4944
Biochemistry 2004, 43, 4944-4954
Studies of the Enzymic Mechanism of Candida tenuis Xylose Reductase (AKR 2B5): X-ray Structure and Catalytic Reaction Profile for the H113A Mutant†,‡ Regina Kratzer,§ Kathryn L. Kavanagh,| David K. Wilson,| and Bernd Nidetzky*,§ Institute of Biotechnology, Graz UniVersity of Technology, Petersgasse 12/I, A-8010 Graz, Austria, and Section of Molecular and Cellular Biology, UniVersity of California, DaVis, California 95616 ReceiVed October 10, 2003; ReVised Manuscript ReceiVed February 10, 2004
ABSTRACT:
Xylose reductase from the yeast Candida tenuis (CtXR) is a family 2 member of the aldoketo reductase (AKR) superfamily of proteins and enzymes. Active site His-113 is conserved among AKRs, but a unified mechanism of how it affects catalytic activity is outstanding. We have replaced His-113 by alanine using site-directed mutagenesis, determined a 2.2 Å structure of H113A mutant bound to NADP+, and compared catalytic reaction profiles of NADH-dependent reduction of different aldehydes catalyzed by the wild type and the mutant. Deuterium kinetic isotope effects (KIEs) on kcat and kcat/ Km xylose show that, relative to the wild type, the hydride transfer rate constant (k7 ≈ 0.16 s-1) has decreased about 1000-fold in H113A whereas xylose binding was not strongly affected. No solvent isotope effect was seen on kcat and kcat/Km xylose for H113A, suggesting that proton transfer has not become rate-limiting as a result of the mutation. The pH profiles of log(kcat/Km xylose) for the wild type and H113A decreased above apparent pKa values of 8.85 and 7.63, respectively. The ∆pKa of -1.2 pH units likely reflects a proximally disruptive character of the mutation, affecting the position of Asp-50. A steady-state kinetic analysis for H113A-catalyzed reduction of a homologous series of meta-substituted benzaldehyde derivatives was carried out, and quantitative structure-reactivity correlations were used to factor the observed kinetic substituent effect on kcat and kcat/Km aldehyde into an electronic effect and bonding effects (which are lacking in the wild type). Using the Hammett σ scale, electronic parameter coefficients (F) of +0.64 (kcat) and +0.78 (kcat/Km aldehyde) were calculated and clearly differ from F(kcat/Kaldehyde) and F(kcat) values of +1.67 and approximately 0.0, respectively, for the wild-type enzyme. Hydride transfer rate constants of H113A, calculated from kinetic parameters and KIE data, display a substituent dependence not seen in the corresponding wild-type enzyme rate constants. An enzymic mechanism is proposed in which His-113, through a hydrogen bond from N2 to aldehyde O1, assists in catalysis by optimizing the CdO bond charge separation and orbital alignment in the ternary complex.
The aldo-keto reductases (AKRs)1 constitute a large protein superfamily and are widespread in nature (1). They all seem to bind to NAD(P) and with few exceptions display † Financial support is acknowledged from the Austrian Science Funds (P-15208-MOB to B.N.), the National Institutes of Health (D.K.W.), and the Keck Foundation. K.L.K. is supported by a grant from the University of California Systemwide Biotechnology Research Program, proposal number 2001-07. The data collection facilities at Stanford Synchrotron Radiation Laboratory are funded by the U.S. Department of Energy and by the National Institutes of Health. ‡ The coordinates and structure factors for the H113A mutant of C. tenuis xylose reductase have been deposited into the Protein Data Bank under the accession number 1R38. Coordinates are numbered according to the DNA sequence with the initiator methionine as residue 1. Numbering within this paper follows the convention of the mature protein in which the initial methionine is processed off; hence residue numbers differ by one. * Corresponding author. Phone: +43-316-873-8400. Fax: +43-316873-8434. E-mail:
[email protected]. § Graz University of Technology. | University of California. 1 Abbreviations: AKR, aldo-keto reductase; AR, aldose reductase; hAR, human AR; XR, xylose reductase [alditol:NAD(P)+ 1-oxidoreductase; EC 1.1.1.21]; CtXR, XR from Candida tenuis; BA, benzaldehyde; 3X-BA, BA substituted in the meta position on the aromatic ring; 3X-BzOH, substituted benzyl alcohol; KIE, kinetic isotope effect; S-KIE, solvent kinetic isotope effect.
