Evidence for a catalytic dyad in the active site of homocitrate synthase from Saccharomyces cerevisiae

Homocitrate synthase (acetyl-coenzyme A: 2-ketoglutarate C-transferase; E.C. 2.3.3.14) (HCS) catalyzes the condensation of acetyl-CoA (AcCoA) and alpha-ketoglutarate (alpha-KG) to give homocitrate and CoA. Although the structure of an HCS has not been solved, the structure of isopropylmalate synthase (IPMS), a homologue, has been solved (Koon, N., Squire, C. J., and Baker, E. N. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 8295-8300). Three active site residues in IPMS, Glu-218, His-379, and Tyr-410, were proposed as candidates for catalytic residues involved in deprotonation of the methyl group of AcCoA prior to the Claisen condensation to give homocitrylCoA. All three of the active site residues in IPMS are conserved in the HCS from Saccharomyces cerevisiae. Site-directed mutagenesis has been carried out to probe the role of the homologous residues, Glu-155, His-309, and Tyr-320, in the S. cerevisiae HCS. No detectable activity was observed for the H309A and H309N mutant enzyme, but a slight increase in activity was observed for H309A in the presence of 300 mM imidazole, which is still 1000-fold lower than that of wild type (wt). The E155Q and E155A mutant enzymes exhibited 1000-fold lower activity than wt. The activity of E155A, but not of E155Q, could be partially rescued by formate; a K act of 60 mM with a modest 4-fold maximum activation was observed. In the presence of formate, E155A gives k cat, K AcCoA, and K alpha-KG values of 0.0031 s (-1), 13 muM, and 39 microM, respectively, while a primary kinetic deuterium isotope effect of about 1.4 was obtained on V, with deuterium in the methyl of AcCoA. The pH dependence of k cat for E155A in the presence of formate gave a p K a of 7.9 for a group that must be protonated for optimum activity, similar to that observed for the wt enzyme. However, a partial change was observed on the acid side of the profile, compared to the all or none change observed for wt giving a p K a of about 6.7. The k cat for E155Q decreased at high pH, similar to the wt enzyme, but was pH independent at low pH. The Y320F mutant enzyme only lost 25-fold activity compared to that of the wt, giving k cat, K AcCoA, and K alpha-KG values of 0.039 s (-1), 33 microM, and 140 microM, respectively, and a primary kinetic deuterium isotope effect of 1.3 and 1.8 on V/ K AcCoA and V, respectively; the pH dependence of k cat was similar to that of the wt. These data, combined with a constant pH molecular dynamics simulation study, suggest that a catalytic dyad comprising Glu-155 and His-309 acts to deprotonate the methyl group of AcCoA, while Tyr320 is likely not directly involved in catalysis, but may aid in orienting the reactant and/or the catalytic dyad.     

Identification of critical residues of choline kinase A2 from Caenorhabditis elegans

Choline kinase catalyzes the phosphorylation of choline by ATP, the first committed step in the CDP-choline pathway for phosphatidylcholine biosynthesis. To begin to elucidate the mechanism of catalysis by this enzyme, choline kinase A-2 from Caenorhabditis elegans was analyzed by systematic mutagenesis of highly conserved residues followed by analysis of kinetic and structural parameters. Specifically, mutants were analyzed with respect to K(m) and k(cat) values for each substrate and Mg(2+), inhibitory constants for Mg(2+) and Ca(2+), secondary structure as monitored by circular dichroism, and sensitivity to unfolding in guanidinium hydrochloride. The most severe impairment of catalysis occurred with the modification of Asp-255 and Asn-260, which are located in the conserved Brenner's phosphotransferase motif, and Asp-301 and Glu-303, in the signature choline kinase motif. For example, mutation of Asp-255 or Asp-301 to Ala eliminated detectable catalytic activity, and mutation of Asn-260 and Glu-303 to Ala decreased k(cat) by 300- and 10-fold, respectively. Additionally, the K(m) for Mg(2+) for mutants N260A and E303A was approximately 30-fold higher than that of wild type. Several other residues (Ser-86, Arg-111, Glu-125, and Trp-387) were identified as being important: Catalytic efficiencies (k(cat)/K(m)) for the enzymes in which these residues were mutated to Ala were reduced to 2-25% of wild type. The high degree of structural similarity among choline kinase A-2, aminoglycoside phosphotransferases, and protein kinases, together with the results from this mutational analysis, indicates it is likely that these conserved residues are located at the catalytic core of choline kinase.     

