Alternate substrate binding modes to two mutant (D98N and H255N) forms of nitrite reductase from Alcaligenes faecalis S-6: structural model of a transient catalytic intermediate

High-resolution nitrite soaked oxidized and reduced crystal structures of two active site mutants, D98N and H255N, of nitrite reductase (NIR) from Alcaligenes faecalis S-6 were determined to better than 2.0 A resolution. In the oxidized D98N nitrite-soaked structures, nitrite is coordinated to the type II copper via its oxygen atoms in an asymmetric bidentate manner; however, elevated B-factors and weak electron density indicate that both nitrite and Asn98 are less ordered than in the native enzyme. This disorder likely results from the inability of the N delta 2 atom of Asn98 to form a hydrogen bond with the bound protonated nitrite, indicating that the hydrogen bond between Asp98 and nitrite in the native NIR structure is essential in anchoring nitrite in the active site for catalysis. In the oxidized nitrite soaked H255N crystal structure, nitrite does not displace the ligand water and is instead coordinated in an alternative mode via a single oxygen to the type II copper. His255 is clearly essential in defining the nitrite binding site despite the lack of direct interaction with the substrate in the native enzyme. The resulting pentacoordinate copper site in the H255N structure also serves as a model for a proposed transient intermediate in the catalytic mechanism consisting of a hydroxyl and nitric oxide molecule coordinated to the copper. The formation of an unusual dinuclear type I copper site in the reduced nitrite soaked D98N and H255N crystal structures may represent an evolutionary link between the mononuclear type I copper centers and dinuclear Cu(A) sites.     

Structure and mutational analysis of the PhoN protein of Salmonella typhimurium provide insight into mechanistic details

The Salmonella typhimurium PhoN protein is a nonspecific acid phosphatase and belongs to the phosphatidic acid phosphatase type 2 (PAP2) superfamily. We report here the crystal structures of phosphate-bound PhoN, the PhoN-tungstate complex, and the T159D mutant of PhoN along with functional characterization of three mutants: L39T, T159D, and D201N. Invariant active site residues, Lys-123, Arg-130, Ser-156, Gly-157, His-158, and Arg-191, interact with phosphate and tungstate oxyanions. Ser-156 also accepts a hydrogen bond from Thr-159. The T159D mutation, surprisingly, severely diminishes phosphatase activity, apparently by disturbing the active site scaffold: Arg-191 is swung out of the active site resulting in conformational changes in His-158 and His-197 residues. Our results reveal a hitherto unknown functional role of Arg-191, namely, restricting the active conformation of catalytic His-158 and His-197 residues. Consistent with the conserved nature of Asp-201 in the PAP2 superfamily, the D201N mutation completely abolished phosphatase activity. On the basis of this observation and in silico analysis we suggest that the crucial mechanistic role of Asp-201 is to stabilize the positive charge on the phosphohistidine intermediate generated by the transfer of phosphoryl to the nucleophile, His-197, located within hydrogen bond distance to the invariant Asp-201. This is in contrast to earlier suggestions that Asp-201 stabilizes His-197 and the His197-Asp201 dyad facilitates formation of the phosphoenzyme intermediate through a charge-relay system. Finally, the L39T mutation in the conserved polyproline motif (39LPPPP43) of dimeric PhoN leads to a marginal reduction in activity, in contrast to the nearly 50-fold reduction observed for monomeric Prevotella intermedia acid phosphatase, suggesting that the varying quaternary structure of PhoN orthologues may have functional significance.     

