy terminus of NADPH-cytochrome P450 oxidoreductase

The motor enzymes that belong to the family of RNA helicases catalyze the strand separation of duplex RNA via ATP hydrolysis. Among these enzymes, Escherichia coli DbpA is a unique RNA helicase because it possesses ATPase-specific activity toward the peptidyl transferase center in 23 S ribosomal RNA. For this reason, it has been the subject of numerous biochemical and structure-function studies. The ATP-stimulated unwinding activity of DbpA toward specific and nonspecific RNA duplexes has been demonstrated. However, the underlying molecular and structural basis, which facilitates its helicase activities, is presently not known. We combined time-dependent limited proteolysis digestion, fluorescence spectroscopy, and three-dimensional structural homology modeling techniques to study the structural conformations of DbpA with respect to its binding to stoichiometric ratios of RNA and cofactors. We show that the conformational state of DbpA is markedly different in the ADP-bound state than in any other state (ATP- or RNA-bound). These results, together with structural homology studies, suggest that a hinge region located in the core domain of DbpA mediates such conformational changes.

We derive approximate expressions for pre- and post-steady state regimes of the velocity-substrate-inhibitor spaces of the Michaelis-Menten enzyme kinetic scheme with fully and partial competitive inhibition. Our refinement over the currently available standard quasi steady state approximation (sQSSA) seems to be valid over wide range of enzyme to substrate and enzyme to inhibitor concentration ratios. Further, we show that the enzyme-inhibitor-substrate system can exhibit temporally well-separated two different steady states with respect to both enzyme-substrate and enzyme-inhibitor complexes under certain conditions. We define the ratios fS = vmax/(KMS + e0) and fI = umax/(KMI + e0) as the acceleration factors with respect to the catalytic conversion of substrate and inhibitor into their respective products. Here KMS and KMI are the Michaelis-Menten parameters associated respectively with the binding of substrate and inhibitor with the enzyme, vmax and umax are the respective maximum reaction velocities and e0, s0, and i0 are total enzyme, substrate and inhibitor levels. When (fS/fI) < 1, then enzyme-substrate complex will show multiple steady states and it reaches the full-fledged steady state only after the depletion of enzyme-inhibitor complex. When (fS/fI) > 1, then the enzyme-inhibitor complex will show multiple steady states and it reaches the full-fledged steady state only after the depletion of enzyme-substrate complex. This multi steady-state behavior especially when (fS/fI) ≠ 1 is the root cause of large amount of error in the estimation of various kinetic parameters of fully and partial competitive inhibition schemes using sQSSA. Remarkably, we show that our refined expressions for the reaction velocities over enzyme-substrate-inhibitor space can control this error more significantly than the currently available sQSSA expressions.

Polyamine oxidases are FAD-dependent enzymes catalyzing the oxidation of polyamines at the secondary amino groups. Zea mays PAO (ZmPAO) oxidizes the carbon on the endo-side of the N5-nitrogen of spermidine (Spd) and spermine (Spm). The structure of ZmPAO revealed that the active site is formed by a catalytic tunnel in which the N5 atom of FAD lies in close proximity to the K300 side chain, the only active-site residue conserved in all PAOs. A water molecule, (HOH309), is hydrogen-bound to the amino group of K300 and mutation of this residue results in a 1400-fold decrease in the rate of flavin reduction. The structural studies on the catalytically impaired ZmPAO-K300M mutant described here show that substrates are bound in an 'out-of-register' mode and the HOH309 water molecule is absent in the enzyme-substrate complexes. Moreover, K300 mutation brings about a 60 mV decrease in the FAD redox potential and a 30-fold decrease in the FAD reoxidation rate, within a virtually unaltered geometry of the catalytic pocket. Taken together, these results indicate that the HOH309-K300 couple plays a major role in multiple steps of ZmPAO catalytic mechanism, such as correct substrate binding geometry as well as FAD reduction and reoxidation kinetics.

Prokaryotes scavenge iron using small molecule chelators called siderophores, made through a novel chemistry of peptide bond formation. One type of siderophore synthesis, increasingly associated with bacterial virulence, uses Non‐ribosomal peptide siderophore Independent Synthesis enzymes (NIS synthetases). Within a sub‐category in this family, an additional remarkable kinetic behavior exists whereby multiple bonds may be catalyzed on the same substrate (iterative.) Prior work in the field has identified a novel protein fold as well as the unique peptide bond chemistry, but no structures exist of an iterative protein. The iterative activity is correlated with broad substrate specificity, but this has never been delineated nor quantified. In fact, no iterative enzyme kinetic parameters have been satisfactorily described. We propose basic science to characterize the structure, function, and elasticity of the under‐described iterative proteins in this novel family of NIS sythetases. To do this, we will utilize the following key methodologies: (1) site‐directed mutagenesis to probe important residues; (2) isothermal titration calorimetry (ITC) to explore the thermodynamics of substrate and cofactor binding; (3) Kinetic assays to quantify and compare iterative vs. single bond formation; and (4) X‐ray crystallography to probe the structural changes of iterative substrate binding. The results of our work will be used as a foundation for future mechanism and inhibitor studies, an effort that could lead to a new class of antibiotics.Support or Funding InformationThis work was supported in part by a grant from the National Science Foundation (MCB‐1716986) and by California Lutheran University.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Calcium Inhibits Human Placental 11β-Hydroxysteroid Dehydrogenase Type 2 Activity