Residues involved in the catalysis,base specificity,and cytotoxicity of ribonuclease from Rana catesbeiana based upon mutagenesis and X-ray crystallography

The Rana catesbeiana (bullfrog) ribonucleases, which belong to the RNase A superfamily, exert cytotoxicity toward tumor cells. RC-RNase, the most active among frog ribonucleases, has a unique base preference for pyrimidine-guanine rather than pyrimidine-adenine in RNase A. Residues of RC-RNase involved in base specificity and catalytic activity were determined by site-directed mutagenesis, k(cat)/K(m) analysis toward dinucleotides, and cleavage site analysis of RNA substrate. The results show that Pyr-1 (N-terminal pyroglutamate), Lys-9, and Asn-38 along with His-10, Lys-35, and His-103 are involved in catalytic activity, whereas Pyr-1, Thr-39, Thr-70, Lys-95, and Glu-97 are involved in base specificity. The cytotoxicity of RC-RNase is correlated, but not proportional to, its catalytic activity. The crystal structure of the RC-RNase.d(ACGA) complex was determined at 1.80 A resolution. Residues Lys-9, His-10, Lys-35, and His-103 interacted directly with catalytic phosphate at the P(1) site, and Lys-9 was stabilized by hydrogen bonds contributed by Pyr-1, Tyr-28, and Asn-38. Thr-70 acts as a hydrogen bond donor for cytosine through Thr-39 and determines B(1) base specificity. Interestingly, Pyr-1 along with Lys-95 and Glu-97 form four hydrogen bonds with guanine at B(2) site and determine B(2) base specificity.     

A double residue substitution in the coenzyme-binding site accounts for the different kinetic properties between yeast and human formaldehyde dehydrogenases

Glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzymatic system for formaldehyde detoxification in all eukaryotic and many prokaryotic organisms. The enzyme of yeasts and some bacteria exhibits about 10-fold higher k(cat) and K(m) values than those of the enzyme from animals and plants. Typically Thr-269 and Glu-267 are found in the coenzyme-binding site of yeast FALDH, but Ile-269 and Asp-267 are present in the FALDH of animals. By site-directed mutagenesis we have prepared the T269I and the D267E mutants and the D267E/T269I double mutant of Saccharomyces cerevisiae FALDH with the aim of investigating the role of these residues in the kinetics. The T269I and the D267E mutants have identical kinetic properties as compared with the wild-type enzyme, although T269I is highly unstable. In contrast, the D267E/T269I double mutant is stable and shows low K(m) (2.5 microM) and low k(cat) (285 min(-1)) values with S-hydroxymethylglutathione, similar to those of the human enzyme. Therefore, the simultaneous exchange at both residues is the structural basis of the two distinct FALDH kinetic types. The local structural perturbations imposed by the substitutions are suggested by molecular modeling studies. Finally, we have studied the effect of FALDH deletion and overexpression on the growth of S. cerevisiae. It is concluded that the FALDH gene is not essential but enhances the resistance against formaldehyde (0.3-1 mM). Moreover, the wild-type enzyme (with high k(cat) and K(m)) provides more resistance than the double mutant (with low k(cat) and K(m)).     

Residues glutamate 216 and aspartate 301 are key determinants of substrate specificity and product regioselectivity in cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes a wide range of therapeutic drugs. CYP2D6 substrates typically contain a basic nitrogen atom, and the active-site residue Asp-301 has been implicated in substrate recognition through electrostatic interactions. Our recent computational models point to a predominantly structural role for Asp-301 in loop positioning (Kirton, S. B., Kemp, C. A., Tomkinson, N. P., St.-Gallay, S., and Sutcliffe, M. J. (2002) Proteins 49, 216-231) and suggest a second acidic residue, Glu-216, as a key determinant in the binding of basic substrates. We have evaluated the role of Glu-216 in substrate recognition, along with Asp-301, by site-directed mutagenesis. Reversal of the Glu-216 charge to Lys or substitution with neutral residues (Gln, Phe, or Leu) greatly decreased the affinity (K(m) values increased 10-100-fold) for the classical basic nitrogen-containing substrates bufuralol and dextromethorphan. Altered binding was also manifested in significant differences in regiospecificity with respect to dextromethorphan, producing enzymes with no preference for N-demethylation versus O-demethylation (E216K and E216F). Neutralization of Asp-301 to Gln and Asn had similarly profound effects on substrate binding and regioselectivity. Intriguingly, removal of the negative charge from either 216 or 301 produced enzymes (E216A, E216K, and D301Q) with elevated levels (50-75-fold) of catalytic activity toward diclofenac, a carboxylate-containing CYP2C9 substrate that lacks a basic nitrogen atom. Activity was increased still further (>1000-fold) upon neutralization of both residues (E216Q/D301Q). The kinetic parameters for diclofenac (K(m) 108 microm, k(cat) 5 min(-1)) along with nifedipine (K(m) 28 microm, k(cat) 2 min(-1)) and tolbutamide (K(m) 315 microm, k(cat) 1 min(-1)), which are not normally substrates for CYP2D6, were within an order of magnitude of those observed with CYP3A4 or CYP2C9. Neutralizing both Glu-216 and Asp-301 thus effectively alters substrate recognition illustrating the central role of the negative charges provided by both residues in defining the specificity of CYP2D6 toward substrates containing a basic nitrogen.     

