Essential histidines of prostaglandin endoperoxide synthase. His-309 is involved in heme binding

Prostaglandin endoperoxide (PGH) synthase has a single iron protoporphyrin IX which is required for both the cyclooxygenase and peroxidase activities of the enzyme. At room temperature, the heme iron is coordinated at the axial position by an imidazole, and about 20% of the heme iron is coordinated at the distal position by an imidazole. We have used site-directed mutagenesis to investigate which histidine residues are involved in PGH synthase catalysis and heme binding. Individual mutant cDNAs for ovine PGH synthases were prepared with amino acid substitutions at each of 13 conserved histidines. cos-1 cells were transfected with each of these cDNAs, and the cyclooxygenase and peroxidase activities of the resulting microsomal PGH synthases were measured. Mutant PGH synthases in which His-207, His-309, or His-388 was replaced with either glutamine or alanine lacked both activities. Gln-386 and Ala-386 PGH synthase mutants exhibited cyclooxygenase but not peroxidase activities. Other mutants exhibited both activities at varying levels. Because binding of heme renders native PGh synthase resistant to cleavage by trypsin, we examined the effects of heme on the relative sensitivities of native, Ala-204, Ala-207, Ala-309, Ala-386, and Ala-388 mutant PGH synthases to trypsin as a measure of the heme-protein interaction. The Ala-309 PGh synthase mutant was notably hypersensitive to tryptic cleavage, even in the presence of exogenous heme; in contrast, the native enzyme and the other alanine mutants exhibited similar, lower sensitivities toward trypsin and, except for the Ala-386 mutant, were partially protected from trypsin cleavage by heme. Preincubation of the native and each of the alanine mutant PGH synthases, including the Ala-309 mutant, with indomethacin protected the proteins from trypsin cleavage. Thus, all the mutant proteins retain sufficient three-dimensional structure to bind cyclooxygenase inhibitors. Our results suggest that His-309 is one of the heme ligands, probably the axial ligand, of PGH synthase. Two other histidines, His-207 and His-388, are essential for both PGH synthase activities suggesting that either His-207 or His-388 can serve as the distal heme ligand; however, the trypsin cleavage measurements imply that neither His-207 nor His-388 is required for heme binding. This is consistent with the fact that only 20% of the distal coordination position of the heme iron of PGH synthase is occupied by an imidazole side chain.     

Identification of active site residues in glucosylceramide synthase. A nucleotide-binding catalytic motif conserved with processive beta-glycosyltransferases

Glucosylceramide synthase (GCS) transfers glucose from UDP-Glc to ceramide, catalyzing the first glycosylation step in the formation of higher order glycosphingolipids. The amino acid sequence of GCS was reported to be dissimilar from other proteins, with no identifiable functional domains. We previously identified His-193 of rat GCS as an important residue in UDP-Glc and GCS inhibitor binding; however, little else is known about the GCS active site. Here, we identify key residues of the GCS active site by performing biochemical and site-directed mutagenesis studies of rat GCS expressed in bacteria. First, we found that Cys-207 was the primary residue involved in GCS N-ethylmaleimide sensitivity. Next, we showed by multiple alignment that the region of GCS flanking His-193 and Cys-207 (amino acids 89-278) contains a D1,D2,D3,(Q/R)XXRW motif found in the putative active site of processive beta-glycosyltransferases (e.g. cellulose, chitin, and hyaluronan synthases). Site-directed mutagenesis studies demonstrated that most of the highly conserved residues were essential for GCS activity. We also note that GCS and processive beta-glycosyltransferases are topologically similar, possessing cytosolic active sites, with putative transmembrane domains immediately N-terminal to the conserved domain. These results provide the first extensive information on the GCS active site and show that GCS and processive beta-glycosyltransferases possess a conserved substrate-binding/catalytic domain.     

Replacement by site-directed mutagenesis indicates a role for histidine 170 in the glutamine amide transfer function of anthranilate synthase

Anthranilate synthase is a glutamine amidotransferase that catalyzes the first reaction in tryptophan biosynthesis. Conserved amino acid residues likely to be essential for glutamine-dependent activity were identified by alignment of the glutamine amide transfer domains in four different enzymes: anthranilate synthase component II (AS II), p-aminobenzoate synthase component II, GMP synthetase, and carbamoyl-P synthetase. Conserved amino acids were mainly localized in three clusters. A single conserved histidine, AS II His-170, was replaced by tyrosine using site-directed mutagenesis. Glutamine-dependent enzyme activity was undetectable in the Tyr-170 mutant, whereas the NH3-dependent activity was unchanged. Affinity labeling of AS II active site Cys-84 by 6-diazo-5-oxonorleucine was used to distinguish whether His-170 has a role in formation or in breakdown of the covalent glutaminyl-Cys-84 intermediate. The data favor the interpretation that His-170 functions as a general base to promote glutaminylation of Cys-84. Reversion analysis was consistent with a proposed role of His-170 in catalysis as opposed to a structural function. These experiments demonstrate the application of combining sequence analyses to identify conserved, possibly functional amino acids, site-directed mutagenesis to replace candidate amino acids, and protein chemistry for analysis of mutationally altered proteins, a regimen that can provide new insights into enzyme function.     