Structure of 6-oxo camphor hydrolase H122A mutant bound to its natural product, (2S,4S)-alpha-campholinic acid: mutant structure suggests an atypical mode of transition state binding for a crotonase homolog

The crotonase homolog, 6-oxo camphor hydrolase (OCH), catalyzes the desymmetrization of bicyclic beta-diketones to optically active keto acids via an enzymatic retro-Claisen reaction, resulting in the cleavage of a carbon-carbon bond. We have previously reported the structure of OCH (Whittingham, J. L., Turkenburg, J. P., Verma, C. S., Walsh, M. A., and Grogan, G. (2003) J. Biol. Chem. 278, 1744-1750), which suggested the involvement of five residues, His-45, His-122, His-145, Asp-154, and Glu-244, in catalysis. Here we report mutation studies on OCH that reveal that H145A and D154N mutants of OCH have greatly reduced values of k(cat)/K(m) derived from a very large increase in K(m) for the native substrate, 6-oxo camphor. In addition, H122A has a greatly reduced value of k(cat), and its K(m) is five times that of the wild-type. The location of the active site is confirmed by the 1.9-A structure of the H122A mutant of OCH complexed with the minor diastereoisomer of (2S,4S)-alpha-campholinic acid, the natural product of the enzyme. This shows the pendant acetate of the product hydrogen bonded to a His-145/Asp-154 dyad and the endocyclic carbonyl of the cyclopentane ring hydrogen bonded to Trp-40. The results are suggestive of a base-catalyzed mechanism of C-C bond cleavage and provide clues to the origin of prochiral selectivity by the enzyme and to the recruitment of the crotonase fold for alternate modes of transition state stabilization to those described for other crotonase superfamily members.     

Functional analysis of conserved aspartate and histidine residues located around the type 2 copper site of copper-containing nitrite reductase

A heterologous expression system of the blue copper-containing nitrite reductase from Alcaligenes xylosoxidans GIFU1051 (AxgNIR) was constructed, and the purified recombinant enzyme was characterized. All the characteristic spectroscopic properties and enzyme activity of native AxgNIR were retained in the copper-reconstituted recombinant protein expressed in Escherichia coli, indicating the correct coordination of two types of Cu (type 1 and 2) in the recombinant enzyme. Moreover, two conserved noncoordinate residues, Asp98 and His255, located near the type 2 Cu site were replaced to elucidate the catalytic residue(s) of NIR. The Asp98 residue hydrogen-bonded to the water molecule ligating the type 2 Cu was changed to Ala, Asn, or Glu, and the His255 residue hydrogen-bonded to Asp98 through the water molecule was replaced with Ala, Lys, or Arg. The catalytic rate constants of all mutants were decreased to 0.4-2% of those of the recombinant enzyme, and the apparent K(m) values for nitrite were greatly increased in the Asp98 mutants. All the steady-state kinetic data of the mutants clearly demonstrate that both Asp98 and His255 are involved not only in the catalytic reaction but also in the substrate anchoring.     

Disruption of an active site hydrogen bond converts human heme oxygenase-1 into a peroxidase

The crystal structure of heme oxygenase-1 suggests that Asp-140 may participate in a hydrogen bonding network involving ligands coordinated to the heme iron atom. To examine this possibility, Asp-140 was mutated to an alanine, phenylalanine, histidine, leucine, or asparagine, and the properties of the purified proteins were investigated. UV-visible and resonance Raman spectroscopy indicate that the distal water ligand is lost from the iron in all the mutants except, to some extent, the D140N mutant. In the D140H mutant, the distal water ligand is replaced by the new His-140 as the sixth iron ligand, giving a bis-histidine complex. The D140A, D140H, and D140N mutants retain a trace (<3%) of biliverdin forming activity, but the D140F and D140L mutants are inactive in this respect. However, the two latter mutants retain a low ability to form verdoheme, an intermediate in the reaction sequence. All the Asp-140 mutants exhibit a new peroxidase activity. The results indicate that disruption of the distal hydrogen bonding environment by mutation of Asp-140 destabilizes the ferrous dioxygen complex and promotes conversion of the ferrous hydroperoxy intermediate obtained by reduction of the ferrous dioxygen complex to a ferryl species at the expense of its normal reaction with the porphyrin ring.     