Glutamate 83 is important for stabilization of domain-domain conformation of Thermus aquaticus glycerol kinase

The gene glpK, encoding glycerol kinase (GlpK) of Thermus aquaticus, has recently been identified. The protein encoded by glpK was found to have an unusually high identity of 81% with the sequence of GlpK from Bacillus subtilis. Three residues (Arg-82, Glu-83, and Asp-244) of T. aquaticus GlpK are conserved in all the known GlpK sequences, including those from various bacteria, yeast and human. The roles that these three residues play in the catalytic mechanism were investigated by using site-directed mutagenesis to produce three mutants: Arg-82-Ala, Glu-83-Ala, and Asp-244-Ala. Replacement of Asp-244 by Ala resulted in a complete loss of activity, thus suggesting that Asp-244 is important for catalysis. Taking k(cat)/K(m) as a simple measure of catalytic efficiency, the mutants Arg-82-Ala and Glu-83-Ala were judged to cause 190- and 37,000-fold decrease, respectively, when compared to the wild-type GlpK. Thus, these three residues play a critical role in the catalytic mechanism. However, only mutant Glu-83-Ala was cleaved by alpha-chymotrypsin, and proteolysis studies showed that the mutant Glu-83-Ala involves a change in the exposure of Tyr-331 at the alpha-chymotrypsin site. This indicates a large domain conformational change, since the residues corresponding to Glu-83 and Tyr-331 in the Escherichia coli GlpK sequence are located in domain IB and domain IIB, respectively. The apparent conformational change caused by replacement of Glu-83 leads us to propose that Glu-83 is an important residue for stabilization of domain conformation.     

Mutational, kinetic, and NMR studies of the roles of conserved glutamate residues and of lysine-39 in the mechanism of the MutT pyrophosphohydrolase