Vanadyl(IV) binding to mammalian ferritins. An EPR study aided by site-directed mutagenesis

During its metabolism, vanadium is known to become associated with the iron storage protein, ferritin. To elucidate probable vanadium binding sites on the protein, VO2+ binding to mammalian ferritins was studied using site-directed mutagenesis and EPR spectroscopy. VO2+-apoferritin EPR spectra of human H-chain (100% H), L-chain (100% L), horse spleen (84% L, 16% H) and sheep spleen (45% L, 55% H) ferritins revealed the presence of alpha and beta VO2+ species in all the proteins, implying that the ligands for these species are conserved between the H- and L-chains. The alpha species is less stable than the beta species and decreases with increasing pH, demonstrating that the two species are not pH-related, a result contrary to earlier proposals. EPR spectra of site-directed HuHF variants of several residues conserved in H- and L-chain ferritins (Asp-131, Glu-134, His-118 and His-128) suggest that His-118 near the outer opening of the three-fold channel is probably a ligand for VO2+ and is responsible for the beta signals in the EPR spectrum. The data indicate that VO2+ does not bind to the Asp-131 and Glu-134 residues within the three-fold channels nor does it bind at the ferroxidase site residues Glu-62 or His-65 or at the putative nucleation site residues Glu-61,64,67. While the ferroxidase site is not a site for VO2+ binding, mutation of residues Glu-62 and His-65 of this site to Ala affects VO2+ binding at His-118, located some 17 A away. Thus, VO2+ spin probe studies provide a window on structural changes in ferritin not seen in most previous work and indicate that long-range effects caused by point mutations must be carefully considered when drawing conclusions from mutagenesis studies of the protein.     

Crystal structure of the CheA histidine phosphotransfer domain that mediates response regulator phosphorylation in bacterial chemotaxis

The x-ray crystal structure of the P1 or H domain of the Salmonella CheA protein has been solved at 2.1-A resolution. The structure is composed of an up-down up-down four-helix bundle that is typical of histidine phosphotransfer or HPt domains such as Escherichia coli ArcB(C) and Saccharomyces cerevisiae Ypd1. Loop regions and additional structural features distinguish all three proteins. The CheA domain has an additional C-terminal helix that lies over the surface formed by the C and D helices. The phosphoaccepting His-48 is located at a solvent-exposed position in the middle of the B helix where it is surrounded by several residues that are characteristic of other HPt domains. Mutagenesis studies indicate that conserved glutamate and lysine residues that are part of a hydrogen-bond network with His-48 are essential for the ATP-dependent phosphorylation reaction but not for the phosphotransfer reaction with CheY. These results suggest that the CheA-P1 domain may serve as a good model for understanding the general function of HPt domains in complex two-component phosphorelay systems.     

Acid-base catalysis in the extradiol catechol dioxygenase reaction mechanism: site-directed mutagenesis of His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB)

The extradiol catechol dioxygenases catalyze the non-heme iron(II)-dependent oxidative cleavage of catechols to 2-hydroxymuconaldehyde products. Previous studies of a biomimetic model reaction for extradiol cleavage have highlighted the importance of acid-base catalysis for this reaction. Two conserved histidine residues were identified in the active site of the class III extradiol dioxygenases, positioned within 4-5 A of the iron(II) cofactor. His-115 and His-179 in Escherichia coli 2,3-dihydroxyphenylpropionate 1,2-dioxygenase (MhpB) were replaced by glutamine, alanine, and tyrosine. Each mutant enzyme was catalytically inactive for extradiol cleavage, indicating the essential nature of these acid-base residues. Replacement of neighboring residues Asp-114 and Pro-181 gave D114N, P181A, and P181H mutant enzymes with reduced catalytic activity and altered pH/rate profiles, indicating the role of His-179 as a base and His-115 as an acid. Mutant H179Q was catalytically active for the lactone hydrolysis half-reaction, whereas mutant H115Q was inactive, implying a role for His-115 in lactone hydrolysis. A catalytic mechanism involving His-179 and His-115 as acid-base catalytic residues is proposed.     