Mechanistic implications of methylglyoxal synthase complexed with phosphoglycolohydroxamic acid as observed by X-ray crystallography and NMR spectroscopy

Methylglyoxal synthase (MGS) and triosephosphate isomerase (TIM) share neither sequence nor structural similarities, yet the reactions catalyzed by both enzymes are similar, in that both initially convert dihydroxyacetone phosphate to a cis-enediolic intermediate. This enediolic intermediate is formed from the abstraction of the pro-S C3 proton of DHAP by Asp-71 of MGS or the pro-R C3 proton of DHAP by Glu-165 of TIM. MGS then catalyzes the elimination of phosphate from this enediolic intermediate to form the enol of methylglyoxal, while TIM catalyzes proton donation to C2 to form D-glyceraldehyde phosphate. A competitive inhibitor of TIM, phosphoglycolohydroxamic acid (PGH) is found to be a tight binding competitive inhibitor of MGS with a K(i) of 39 nM. PGH's high affinity for MGS may be due in part to a short, strong hydrogen bond (SSHB) from the NOH of PGH to the carboxylate of Asp-71. Evidence for this SSHB is found in X-ray, 1H NMR, and fractionation factor data. The X-ray structure of the MGS homohexamer complexed with PGH at 2.0 A resolution shows this distance to be 2.30-2.37 +/- 0.24 A. 1H NMR shows a PGH-dependent 18.1 ppm signal that is consistent with a hydrogen bond length of 2.49 +/- 0.02 A. The D/H fractionation factor (phi = 0.43 +/- 0.02) is consistent with a hydrogen bond length of 2.53 +/- 0.01 A. Further, 15N NMR suggests a significant partial positive charge on the nitrogen atom of bound PGH, which could strengthen hydrogen bond donation to Asp-71. Both His-98 and His-19 are uncharged in the MGS-PGH complex on the basis of the chemical shifts of their Cdelta and C(epsilon) protons. The crystal structure reveals that Asp-71, on the re face of PGH, and His-19, on the si face of PGH, both approach the NO group of the analogue, while His-98, in the plane of PGH, approaches the carbonyl oxygen of the analogue. The phosphate group of PGH accepts nine hydrogen bonds from seven residues and is tilted out of the imidate plane of PGH toward the re face. Asp-71 and phosphate are thus positioned to function as the base and leaving group, respectively, in a concerted suprafacial 1,4-elimination of phosphate from the enediolic intermediate in the second step of the MGS reaction. Combined, these data suggest that Asp-71 is the one base that initially abstracts the C3 pro-S proton from DHAP and subsequently the 3-OH proton from the enediolic intermediate. This mechanism is compared to an alternative TIM-like mechanism for MGS, and the relative merits of both mechanisms are discussed.     

Stability of TEM β-lactamase mutants hydrolyzing third generation cephalosporins

The stability properties of six natural mutants of the TEM-1 β-lactamase have been studied. The glutamate to lysine substitution at positions 104 and 240 stabilize the enzyme. Conversely, the G238S mutant's decreased stability might reflect an altered conformation of the active site and thus be related to the modified substrate profile. The relative stability of the R164S and R164H mutants is explained by the formation of a hydrogen bond between these residues and Asp-179 conferring a somewhat different structure to the omega loop and thus also explaining the extended substrate profile of these mutants. The loss of stability of the R164H mutant with increasing pH values can be explained by the titration of a hydrogen bond between the Nδ of His-164 and the Oδ of Asp-179. The properties of the G238S+E104K double mutant which is the most active against third-generation cephalosporins result from a balance of destabilizing and stabilizing substitutions, and their effects seem to be additive. The behavior of the R164S + E240K mutant might be explained on the basis of a similar compensation phenomenon. © 1995 Wiley-Liss, Inc.     