The MutT enzyme catalyzes the hydrolysis of nucleoside triphosphates (NTP) to NMP and PP(i) by nucleophilic substitution at the rarely attacked beta-phosphorus. The solution structure of the quaternary E-M(2+)-AMPCPP-M(2+) complex indicated that conserved residues Glu-53, -56, -57, and -98 are at the active site near the bound divalent cation possibly serving as metal ligands, Lys-39 is positioned to promote departure of the NMP leaving group, and Glu-44 precedes helix I (residues 47-59) possibly stabilizing this helix which contributes four catalytic residues to the active site [Lin, J. , Abeygunawardana, C., Frick, D. N., Bessman, M. J., and Mildvan, A. S. (1997) Biochemistry 36, 1199-1211]. To test these proposed roles, the effects of mutations of each of these residues on the kinetic parameters and on the Mn(2+), Mg(2+), and substrate binding properties were examined. The largest decreases in k(cat) for the Mg(2+)-activated enzyme of 10(4.7)- and 10(2.6)-fold were observed for the E53Q and E53D mutants, respectively, while 97-, 48-, 25-, and 14-fold decreases were observed for the E44D, E56D, E56Q, and E44Q mutations, respectively. Smaller effects on k(cat) were observed for mutations of Glu-98 and Lys-39. For wild type MutT and its E53D and E44D mutants, plots of log(k(cat)) versus pH exhibited a limiting slope of 1 on the ascending limb and then a hump, i.e., a sharply defined maximum near pH 8 followed by a plateau, yielding apparent pK(a) values of 7.6 +/- 0.3 and 8.4 +/- 0.4 for an essential base and a nonessential acid catalyst, respectively, in the active quaternary MutT-Mg(2+)-dGTP-Mg(2+) complex. The pK(a) of 7.6 is assigned to Glu-53, functioning as a base catalyst in the active quaternary complex, on the basis of the disappearance of the ascending limb of the pH-rate profile of the E53Q mutant, and its restoration in the E53D mutant with a 10(1.9)-fold increase in (k(cat))(max). The pK(a) of 8.4 is assigned to Lys-39 on the basis of the disappearance of the descending limb of the pH-rate profile of the K39Q mutant, and the observation that removal of the positive charge of Lys-39, by either deprotonation or mutation, results in the same 8.7-fold decrease in k(cat). Values of k(cat) of both wild type MutT and the E53Q mutant were independent of solvent viscosity, indicating that a chemical step is likely to be rate-limiting with both. A liganding role for Glu-53 and Glu-56, but not Glu-98, in the binary E-M(2+) complex is indicated by the observation that the E53Q, E53D, E56Q, and E56D mutants bound Mn(2+) at the active site 36-, 27-, 4.7-, and 1.9-fold weaker, and exhibited 2.10-, 1.50-, 1.12-, and 1.24-fold lower enhanced paramagnetic effects of Mn(2+), respectively, than the wild type enzyme as detected by 1/T(1) values of water protons, consistent with the loss of a metal ligand. However, the K(m) values of Mg(2+) and Mn(2+) indicate that Glu-56, and to a lesser degree Glu-98, contribute to metal binding in the active quaternary complex. Mutations of the more distant but conserved residue Glu-44 had little effect on metal binding or enhancement factors in the binary E-M(2+) complexes. Two-dimensional (1)H-(15)N HSQC and three-dimensional (1)H-(15)N NOESY-HSQC spectra of the kinetically damaged E53Q and E56Q mutants showed largely intact proteins with structural changes near the mutated residues. Structural changes in the kinetically more damaged E44D mutant detected in (1)H-(15)N HSQC spectra were largely limited to the loop I-helix I motif, suggesting that Glu-44 stabilizes the active site region. (1)H-(15)N HSQC titrations of the E53Q, E56Q, and E44D mutants with dGTP showed changes in chemical shifts of residues lining the active site cleft, and revealed tighter nucleotide binding by these mutants, indicating an intact substrate binding site. (ABSTRACT TRUNCATED)     

Identification of important residues in diketoreductase from Acinetobacter baylyi by molecular modeling and site-directed mutagenesis

Diketoreductase (DKR) from Acinetobacter baylyi exhibits a unique property of double reduction of a β, δ-diketo ester with excellent stereoselectivity, which can serve as an efficient biocatalyst for the preparation of an important chiral intermediate for cholesterol lowering statin drugs. Taken the advantage of high homology between DKR and human heart 3-hydroxyacyl-CoA dehydrogenase (HAD), a molecular model was created to compare the tertiary structures of DKR and HAD. In addition to the possible participation of His-143 in the enzyme catalysis by pH profile, three key amino acid residues, Ser-122, His-143 and Glu-155, were identified and mutated to explore the possibility of involving in the catalytic process. The catalytic activities for mutants S122A/C, H143A/K and E155Q were below detectable level, while their binding affinities to the diketo ester substrate and cofactor NADH did not change obviously. The experimental results were further supported by molecular docking, suggesting that Ser-122 and His-143 were essential for the proton transfer to the carbonyl functional groups of the substrate. Moreover, Glu-155 was crucial for maintaining the proper orientation and protonation of the imidazole ring of His-143 for efficient catalysis.     

The fourth transmembrane domain of the Helicobacter pylori Na+/H+ antiporter NhaA faces a water-filled channel required for ion transport

Cysteine-scanning mutagenesis was performed from Ser-130 to Leu-160 in the fourth transmembrane domain (TM4) of the Na+/H+ antiporter NhaA from Helicobacter pylori to determine the topology of each residue and to identify functionally important residues. All of the mutants were based on cysteine-less NhaA (Cys-less NhaA), which functions very similarly to the wild-type protein, and were expressed at a level similar to Cys-less NhaA. Discontinuity of [14C]N-ethylmaleimide (NEM)-reactive residues suggested that TM4 comprises residues Gly-135 to Val-156. Even within TM4, NEM reactivity was high for I136C, D141C to A143C, L146C, M150C, and G153C to R155C. These residues are thought to be located on one side of the -helical structure of TM4 and to face a putative water-filled channel. Pretreatment of intact cells with membrane-impermeable maleimide did not inhibit [14C]NEM binding to the NEM-reactive residues within TM4, suggesting that the putative channel opens toward the cytoplasm. NEM reactivity of the A143C mutant was significantly inhibited by Li+. The T140C and D141C mutants showed lower affinity for Na+ and Li+ as transport substrates, but their maximal antiporter velocities (Vmax) were relatively unaffected. Whereas the I142C and F144C mutants completely lost their Li+/H+ antiporter activity, I142C had a lower Vmax for the Na+/H+ antiporter. F144C exhibited a markedly lower Vmax and a partially reduced affinity for Na+. These results suggest that Thr-140, Asp-141, and Phe-144 are located in the end portion of a putative water-filled channel and may provide the binding site for Na+, Li+, and/or H+. Furthermore, residues Ile-142 to Phe-144 may be important for the conformational change that accompanies ion transport in NhaA.     