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.     

Identification of histidine residues in Wolinella succinogenes hydrogenase that are essential for menaquinone reduction by H2

The cytochrome b subunit (HydC) of Wolinella succinogenes hydrogenase binds two haem B groups. This is concluded from the haem B content of the isolated hydrogenase and is confirmed by the response of its cytochrome b to redox titration. In addition, three of the four haem B ligands were identified by characterizing mutants with the corresponding histidine residues replaced by alanine or methionine. Substitution in HydC of His-25, His-67 or His-186, which are, in addition to His-200, predicted to be haem B ligands, caused the loss of quinone reactivity of the hydrogenase, while the activity of benzylviologen reduction was retained. The corresponding mutants did not grow with H₂ as electron donor and either fumarate or polysulphide as terminal electron acceptor. The mutants grown with formate and fumarate did not catalyse electron transport from H₂ to fumarate or to polysulphide, or quinone reduction by H₂, in contrast to the wild-type strain. Cytochrome b was not reduced by H₂ in the Triton X-100 extract of the mutant membranes, which contained wild-type amounts of the mutated HydC protein. Substitution in HydC of His-122, His-158 or His-187, which are predicted not to be haem B ligands, yielded mutants with wild-type properties. Substitution in HydA of His-188 or of His-305 resulted in mutants with the same properties as those lacking one of the haem B ligands of HydC. His-305 is located in the membrane-integrated C-terminal helix of HydA. His-188 of HydA is predicted to be a ligand of the distal iron–sulphur centre that may serve as the direct electron donor to the haem B groups of HydC. The results suggest that each of the three predicted haem B ligands of HydC tested (out of four) is required for electron transport from H₂ to either fumarate or polysulphide, and for quinone reactivity. This also holds true for the two conserved histidine residues of HydA.     

Identification of the stable free radical tyrosine residue in ribonucleotide reductase. A sequence comparison

The small subunit of ribonucleoside diphosphate reductase contains a unique tyrosine radical and a binuclear iron center. An alignment of different primary structures of the small subunit in Escherichia coli, the marine mollusc Spisula solidissima, Epstein Barr and Herpes simplex viruses shows that regions comprising residues 115-122, 204-212 and 234-241 (in E.coli numbering) are strikingly similar and are likely to be recognized as functionally important. Two of 16 tyrosine residues and 2 of 8 histidine residues are conserved. We propose that Tyr-122 is responsible for radical stabilization and that His-118 and His-241 together with Glu-115 and Asp-237 or Glu-238 are ligands of the iron center.     

Interaction between collagen and the alpha(2) I-domain of integrin alpha(2)beta(1). Critical role of conserved residues in the metal ion-dependent adhesion site (MIDAS) region.

A docking model of the alpha(2) I-domain and collagen has been proposed based on their crystal structures (Emsley, J., King, S., Bergelson, J., and Liddington, R. C. (1997) J. Biol. Chem. 272, 28512-28517). In this model, several amino acid residues in the I-domain make direct contact with collagen (Asn-154, Asp-219, Leu-220, Glu-256, His-258, Tyr-285, Asn-289, Leu-291, Asn-295, and Lys-298), and the protruding C-helix of alpha(2) (residues 284-288) determines ligand specificity. Because most of the proposed critical residues are not conserved, different I-domains are predicted to bind to collagen differently. We found that deleting the entire C-helix or mutating the predicted critical residues had no effect on collagen binding to whole alpha(2)beta(1), with the exception that mutating Asn-154, Asp-219, and His-258 had a moderate effect. We performed further studies and found that mutating the conserved surface-exposed residues in the metal ion-dependent adhesion site (MIDAS) (Tyr-157 and Gln-215) significantly blocks collagen binding. We have revised the docking model based on the mutagenesis data. In the revised model, conserved Tyr-157 makes contact with collagen in addition to the previously proposed Asn-154, Asp-219, His-258, and Tyr-285 residues. These results suggest that the collagen-binding I-domains (e.g. alpha(1), alpha(2), and alpha(10)) bind to collagen in a similar fashion.     