Zinc-coordination of aspartic acid-76 in Sulfolobus ferredoxin is not required for thermal stability of the molecule

The highly thermostable 7Fe-ferredoxin from Sulfolobus sp. strain 7 has tightly bound zinc at the interface between the N-terminal extra domain and the C-terminal core. The zinc is tetrahedrally ligated by His-16, His-19, His-34, and Asp-76. Previous studies on truncated mutants have shown that the zinc and certain parts, i.e. not all, of the N-terminal extra stretch are responsible for the thermal stabilization of the molecule. To study the role of Asp-76, a series of mutants were constructed with Asp-76 replaced by Glu (D76E), Asn (D76N), or Ala (D76A). All the mutants, as well as wild type ferredoxin, bound 1 mol zinc/mol protein, and showed similar kinetics for 2-oxoacid:ferredoxin oxidoreductase. The stability of the protein was examined by thermal degradation of the clusters. In the absence of guanidium thiocyanate, the T(m), defined as the mid-point temperature of the thermal transition from the native to the denatured state, for every mutant was above 100 degrees C. The T(m) values in the presence of 1 M guanidium thiocyanate were determined to be 90.8, 90.2, 87.1, 84.4, and 72.9 degrees C for the natural, recombinant, D76N-, D76A-, and D76E-ferredoxins, respectively. These results indicate that the interaction between zinc and the carboxyl oxygen of Asp-76 has subtle effects on both the zinc-ligation and stability, although the native zinc center is liganded with high symmetry, suggesting that the three His residues are more important for zinc-binding.     

Specificity and catalysis of uracil DNA glycosylase. A molecular dynamics study of reactant and product complexes with DNA.

The structure of uracil DNA glycosylase (UDG) in complex with a nonamer duplex DNA containing a uracil has been determined only in the product state. The reactant state was constructed by reattaching uracil to the deoxyribose, and both complexes were studied by molecular dynamics simulations. Significant changes in the positions of secondary structural elements in the enzyme are induced by the hydrolysis of the glycosidic bond. The simulations show that the specificity of the uracil pocket in the enzyme is largely retained in both complexes with the exception of Asn-204, which has been identified as a residue that contributes to discrimination between uracil and cytosine. The hydrogen bond between the amide group of Asn-204 and O(4) of uracil is disrupted by fluctuations of the side chain in the reactant state and is replaced by a hydrogen bond to water molecules trapped in the interior of the protein behind the uracil binding pocket. The role of two residues implicated by mutation experiments to be important in catalysis, His-268 and Asp-145, is clarified by the simulations. In the reactant state, His-268 is found 3.45 +/- 0.34 A from the uracil, allowing a water molecule to form a bridge to O(2). The environment in the enzyme raises the pK(a) value of His-268 to 7.1, establishing a protonated residue for assisting in the hydrolysis of the glycosidic bond. In agreement with the crystallographic structure, the DNA backbone retracts after the hydrolysis to allow His-268 to approach the O(2) of uracil with a concomitant release of the bridging water molecule and a reduction in the pK(a) to 5.5, which releases the proton to the product. The side chain of Asp-145 is fully solvated in the reactant state and H-bonded through a water molecule to the 3'-phosphate of uridine. Both the proximity of Asp-145 to the negatively charged phosphate and its pK(a) of 4.4 indicate that it cannot act as a general base catalyst. We propose a mechanism in which the bridging water between Asp-145 and the 3'-phosphate accepts a proton from another water to stabilize the bridge through a hydronium ion as well as to produce the hydroxide anion required for the hydrolytic step. The mechanism is consistent with known experimental data.     

Biochemical and crystallographic studies of the Met144Ala, Asp92Asn and His254Phe mutants of the nitrite reductase from Alcaligenes xylosoxidans provide insight into the enzyme mechanism