Identification of a specific tyrosine residue in Bryodin 1 distinct from the active site but required for full catalytic and cytotoxic activity.

Bryodin 1 (BD1) is a type I ribosome-inactivating protein (RIP) with low inherent animal toxicity. It has been cloned recently and the recombinant protein (rBD1) has been produced and crystallized. To gain insight into the relationship of rBD1 structure and function, we investigated the role of sequences in a region (residues 128-156) that exhibits homology with membrane interactive sequences and is not part of the enzymatically defined active site. Progressive deletions representing alpha-helical tums within these residues were generated; mutant rBD1 proteins were expressed in Escherichia coli and demonstrated increasing losses of enzymatic activity. Point mutations were also generated within this region to replace Y140, Y141, and Y142 with either alanine or lysine. Mutants at position 140 or 142 retained full enzymatic activity, whereas A141 and K141 mutants were >19-fold less potent. In cytotoxicity assays, the rBD1 point mutants at Y141 were >80-fold less potent than either rBD1 or mutants at residues 140 or 142. However, when introduced into the anti-CD40 single-chain immunotoxin rBD1-G28-5 sFv, the A140 and A141 point mutations led to decreased cytotoxicity toward CD40 positive cell lines. These data indicate that Y141 plays an important role in the enzymatic activity of BD1 and that Y140, although not essential for catalytic activity, is required for full BD1 function. Because residues 140 and 141 are distinct from residues implicit in the active site, they may be involved in ribosomal and/or membrane interactions or in intracellular trafficking of the toxin and immunotoxin.     

Two glutamic acids in chitosanase A from Matsuebacter chitosanotabidus 3001 are the catalytically important residues.

Chitosanase is the glycolytic enzyme that hydrolyzes the glucosamine GlcN-GlcN bonds of chitosan. To determine the catalytically important residues of chitosanase A (ChoA) from Matsuebacter chitosanotabidus 3001, we performed both site-directed and random mutagenesis of choA, obtaining 31 mutants. These mutations indicated that Glu-121 and Glu-141 were catalytically important residues, as mutation at these sites to Ala or Asp drastically decreased the enzymatic activity to 0.1-0.3% of that of the wild type enzyme. Glu-141 mutations remarkably decreased kinetic constant k(cat) for hydrolysis of chitosan, meanwhile Glu-121 mutations decreased the activities to undeterminable levels, precluding parameter analysis. No hydrolysis of (GlcN)(6) was observed with the purified Glu-121 mutant and extremely slow hydrolysis with the Glu-141 mutant. We also found that Asp-139, Asp-148, Arg-150, Gly-151, Asp-164, and Gly-280 were important residues for enzymatic activities, although they are not directly involved in catalysis. In addition, mutation of any of the six cysteine residues of ChoA abrogated the enzymatic activity, and Cys-136 and Cys-231 were found to form a disulfide bond. In support of the significance of the disulfide bond of ChoA, chitosanase activity was impaired on incubation with a reducing agent. Thus, ChoA from M. chitosanotabidus 3001 uses two glutamic acid residues as putative catalytic residues and has at least one disulfide bond.     