NAD(P)-dependent oxidoreductase activity (1, 2). AKRs are single-domain proteins of ≈36 kDa mass and share a common (β/R)8 barrel fold. The canonical active site conformation of AKRs involves a tetrad of Tyr-51, Lys-80, Asp-46, and His-113 [numbered according to CtXR (3); Figure 1A] (2). In a multiple sequence alignment across all current 14 AKR families, the positions of the tetrad residues are highly conserved, suggesting a common mechanistic basis of catalytic action (1). An enzymic mechanism for AKRs has been suggested which assigns roles in catalysis to Tyr-51, Lys-80, and Asp46 (4-10). Tyr-51 is widely believed to function as the general acid/base catalyst of the reaction (4-10). The -NH2 group of Lys-80 is protonated under the physiological pH conditions and brought into position through a salt link interaction with the carboxylate side chain of Asp-46. It is thought to donate a hydrogen for bonding with a lone electron pair on the oxygen of the phenolic hydroxyl of the tyrosine. The pKa of Tyr-OH is thus depressed from a value of ≈10.5 in aqueous solution to a value between about 7 and 9 in both the enzyme-NADPH complex (6-8, 11) and the ternary complex (4, 5). The pKa shift by g1.5 pH units is required for Tyr-51 to function as an effective proton donor
10.1021/bi035833r CCC: $27.50 © 2004 American Chemical Society Published on Web 04/07/2004
Catalytic Mechanism of C. tenuis Xylose Reductase
Biochemistry, Vol. 43, No. 17, 2004 4945
FIGURE 1: (A) Overlap of wild-type CtXR (green bonds) and H113A (gray bonds) that includes the nicotinamide from the mutant structure. Hydrogen bonds between catalytically important residues are depicted as gold dotted lines. (B) Representative 2Fo - Fc (blue; contoured at 1.4σ) and Fo - Fc (green; contoured at 6σ) electron density. Phases were calculated from the final refined H113A model with the side-chain atoms of Asp-50 omitted. Side chains are colored as in panel A.
for carbonyl group reduction. Characterization of CtXR mutants Y51F and K80A yielded results supporting these basic components of the AKR catalytic mechanism (12). The question of the exact role of His-113 has been a topic of controversy over the years. Site-directed replacements of His-113 by Asn, Gln, Glu, or Ala in different AKRs gave mutant enzymes that displayed low, however, robust activity (4-11), indicating an auxiliary function for the histidine in the AKR catalytic mechanism. Analysis of kinetic consequences of the mutations using comparative pH and isotope effect studies led to consideration of different mechanistic proposals. First, His-113 is required for substrate orientation in the binding pocket (6, 8, 11), likely by donating a hydrogen for bonding from the N2-H of its neutral imidazole ring. Second, it assists in proton transfer from TyrOH to the oxygen atom of the reactive carbonyl group in the reductive direction of the reaction (4, 5, 11). In this scenario, the side chain of His-113 is doubly protonated at the optimum pH for reduction. A number of computational studies have suggested, however, that formation of a positively charged imidazolium ring is energetically disfavored at AKR active sites (13, 14), although this notion is not without criticism (15). Penning and co-workers argue that the delocalized positive charge on the imidazole ring is part of a catalytic proton relay between His-113 and Tyr-51 that facilitates carbonyl group reduction (4, 5, 11). Considering the available evidence from previous experimental and theoretical investigations, neither of the two mechanisms for His-113 is ultimately persuasive, and a unified description of how His-113 affects AKR catalytic activity is still outstanding.
The present paper describes experiments that were designed to explore the role of His-113 in the NADH-dependent reduction of aldehydes by CtXR. We report enzyme kinetics and the X-ray structure of a H113A mutant and show that, in CtXR, His-113 facilitates the reaction through the precise positioning of the substrate carbonyl group at the active site. This mechanism explains the high conservation of the histidine throughout the superfamily, and we will discuss that it is consistent with observations made by others with different AKRs. MATERIALS AND METHODS Materials. All chemicals and reagents have been described elsewhere (16). Mutagenesis, Enzyme Production, and Purification. An inverse PCR method was employed to mutate His-113 into alanine (17). The mutagenic oligonucleotide primers used are listed below with the mismatched bases underlined: H113A (forward), 5′-GTTCTTGATCGCTTTCCCAATTG3′; H113A (reverse), 5′-AAGTCAACGTAGTCAACCTTAAG-3′. Plasmid miniprep DNA was subjected to dideoxy sequencing to verify the introduction of the desired mutations and that no misincorporations of nucleotides had occurred because of DNA polymerase errors. Expression of the mutagenized gene in Escherichia coli was carried out by reported methods (18). SDS-PAGE analysis of induced and noninduced cell extracts was used to confirm the synthesis of the recombinant protein. H113A was purified by Red 31 dye ligand chromatography (18). MonoQ anion-exchange chromatography that is used as the polishing step in the purification of the wild-type enzyme (18) was omitted
4946 Biochemistry, Vol. 43, No. 17, 2004 Table 1: Summary of H113A CtXR Data Collection, Refinement, and Models Data Collection space group unit cell a (Å) b (Å) c (Å) β (deg) monomers per asymmetric unit resolution range (Å) no. of observations/unique reflections Rmerge (overall/high-resolution shell) completeness (overall/high-resolution shell) (%) I/σ(I) (overall/high-resolution shell)