Dissimilatory nitrite reductase catalyses the reduction of nitrite (NO(2)(-)) to nitric oxide (NO). Copper-containing nitrite reductases contain both type 1 and type 2 Cu sites. Electron transfer from redox partners is presumed to be mediated via the type 1 Cu site and used at the catalytic type 2 Cu centre along with the substrate nitrite. At the type 2 Cu site, Asp92 has been identified as a key residue in substrate utilisation, since it hydrogen bonds to the water molecule at the nitrite binding site. We have also suggested that protons enter the catalytic site via Asp92, through a water network that is mediated by His254. The role of these residues has been investigated in the blue copper nitrite reductase from Alcaligenes xylosoxidans (NCIMB 11015) by a combination of point mutation, enzymatic activity measurement and structure determination.In addition, it has been suggested that the enzyme operates via an ordered mechanism where an electron is transferred to the type 2 Cu site largely when the second substrate nitrite is bound and that this is controlled via the lowering of the redox potential of the type 2 site when it is loaded with nitrite. Thus, a small perturbation of the type 1 Cu site should result in a significant effect on the activity of the enzyme. For this reason a mutation of Met144, which is the weakest ligand of the type 1 Cu, is investigated. The structures of H254F, D92N and M144A have been determined to 1.85 A, 1.9 A and 2.2 A resolution, respectively. The D92N and H254F mutants have negligible or no activity, while the M144A mutant has 30 % activity of the native enzyme. Structural and spectroscopic data show that the loss of activity in H254F is due to the catalytic site being occupied by Zn while the loss/reduction of activity in D92N/M144A are due to structural reasons. The D92N mutation results in the loss of the Asp92 hydrogen bond to the Cu-ligated water. Therefore, the ligand is no longer able to perform proton abstraction. Even though the loss of activity in H254F is due to lack of catalytic Cu, the mutation does cause the disruption of the water network, confirming its key role in proton channel. The structure of the H254F mutant is the first case where full occupancy Zn at the type 2 Cu site is observed, but despite the previously noted similarity of this site to the carbonic anhydrase catalytic site, no carbonic anhydrase activity is observed. The H254F and D92N mutant structures provide, for the first time, observation of surface Zn sites which may act as a Zn sink and prevent binding of Zn at the catalytic Cu site in the native enzyme.     

Structure and function of the catalytic site mutant Asp 99 Asn of phospholipase A2: absence of the conserved structural water.

To probe the role of the Asp-99 ... His-48 pair in phospholipase A2 (PLA2) catalysis, the X-ray structure and kinetic characterization of the mutant Asp-99-->Asn-99 (D99N) of bovine pancreatic PLA2 was undertaken. Crystals of D99N belong to the trigonal space group P3(1)21 and were isomorphous to the wild type (WT) (Noel JP et al., 1991, Biochemistry 30:11801-11811). The 1.9-A X-ray structure of the mutant showed that the carbonyl group of Asn-99 side chain is hydrogen bonded to His-48 in the same way as that of Asp-99 in the WT, thus retaining the tautomeric form of His-48 and the function of the enzyme. The NH2 group of Asn-99 points away from His-48. In contrast, in the D102N mutant of the protease enzyme trypsin, the NH2 group of Asn-102 is hydrogen bonded to His-57 resulting in the inactive tautomeric form and hence the loss of enzymatic activity. Although the geometry of the catalytic triad in the PLA2 mutant remains the same as in the WT, we were surprised that the conserved structural water, linking the catalytic site with the ammonium group of Ala-1 of the interfacial site, was ejected by the proximity of the NH2 group of Asn-99. The NH2 group now forms a direct hydrogen bond with the carbonyl group of Ala-1.     

An Alternative Mechanism for the Methylation of Phosphoethanolamine Catalyzed by Plasmodium falciparum Phosphoethanolamine Methyltransferase

The phosphobase methylation pathway catalyzed by the phosphoethanolamine methyltransferase in Plasmodium falciparum (PfPMT), the malaria parasite, offers an attractive target for anti-parasitic drug development. PfPMT methylates phosphoethanolamine (pEA) to phosphocholine for use in membrane biogenesis. Quantum mechanics and molecular mechanics (QM/MM) calculations tested the proposed reaction mechanism for methylation of pEA involving the previously identified Tyr-19–His-132 dyad, which indicated an energetically unfavorable mechanism. Instead, the QM/MM calculations suggested an alternative mechanism involving Asp-128. The reaction coordinate involves the stepwise transfer of a proton to Asp-128 via a bridging water molecule followed by a typical S_n2-type methyl transfer from S-adenosylmethionine to pEA. Functional analysis of the D128A, D128E, D128Q, and D128N PfPMT mutants shows a loss of activity with pEA but not with the final substrate of the methylation pathway. X-ray crystal structures of the PfPMT-D128A mutant in complex with S-adenosylhomocysteine and either pEA or phosphocholine reveal how mutation of Asp-128 disrupts a hydrogen bond network in the active site. The combined QM/MM, biochemical, and structural studies identify a key role for Asp-128 in the initial step of the phosphobase methylation pathway in Plasmodium and provide molecular insight on the evolution of multiple activities in the active site of the PMT.     