H(+)-ATPase gamma subunit of Escherichia coli. Role of the conserved carboxyl-terminal region

Cloned uncG genes (wild-type or in vitro mutagenized) for the Escherichia coli gamma subunit were introduced into the uncG mutant Gln-14----end), and the functions of the mutant subunits were studied. The F1's with Ala-283----end and Thr-277----end mutant gamma subunits had 63 and 14% of the wild-type ATPase activity, respectively, and mutants with these subunits showed reduced growth by oxidative phosphorylation, indicating that the 10 residues at the carboxyl terminus (286th residue) are important, but dispensable, for catalysis. On the other hand, F1 with a Gln-269----end gamma subunit was inactive. Replacement of conserved residues (Gln-269, Thr-273, or Glu-275) between Gln-269 and Leu-276 gave enzymes with significantly reduced ATPase activity (2-41% of that of the wild-type) and lower ATP-driven proton conduction. Thus these residues are required for the normal catalytic activity of F1, although they are not absolutely essential. Membranes with amino acid replacements (Thr-277----end, Gln-269----Leu, or Glu-275----Lys) and the frameshift mutation (downstream of Thr-277) had about 15% of the wild-type ATPase activity, but showed different degrees of ATP-dependent H+ translocation and growth yield by oxidative phosphorylation, suggesting that the gamma subunit, especially its carboxyl-terminal region, functions in coupling between catalysis and H+ translocation.     

Mutagenesis of Trp(54) and Trp(203) residues on Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase significantly affects catalytic activities of the enzyme

The possible structural and catalytic functions of the nine tryptophan amino acid residues, including Trp(54), Trp(105), Trp(112), Trp(141), Trp(148), Trp(165), Trp(186), Trp(198), and Trp(203) in Fibrobacter succinogenes 1,3-1,4-beta-D-glucanase (Fs beta-glucanase), were characterized using site-directed mutagenesis, initial rate kinetics, fluorescence spectrometry, and structural modeling analysis. Kinetic studies showed that a 5-7-fold increase in K(m) value for lichenan was observed for W141F, W141H, and W203R mutant Fs beta-glucanases, and approximately 72-, 56-, 30-, 29.5-, 4.9-, and 4.3-fold decreases in k(cat) relative to that for the wild-type enzyme were observed for the W54F, W54Y, W141H, W203R, W141F, and W148F mutants, respectively. In contrast, W186F and W203F, unlike the other 12 mutants, exhibited a 1.4- and 4.2-fold increase in k(cat), respectively. W165F and W203R were the only two mutants that exhibited a 4-7-fold higher activity relative to the wild-type enzyme after they were incubated at pH 3.0 for 1 h. Fluorescence spectrometry indicated that all of the mutations on the nine tryptophan amino acid residues retained a folding similar to that of the wild-type enzyme. Structural modeling and kinetic studies suggest that Trp(54), Trp(141), Trp(148), and Trp(203) play important roles in maintaining structural integrity in the substrate-binding cleft and the catalytic efficiency of the enzyme.     

The bifunctional active site of s-adenosylmethionine synthetase. Roles of the active site aspartates.

S-Adenosylmethionine (AdoMet) synthetase catalyzes the biosynthesis of AdoMet in a unique enzymatic reaction. Initially the sulfur of methionine displaces the intact tripolyphosphate chain (PPP(i)) from ATP, and subsequently PPP(i) is hydrolyzed to PP(i) and P(i) before product release. The crystal structure of Escherichia coli AdoMet synthetase shows that the active site contains four aspartate residues. Aspartate residues Asp-16* and Asp-271 individually provide the sole protein ligand to one of the two required Mg(2+) ions (* denotes a residue from a second subunit); aspartates Asp-118 and Asp-238* are proposed to interact with methionine. Each aspartate has been changed to an uncharged asparagine, and the metal binding residues were also changed to alanine, to assess the roles of charge and ligation ability on catalytic efficiency. The resultant enzyme variants all structurally resemble the wild type enzyme as indicated by circular dichroism spectra and are tetramers. However, all have k(cat) reductions of approximately 10(3)-fold in AdoMet synthesis, whereas the MgATP and methionine K(m) values change by less than 3- and 8-fold, respectively. In the partial reaction of PPP(i) hydrolysis, mutants of the Mg(2+) binding residues have >700-fold reduced catalytic efficiency (k(cat)/K(m)), whereas the D118N and D238*N mutants are impaired less than 35-fold. The catalytic efficiency for PPP(i) hydrolysis by Mg(2+) site mutants is improved by AdoMet, like the wild type enzyme. In contrast AdoMet reduces the catalytic efficiency for PPP(i) hydrolysis by the D118N and D238*N mutants, indicating that the events involved in AdoMet activation are hindered in these methionyl binding site mutants. Ca(2+) uniquely activates the D271A mutant enzyme to 15% of the level of Mg(2+), in contrast to the approximately 1% Ca(2+) activation of the wild type enzyme. This indicates that the Asp-271 side chain size is a discriminator between the activating ability of Ca(2+) and the smaller Mg(2+).     

Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K(m) for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K(m) (0.72 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K(m) for testosterone 16alpha-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K(m) than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a approximately 4-fold decrease in K(m). Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k(cat)/K(m). Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.     

Coenzyme specificity of human monomeric carbonyl reductase: contribution of Lys-15, Ala-37 and Arg-38

Short-chain dehydrogenases/reductases catalyze the oxidoreduction of alcohol and carbonyl compounds using either NAD or NADPH as coenzyme. Structural analysis suggests that specificity for NADPH is conferred by two highly conserved basic residues in the N-terminal part of the peptide chain, whereas specificity for NAD correlates with the presence of an Asp adjacent to the position of the distal basic residue in NADP-dependent enzymes. We carried out site-directed mutagenesis of the two basic residues: Lys-15 and Arg-38, as well as of Ala-37 of human monomeric carbonyl reductase in order to investigate their contribution to coenzyme binding and specificity. Substitution of Lys-15 or Arg-38 by Gln and, even more pronounced Asp decreased the catalytic efficiency (k(cat)/K(m,NADPH)) by more than three orders of magnitude. Similarly, substitution of Asp for Ala-37 decreased k(cat)/K(m,NADPH) 1000-fold but had little effect on k(cat)/K(m,NADH). The results demonstrate the importance of basic residues at positions 15 and 38 and the absence of an acidic residue at position 37 for NADPH binding and catalysis.     

Giardia lamblia RNA cap guanine-N2 methyltransferase (Tgs2)

Tgs1 is the enzyme responsible for converting 7-methylguanosine RNA caps to the 2,2,7-trimethylguanosine cap structures of small nuclear and small nucleolar RNAs. Whereas budding yeast Saccharomyces cerevisiae and fission yeast Schizosaccharomyces pombe encode a single Tgs1 protein, the primitive eukaryote Giardia lamblia encodes two paralogs, Tgs1 and Tgs2. Here we show that purified Tgs2 is a monomeric enzyme that catalyzes methyl transfer from AdoMet (K(m) of 6 microm) to m(7)GDP (K(m) of 65 microm; k(cat) of 14 min(-1)) to form m(2,7)GDP. Tgs2 also methylates m(7)GTP (K(m) of 30 microm; k(cat) of 13 min(-1)) and m(7)GpppA (K(m) of 7 microm; k(cat)) of 14 min(-1) but is unreactive with GDP, GTP, GpppA, ATP, CTP, or UTP. We find that the conserved residues Asp-68, Glu-91, and Trp-143 are essential for Tgs2 methyltransferase activity in vitro. The m(2,7)GDP product formed by Tgs2 can be converted to m(2,2,7)GDP by S. pombe Tgs1 in the presence of excess AdoMet. However, Giardia Tgs2 itself is apparently unable to add a second methyl group at guanine-N2. This result implies that 2,2,7-trimethylguanosine caps in Giardia are either synthesized by Tgs1 alone or by the sequential action of Tgs2 and Tgs1. The specificity of Tgs2 raises the prospect that some Giardia mRNAs might contain dimethylguanosine caps.     

Studies on reduction of S-nitrosoglutathione by human carbonyl reductases 1 and 3

Human carbonyl reductases 1 and 3 (CBR1 and CBR3) are monomeric NADPH-dependent enzymes of the short-chain dehydrogenase/reductase superfamily. Despite 72% identity in primary structure they exhibit substantial differences in substrate specificity. Recently, the endogenous low molecular weight S-nitrosothiol S-nitrosoglutathione (GSNO) has been added to the broad substrate spectrum of CBR1. The current study initially addressed whether CBR3 could equally reduce GSNO which was not the case. Neither the introduction of residues which contribute to glutathione binding in CBR1, i.e. K106Q and S97V/D98A, nor the exchange C143S, which prevents a theoretical disulfide bond with C227 in CBR3, could engender activity towards GSNO. However, exchanging amino acids 236-244 in CBR3 to correspond to CBR1 was sufficient to engender catalytic activity towards GSNO. Catalytic efficiency was further improved by the exchanges Q142M, C143S, P230W and H270S. Hence, the same residues previously reported as important for reduction of carbonyl compounds appear to be key to CBR1-mediated reduction of GSNO. Furthermore, for CBR1-mediated reduction of GSNO, considerable substrate inhibition at concentrations >5 K(m) was observed. Treatment of CBR1 with GSNO followed by removal of low molecular weight compounds decreased the GSNO reducing activity, suggesting a covalent modification. Treatment with dithiothreitol, but not with ascorbic acid, could rescue the activity, indicating S-glutathionylation rather than S-nitrosation as the underlying mechanism. As C227 has previously been identified as the reactive cysteine in CBR1, the variant CBR1 C227S was generated, which, in comparison to the wild-type protein, displayed a similar k(cat), but a 30-fold higher K(m), and did not show substrate inhibition. Collectively, the results clearly argue for a physiological role of CBR1, but not for CBR3, in GSNO reduction and thus ultimately in regulation of NO signaling. Furthermore, at higher concentrations, GSNO appears to work as a suicide inhibitor for CBR1, probably through glutathionylation of C227.     