Identification of putative active site residues of ACAT enzymes

In this report, we sought to determine the putative active site residues of ACAT enzymes. For experimental purposes, a particular region of the C-terminal end of the ACAT protein was selected as the putative active site domain due to its high degree of sequence conservation from yeast to humans. Because ACAT enzymes have an intrinsic thioesterase activity, we hypothesized that by analogy with the thioesterase domain of fatty acid synthase, the active site of ACAT enzymes may comprise a catalytic triad of ser-his-asp (S-H-D) amino acid residues. Mutagenesis studies revealed that in ACAT1, S456, H460, and D400 were essential for activity. In ACAT2, H438 was required for enzymatic activity. However, mutation of D378 destabilized the enzyme. Surprisingly, we were unable to identify any S mutations of ACAT2 that abolished catalytic activity. Moreover, ACAT2 was insensitive to serine-modifying reagents, whereas ACAT1 was not. Further studies indicated that tyrosine residues may be important for ACAT activity. Mutational analysis showed that the tyrosine residue of the highly conserved FYXDWWN motif was important for ACAT activity. Furthermore, Y518 was necessary for ACAT1 activity, whereas the analogous residue in ACAT2, Y496, was not. The available data suggest that the amino acid requirement for ACAT activity may be different for the two ACAT isozymes.     

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.     

Mutagenesis of histidinol dehydrogenase reveals roles for conserved histidine residues.

The dimeric zinc metalloenzyme L-histidinol dehydrogenase (HDH) catalyzes an unusual four-electron oxidation of the amino alcohol histidinol via the histidinaldehyde intermediate to the acid product histidine with the reduction of two molecules of NAD. An essential base, with pKa about 8, is involved in catalysis. Here we report site-directed mutagenesis studies to replace each of the five histidine residues (His-98, His-261, His-326, His-366, and His-418) in Salmonella typhimurium with either asparagine or glutamine. In all cases, the overexpressed enzymes were readily purified and behaved as dimers. Substitution of His-261 and His-326 by asparagine caused about 7000- and 500-fold decreases in kcat, respectively, with little change in KM values. Similar loss of activity was also reported for a H261N mutant Brassica HDH [Nagai, A., and Ohta, D. (1994) J. Biochem. 115, 22-25]. Kinetic isotope effects, pH profiles, substrate rescue, and stopped-flow experiments suggested that His-261 and His-326 are involved in proton transfers during catalysis. Sensitivity to metal ion chelator and decreased affinities for metal ions with substitutions at His-261 and His-418 suggested that these two residues are candidates for zinc ion ligands.     

Identification of catalytically important amino acid residues of Streptomyces lividans acetylxylan esterase A from carbohydrate esterase family 4

Multiple sequence alignment of Streptomyces lividans acetylxylan esterase A and other carbohydrate esterase family 4 enzymes revealed the following conserved amino acid residues: Asp-12, Asp-13, His-62, His-66, Asp-130, and His-155. These amino acids were mutated in order to investigate a functional role of these residues in catalysis. Replacement of the conserved histidine residues by alanine caused significant reduction of enzymatic activity. Maintenance of ionizable carboxylic group in side chains of amino acids at positions 12, 13, and 130 seems to be necessary for catalytic efficiency. The absence of conserved serine excludes a possibility that the enzyme is a serine esterase, in contrast to acetylxylan esterases of carbohydrate esterase families 1, 5, and 7. On the contrary, total conservation of Asp-12, Asp-13, Asp-130, and His-155 along with dramatic decrease in enzyme activity of mutants of either of these residues lead us to a suggestion that acetylxylan esterase A from Streptomyces lividans and, by inference, other members of carbohydrate esterase family 4 are aspartic deacetylases. We propose that one component of the aspartate dyad/triad functions as a catalytic nucleophile and the other one(s) as a catalytic acid/base. The ester/amide bond cleavage would proceed via a double displacement mechanism through covalently linked acetyl-enzyme intermediate of mixed anhydride type.     

Evidence that conserved residues Cys-62 and Cys-154 within the Azotobacter vinelandii nitrogenase MoFe protein α-subunit are essential for nitrogenase activity but conserved residues His-83 and Cys-88 are not

Metallocluster extrusion requirements, interspecies MoFe-protein primary sequence comparisons and comparison of the primary sequences of the MoFe-protein subunits with each other have been used to assign potential P-cluster (Fe-S cluster) domains within the MoFe protein. In each alpha-beta unit of the MoFe protein, alpha-subunit domains, which include potential Fe-S cluster ligands Cys-62, His-83, Cys-88 and Cys-154, and beta-subunit domains, which include potential Fe-S cluster ligands Cys-70, His-90, Cys-95 and Cys-153, are proposed to comprise nearly equivalent P-cluster environments located adjacent to each other in the native protein. As an approach to test this model and to probe the functional properties of the P clusters, amino acid residue substitutions were placed at the alpha-subunit Cys-62, His-83, Cys-88 and Cys-154 positions by site-directed mutagenesis of the Azotobacter vinelandii nifD gene. The diazotrophic growth rates, MoFe-protein acetylene-reduction activities, and whole-cell S = 3/2 electron paramagnetic resonance spectra of these mutants were examined. Results of these experiments show that MoFe-protein alpha-subunit residues, Cys-62 and Cys-154, are probably essential for MoFe-protein activity but that His-83 and Cys-88 residues are not. These results indicate either that His-83 and Cys-88 do not provide essential P-cluster ligands or that a new cluster-ligand arrangement is formed in their absence.     