Mutational analysis of basic residues in the rat vesicular acetylcholine transporter. Identification of a transmembrane ion pair and evidence that histidine is not involved in proton translocation

The function of positively charged residues and the interaction of positively and negatively charged residues of the rat vesicular acetylcholine transporter (rVAChT) were studied. Changing Lys-131 in transmembrane domain helix 2 (TM2) to Ala or Leu eliminated transport activity, with no effect on vesamicol binding. However, replacement by His or Arg retained transport activity, suggesting a positive charge in this position is critical. Mutation of His-444 in TM12 or His-413 in the cytoplasmic loop between TM10 and TM11 was without effect on ACh transport, but vesamicol binding was reduced with His-413 mutants. Changing His-338 in TM8 to Ala or Lys did not effect ACh transport, however replacement with Cys or Arg abolished activity. Mutation of both of the transmembrane histidines or all three of the luminal loop histidines showed no change in acetylcholine transport. The mutant H338A/D398N between oppositely charged residues in transmembrane domains showed no vesamicol binding, however the charge reversal mutant H338D/D398H restored binding. This suggests that His-338 forms an ion pair with Asp-398. The charge neutralizing mutant K131A/D425N or the charge exchanged mutant K131D/D425K did not restore ACh transport. Taken together these results provide new insights into the tertiary structure in VAChT.     

Studies on the active sites ofBacillus cereus sphingomyelinase substitution of some amino acids by site-directed mutagenesis

Summary Chemical modifications suggested that acidic amino acids such as aspartic and glutamic acids are involved in the active sites ofBacillus cereus sphingomyelinase. Among aspartic acid residues in the conserved regions of this enzyme, Asp-126, Asp-156, Asp-233 and Asp-295 were converted to glycine by site-directed mutagenesis. According to prediction on structural similarity to pancreatic DNase I, His-151 and His-296 were also converted to alanine. The Asp and His mutants, D126G, D156G, D233G, D295G, H151A and H296A, were produced inBacillus brevis 47, a protein-hyperproducing strain. The catalytic activities of D295G, H151A and H296A were completely abolished, and sphingomyelin-hydrolyzing activity of D126G or D156G was reduced by more than 50%. The activity of D126G towardp-NPPC was comparable to that of the wild-type, while D156G catalyzed the hydrolysis of HNP andp-NPPC more efficiently than the wild-type. Hemolytic activities of the mutants were parallel to their sphingomyelin-hydrolyzing activities.     

Role of aspartate-133 and histidine-458 in the mechanism of tryptophan indole-lyase from Proteus vulgaris