Role of enzyme-ribofuranosyl contacts in the ground state and transition state for orotidine 5'-phosphate decarboxylase: a role for substrate destabilization?

The crystal structure of yeast orotidine 5'-monophosphate decarboxylase (ODCase) complexed with the inhibitor 6-hydroxyuridine 5'-phosphate (BMP) reveals the presence of a series of strong interactions between enzyme residues and functional groups of this ligand. Enzyme contacts with the phosphoribofuranosyl moiety of orotidine 5'-phosphate (OMP) have been shown to contribute at least 16.6 kcal/mol of intrinsic binding free energy to the stabilization of the transition state for the reaction catalyzed by yeast ODCase. In addition to these enzyme-ligand contacts, active site residues contributed by both subunits of the dimeric enzyme are positioned to form hydrogen bonds with the 2'- and 3'-OH groups of the ligand's ribosyl moiety. These involve Thr-100 of one subunit and Asp-37 of the opposite subunit, respectively. To evaluate the contributions of these ribofuranosyl contacts to ground state and transition state stabilization, Thr-100 and Asp-37 were each mutated to alanine. Elimination of the enzyme's capacity to contact individual ribosyl OH groups reduced the k(cat)/K(m) value of the T100A enzyme by 60-fold and that of the D37A enzyme by 300-fold. Removal of the 2'-OH group from the substrate OMP decreased the binding affinity by less than a factor of 10, but decreased k(cat) by more that 2 orders of magnitude. Upon removal of the complementary hydroxymethyl group from the enzyme, little further reduction in k(cat)/K(m) for 2'-deoxyOMP was observed. To assess the contribution made by contacts involving both ribosyl hydroxyl groups at once, the ability of the D37A mutant enzyme to decarboxylate 2'-deoxyOMP was measured. The value of k(cat)/K(m) for this enzyme-substrate pair was 170 M(-1) s(-1), representing a decrease of more than 7.6 kcal/mol of binding free energy in the transition state. To the extent that electrostatic repulsion in the ground state can be tested by these simple alterations, the results do not lend obvious support to the view that electrostatic destabilization in the ground state enzyme-substrate complex plays a major role in catalysis.     

Directed evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide

Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymerase chain reaction and activity screening were optimized using the L209A mutant of P450 2B1 in an N-terminally modified construct with a C-terminal His tag (P450 2B1dH). Two rounds of mutagenesis and screening and one subcloning step yielded the P450 2B1 quadruple mutant V183L/F202L/L209A/S334P, which demonstrated a 6-fold higher k(cat) than L209A. Further random or site-directed mutagenesis did not improve the activity. When assayed in an NADPH-supported reconstituted system, V183L/L209A demonstrated lower 7-EFC oxidation than L209A. Therefore, F202L/L209A/S334P was generated, which showed a 2.5-fold higher k(cat)/K(m) for NADPH-dependent 7-EFC oxidation than L209A. F202L/L209A/S334P also showed enhanced catalytic efficiency with 7-benzyloxyresorufin, benzphetamine, and testosterone, and a 10-fold increase in stereoselectivity for testosterone 16alpha-versus 16beta-hydroxylation compared with 2B1dH. Enhanced catalytic efficiency of F202L/L209A/S334P was also retained in the full-length P450 2B1 background with 7-EFC and testosterone as substrates. Finally, the individual mutants were tested for metabolism of the anti-cancer prodrugs cyclophosphamide and ifosfamide. Several of the mutants showed increased metabolism via the therapeutically beneficial 4-hydroxylation pathway, with L209A/S334P showing 2.8-fold enhancement of k(cat)/K(m) with cyclophosphamide and V183L/L209A showing 3.5-fold enhancement with ifosfamide. Directed evolution can thus be used to enhance P450 2B1 catalytic efficiency across a panel of substrates and to identify functionally important residues distant from the active site.