Replacement of putative axial ligands of heme iron in yeast cytochrome c1 by site-directed mutagenesis

The His-44 and Met-164 residues of yeast cytochrome c1 are evolutionally conserved and regarded as heme axial ligands bonding to the fifth and sixth coordination sites of the heme iron, which is directly involved in the electron transfer mechanism. Oligonucleotide-directed mutagenesis was used to generate mutant forms of cytochrome c1 of yeast having amino acid replacements of the putative axial ligands of the heme iron. When a cytochrome c1-deficiency yeast strain was transformed with a gene encoding the Phe-44, Tyr-44, Leu-164, or Lys-164 protein, none of these transformants could grow on the non-fermentable carbon source. These results suggest that the His-44 and Met-164 residues have a critical role in the function of cytochrome c1 in vivo, most probably as axial ligands of the heme iron. Further analysis revealed that the mutant yeast cells with the Phe-44, Tyr-44, or Leu-164 protein lacked the characteristic difference spectroscopic signal of cytochrome c1. However, in the Lys-164 mutant cells, partial recovery of the cytochrome c1 signal was observed. Moreover, the Lys-164 protein retained a low but significant level of succinate-cytochrome c reductase activity in vitro. The possibility that the nitrogen of Lys-164 served as the sixth heme ligand is discussed in comparison with cytochrome f of a photosynthetic electron-transfer complex, in which lysine has been proposed to be the sixth ligand.     

Mutation analysis of the human 5-lipoxygenase C-terminus: support for a stabilizing C-terminal loop

Lipoxygenases contain prosthetic iron, in human 5-lipoxygenase (5LO) the C-terminal isoleucine carboxylate constitutes one of five identified ligands. ATP is one of several factors determining 5LO activity. We compared properties of a series of 5LO C-terminal deletion mutants (one to six amino acid residues deleted). All mutants were enzymatically inactive (expected due to loss of iron), but expression yield (in E. coli) and affinity to ATP-agarose was markedly different. Deletion of up to four C-terminal residues was compatible with good expression and retained affinity to the ATP-column, as for wild-type 5LO. However when also the fifth residue was deleted (Asn-669) expression yield decreased and the affinity to ATP was markedly diminished. This was interpreted as a result of deranged structure and stability, due to loss of a hydrogen bond between Asn-669 and His-399. Mutagenesis of these residues supported this conclusion. In the structure of soybean lipoxygenase-1, a C-terminal loop was pointed out as important for correct orientation of the C-terminus. Accordingly, a hydrogen bond appears to stabilize such a C-terminal loop also in 5LO.     

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.     

Identification of key amino acid residues in Neisseria polysaccharea amylosucrase

Amylosucrase from Neisseria polysaccharea catalyzes the synthesis of an amylose-like polymer from sucrose. Sequence alignment revealed that it belongs to the glycoside hydrolase family 13. Site-directed mutagenesis enabled the identification of functionally important amino acid residues located at the active center. Asp-294 is proposed to act as the catalytic nucleophile and Glu-336 as general acid base catalyst in amylosucrase. The conserved Asp-401, His-195 and His-400 residues are critical for the enzymatic activity. These results provide strong support for the predicted close structural and functional relationship between the sucrose-glucosyltransferases and enzymes of the alpha-amylase family.     

Structural and chemical requirements for histidine phosphorylation by the chemotaxis kinase CheA

The CheA histidine kinase initiates the signal transduction pathway of bacterial chemotaxis by autophosphorylating a conserved histidine on its phosphotransferase domain (P1). Site-directed mutations of neighboring conserved P1 residues (Glu-67, Lys-48, and His-64) show that a hydrogen-bonding network controls the reactivity of the phospho-accepting His (His-45) in Thermotoga maritima CheA. In particular, the conservative mutation E67Q dramatically reduces phosphotransfer to P1 without significantly affecting the affinity of P1 for the CheA ATP-binding domain. High resolution crystallographic studies revealed that although all mutants disrupt the hydrogen-bonding network to varying degrees, none affect the conformation of His-45. 15N-NMR chemical shift studies instead showed that Glu-67 functions to stabilize the unfavored N(delta1)H tautomer of His-45, thereby rendering the N(epsilon2) imidazole unprotonated and well positioned for accepting the ATP phosphoryl group.