Tryptophan indole-lyase (Trpase) from Proteus vulgaris is a pyridoxal 5'-phosphate dependent enzyme that catalyzes the reversible hydrolytic cleavage of L-Trp to yield indole and ammonium pyruvate. Asp-133 and His-458 are strictly conserved in all sequences of Trpase, and they are located in the proposed substrate-binding region of Trpase. These residues were mutated to alanine to probe their role in substrate binding and catalysis. D133A mutant Trpase has no measurable activity with L-Trp as substrate, but still retains activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase has 1.6% of wild-type Trpase activity with L-Trp, and high activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines, and beta-chloro-L-alanine. H458A mutant Trpase does not exhibit the pK(a) of 5.3 seen in the pH dependence of k(cat)/K(m) of L-Trp for wild-type Trpase. Both mutant enzymes are inhibited by L-Ala, L-Met, and L-Phe, with K(i) values similar to those of wild-type Trpase, but oxindolyl-L-alanine and beta-phenyl-DL-serine show much weaker binding to the mutant enzymes, suggesting that Asp-133 and His-458 are involved in the binding of these ligands. D133A and H458A mutant Trpase exhibit absorption and CD spectra in the presence of substrates and inhibitors that are similar to wild-type Trpase, with peaks at about 420 and 500 nm. The rate constants for formation of the 500 nm bands for the mutant enzymes are equal to or greater than those of wild-type Trpase, indicating that Asp-133 and His-458 do not play a role in the formation of quinonoid intermediates. In constrast to wild-type and H458A mutant Trpase, D133A mutant Trpase forms an intermediate from S-ethyl-L-Cys that absorbs at 345 nm, and is likely to be an alpha-aminoacrylate. Crystals of D133A and H458A mutant Trpase bind amino acids with similar affinity as the proteins in solution, except for L-Ala, which binds to D133A mutant Trpase crystals about 20-fold stronger than in solution. These results suggest that Asp-133 and His-458 play an important role in the elimination reaction of L-Trp. Asp-133 likely forms a hydrogen bond directly to the indole NH of the substrate, while His-458 probably is hydrogen bonded to Asp-133.     

An examination of the role of asp-177 in the His-Asp catalytic dyad of Leuconostoc mesenteroides glucose 6-phosphate dehydrogenase: X-ray structure and pH dependence of kinetic parameters of the D177N mutant enzyme

The role of Asp-177 in the His-Asp catalytic dyad of glucose 6-phosphate dehydrogenase from Leuconostoc mesenteroides has been investigated by a structural and functional characterization of the D177N mutant enzyme. Its three-dimensional structure has been determined by X-ray cryocrystallography in the presence of NAD(+) and in the presence of glucose 6-phosphate plus NADPH. The structure of a glucose 6-phosphate complex of a mutant (Q365C) with normal enzyme activity has also been determined and substrate binding compared. To understand the effect of Asp-177 on the ionization properties of the catalytic base His-240, the pH dependence of kinetic parameters has been determined for the D177N mutant and compared to that of the wild-type enzyme. The structures give details of glucose 6-phosphate binding and show that replacement of the Asp-177 of the catalytic dyad with asparagine does not affect the overall structure of glucose 6-phosphate dehydrogenase. Additionally, the evidence suggests that the productive tautomer of His-240 in the D177N mutant enzyme is stabilized by a hydrogen bond with Asn-177; hence, the mutation does not affect tautomer stabilization. We conclude, therefore, that the absence of a negatively charged aspartate at 177 accounts for the decrease in catalytic activity at pH 7.8. Structural analysis suggests that the pH dependence of the kinetic parameters of D177N glucose 6-phosphate dehydrogenase results from an ionized water molecule replacing the missing negative charge of the mutated Asp-177 at high pH. Glucose 6-phosphate binding orders and orients His-178 in the D177N-glucose 6-phosphate-NADPH ternary complex and appears to be necessary to form this water-binding site.     

Characterization of mutants that change the hydrogen bonding of the semiquinone radical at the QH site of the cytochrome bo3 from Escherichia coli

The cytochrome bo3 ubiquinol oxidase catalyzes the two-electron oxidation of ubiquinol in the cytoplasmic membrane of Escherichia coli, and reduces O2 to water. This enzyme has a high affinity quinone binding site (QH), and the quinone bound to this site acts as a cofactor, necessary for rapid electron transfer from substrate ubiquinol, which binds at a separate site (QL), to heme b. Previous pulsed EPR studies have shown that a semiquinone at the QH site formed during the catalytic cycle is a neutral species, with two strong hydrogen bonds to Asp-75 and either Arg-71 or Gln-101. In the current work, pulsed EPR studies have been extended to two mutants at the QH site. The D75E mutation has little influence on the catalytic activity, and the pattern of hydrogen bonding is similar to the wild type. In contrast, the D75H mutant is virtually inactive. Pulsed EPR revealed significant structural changes in this mutant. The hydrogen bond to Arg-71 or Gln-101 that is present in both the wild type and D75E mutant oxidases is missing in the D75H mutant. Instead, the D75H has a single, strong hydrogen bond to a histidine, likely His-75. The D75H mutant stabilizes an anionic form of the semiquinone as a result of the altered hydrogen bond network. Either the redistribution of charge density in the semiquinone species, or the altered hydrogen bonding network is responsible for the loss of catalytic function.     