C2 179.98 129.09 79.87 90.82 4 30-2.2 (2.24-2.2) 306084/93679 0.074/0.294 99.4/100 15.3/3.9
Model protein atoms NADP+ atoms water molecules
10108 192 827
Refinement reflections used (I > 0) Rcryst Rfree rmsd from ideal bond length (Å) rmsd from ideal bond angle (deg)
91,348 0.185 0.226 0.011 1.50
because the mutant precipitated on the column. Therefore, only the peak fractions of H113A eluting from the dye column collection were used further. The H113A preparation was electrophoretically pure (see Supporting Information). Protein concentrations were determined with the BCA protein assay (Pierce) using BSA as the reference. Crystallization and Structure Determination. Crystals of the mutant holoenzyme were obtained using the vapor diffusion technique in conditions similar to those of the wild type. The protein stock solution used for crystallization was 17 mg/mL mutant protein in 10 mM Hepes and 3 mM NADP+, pH 7.4. A drop composed of 2 µL of protein solution and 1 µL of well solution was suspended over a well containing 0.35 M ammonium sulfate, 32% (w/v) poly(ethylene glycol) methyl ether 5000, and 100 mM citrate, pH 5.6. These were harvested into a cryoprotectant containing 75% well solution and 25% ethylene glycol and flash cooled prior to data collection. Diffraction data were collected on a Mar Research 345 imaging plate detector mounted on beamline 7-1 at Stanford Synchrotron Radiation Laboratory (SSRL). The crystals were indexed, and reflections were integrated using DENZO (19). Data were merged using SCALEPACK, yielding a 2.2 Å data set with an Rmerge of 7.4%. Other relevant data collection statistics are given in Table 1. The structure of the mutant was determined using molecular replacement as implemented by EPMR (20). The search object was the dimer of the wild-type apoenzyme [PDB accession code 1JEZ (21)] and was used to locate the two dimers that compose the asymmetric unit. A correlation coefficient of 0.56 and a crystallographic R-factor of 41.5% were observed for the initial model. Alternating cycles of manual refitting using O (22) and crystallographic refinement using CNS (23) were used to produce the final model. Final statistics describing the refinement and model quality are given in Table 1. 3F-BA Modeling. Polar hydrogens were added to the crystal structures, and molecules of m-fluorobenzaldehyde (3F-BA) were manually placed into the active site of wild-
Kratzer et al. type CtXR and H113A holoenzymes. Resultant structures were energy minimized for 100 steps using the program CNS (23). The final models were analyzed for substrate orientation conducive to catalysis as well as potential polar and hydrophobic interactions. Steady-State Enzyme Kinetics and Ligand Binding. All experiments were carried out in 50 mM potassium phosphate buffer, pH 7.0, and at 25 °C unless otherwise mentioned. Initial rates of aldehyde reduction or alcohol oxidation by H113A and the wild-type enzyme were measured with a Beckman DU-800 spectrophotometer monitoring the consumption and formation of NAD(P)H. Typical reaction mixtures contained a concentration of H113A in the range 0.5-11.5 µM, which is between 10- and 1000-fold above the corresponding wild-type enzyme levels used. Time courses of H113A-catalyzed reactions were monitored for g15 min, compared to e5 min for the wild-type enzyme. Other methods of the initial rate analysis have been reported elsewhere (16). Apparent dissociation constants (Kd NAD(P)) of the wildtype enzyme and H113A complexes with NAD(P)+ were determined by fluorescence titration. Quenching of tryptophan fluorescence (excitation 295 nm, emission 340 nm) upon the addition of NADP+ to a solution of the wild-type enzyme or H113A (3-5 µM) served as a reporter of binding and was recorded with a Hitachi F-4500 fluorescence spectrophotometer equipped with a temperature-controlled cell holder. Typically, 2-4 µL aliquots of the stock solution of NAD+ (2 mM) or NADP+ (0.1 mM) were added incrementally to yield a final nucleotide level of >6Kd. pH and Kinetic Isotope Effects. Studies of pH effects on the catalytic rates of the wild-type enzyme and H113A in the direction of NADH-dependent reduction of xylose were carried out at 25 °C in potassium phosphate buffer (pH range 6.0-8.0) and Tris-HCl buffer (pH range 8.0-9.0) at a constant value of I ) 0.02 for the molar ionic strength. Initial rates were obtained under conditions in which [xylose] was varied and [coenzyme] was constant and saturating (>5Km NAD(P)H; 230 µM). Suitable control experiments showed that the wild-type enzyme and mutant were stable in the chosen pH range over the time of the assays. Primary deuterium kinetic isotope effects (KIEs) and solvent KIEs (S-KIEs) on apparent kinetic parameters of aldehyde reduction by the wild-type enzyme and H113A were obtained under conditions described elsewhere in detail (16). Data Processing. Kinetic parameters or ligand binding constants were obtained from unweighted nonlinear leastsquares fits of experimental data to eqs 1-8 using the program Sigmaplot 2001 (for Windows, version 7.0). Initial rate data yielding linear Lineweaver-Burk plots were fitted to the equation
V ) kcat[E][A]/(Km A + [A])
(1)
where V is the initial rate, [E] is the molar concentration of the enzyme subunit (36 kDa), [A] is the substrate or coenzyme concentration, kcat is the turnover number (s-1), and Km A is an apparent Michaelis-Menten constant. Unless otherwise stated, estimates of kinetic parameters had standard errors of