The role of the conserved residues His-246, His-199, and Tyr-255 in the catalysis of catechol 2,3-dioxygenase from Pseudomonas stutzeri OX1

Catechol 2,3-dioxygenase (C2,3O) from Pseudomonas stutzeri OX1, which is able to grow on various aromatic substrates as the sole source of carbon and energy, has been expressed in Escherichia coli, purified, characterized, and found to be very similar to other dioxygenases from Pseudomonas species. Interestingly, the activity of the protein shows a rather unusual pH dependence when assayed on catechol. A model of the catalytic mechanism was developed that is able to reproduce the catalytic behavior of the protein as a function of the pH. The model includes multiple equilibria and four productive intermediates with different ionization states of the enzyme-substrate complex. The fitting of the theoretical curve to the experimental data suggests that a tyrosine and two histidine residues are involved in catalysis. Mutants (H246N)-, (H246A)-, (H199N)- and (Y255F)-C2,3O were produced to investigate the role of highly conserved His-199, His-246, and Tyr-255. The strongly reduced activity of the mutants suggests a primary catalytic role for each of these residues. Moreover, mutants at positions 199 and 246 display pH profiles different from that of the wild-type protein, thus indicating that residues His-246 and His-199 play a role in determining the unusual pH dependence of the enzyme. In addition, electron-withdrawing groups on catechol, which increase the acidity of the phenolic hydroxyl group, are able to counterbalance the effect of the mutation H246N in reducing catalytic activity but cause a further reduction of the activity of (H199N)-C2,3O. This finding suggests that His-246 is involved in the initial catechol deprotonation, whereas His-199 promotes the reaction between oxygen and the aromatic ring.     

Distinct enzymic functional groups are required for the phosphomonoesterase and phosphodiesterase activities of Clostridium thermocellum polynucleotide kinase/phosphatase

The central phosphatase domain of Clostridium thermocellum polynucleotide kinase/phosphatase (CthPnkp) belongs to the dinuclear metallophosphoesterase superfamily. Prior mutational studies of CthPnkp identified 7 individual active site side chains (Asp-187, His-189, Asp-233, Asn-263, His-323, His-376, and Asp-392) required for Ni2+-dependent hydrolysis of p-nitrophenyl phosphate. Here we find that Mn2+-dependent phosphomonoesterase activity requires two additional residues, Arg-237 and His-264. We report that CthPnkp also converts bis-p-nitrophenyl phosphate to p-nitrophenol and inorganic phosphate via a processive two-step mechanism. The Ni2+-dependent phosphodiesterase activity of CthPnkp requires the same seven side chains as the Ni2+-dependent phosphomonoesterase. However, the Mn2+-dependent phosphodiesterase activity does not require His-189, Arg-237, or His-264, each of which is critical for the Mn2+-dependent phosphomonoesterase. Mutations H189A, H189D, and D392N transform the metal and substrate specificity of CthPnkp such that it becomes a Mn2+-dependent phosphodiesterase. The H189E change results in a Mn2+/Ni2+-dependent phosphodiesterase. Mutations H376N, H376D, and D392E convert the enzyme into a Mn2+-dependent phosphodiesterase-monoesterase. The phosphodiesterase activity is strongly stimulated compared with wild-type CthPnkp when His-189 is changed to Asp, Arg-237 is replaced by Ala or Gln, and His-264 is replaced by Ala, Asn, or Gln. Steady-state kinetic analysis of wild-type and mutated enzymes illuminates the structural features that affect substrate affinity and kcat. Our results highlight CthPnkp as an "undifferentiated" diesterase-monoesterase that can evolve toward narrower metal and substrate specificities via alterations of the active site milieu.