Biochemical characterization and mutational analysis of the mononuclear non-haem Fe2+ site in Dke1, a cupin-type dioxygenase from Acinetobacter johnsonii

beta-diketone-cleaving enzyme Dke1 is a homotetrameric Fe2+-dependent dioxygenase from Acinetobacter johnsonii. The Dke1protomer adopts a single-domain beta-barrel fold characteristic of the cupin superfamily of proteins and features a mononuclear non-haem Fe2+ centre where a triad of histidine residues, His-62, His-64 and His-104, co-ordinate the catalytic metal. To provide structure-function relationships for the peculiar metal site of Dke1 in relation to the more widespread 2-His-1-Glu/Asp binding site for non-haem Fe2+,we replaced each histidine residue individually with glutamate and asparagine and compared binding of Fe2+ and four non-native catalytically inactive metals with purified apo-forms of wild-type and mutant enzymes. Results from anaerobic equilibrium microdialysis (Fe2+) and fluorescence titration (Fe2+, Cu2+, Ni2+, Mn2+ and Zn2+) experiments revealed the presence of two broadly specific metal-binding sites in native Dke1 that bind Fe2+ with a dissociation constant (Kd) of 5 microM (site I) and approximately 0.3 mM (site II). Each mutation, except for the substitution of asparagine for His-104, disrupted binding of Fe2+, but not that of the other bivalent metal ions, at site I,while leaving metal binding at site II largely unaffected. Dke1 mutants harbouring glutamate substitutions were completely inactive and not functionally complemented by external Fe2+.The Fe2+ catalytic centre activity (kcat) of mutants with asparagine substitution of His-62 and His-104 was decreased 140- and 220-fold respectively, compared with the kcat value of 8.5 s(-1) for the wild-type enzyme in the reaction with pentane-2,4-dione.The H64N mutant was not catalytically competent, except in the presence of external Fe2+ (1 mM) which elicited about 1/1000 of wild-type activity. Therefore co-ordination of Fe2+ by Dke1 requires an uncharged metallocentre, and three histidine ligands are needed for the assembly of a fully functional catalytic site. Oxidative inactivation of Dke1 was shown to involve conversion of enzyme-bound Fe2+ into Fe3+, which is then released from the metal centre.     

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 the iron-binding histidine residues in soybean lipoxygenase L-1.

Lipoxygenases constitute a class of non-heme, non-sulfur iron dioxygenases acting upon lipids possessing a 1,4-cis-cis-pentadiene moiety. The iron is known to be essential for activity. A motif of six histidine residues has been found in all of the thirteen lipoxygenases, from both plant and animal sources, whose sequences have been reported. We had previously obtained mutant proteins in which each of the 6 conserved histidines of soybean lipoxygenase L-1 had been replaced and found that the mutants H499Q, H504Q (or H504S) and H690Q had no detectable enzymatic activity. We have now found that these inactive proteins contain no Fe, although they have the same electrophoretic mobility as wild-type L-1 under both denaturing and non-denaturing conditions and react with anti-L-1 antibodies.     

Characterization of the outer membrane receptor ShuA from the heme uptake system of Shigella dysenteriae. Substrate specificity and identification of the heme protein ligands

Shigella dysenteriae, like many bacterial pathogens, has evolved outer membrane receptor-mediated pathways for the uptake and utilization of heme as an iron source. As a first step toward understanding the mechanism of heme uptake we have undertaken a site-directed mutagenesis, spectroscopic, and kinetic analysis of the outer membrane receptor ShuA of S. dysenteriae. Purification of the outer membrane receptor gave a single band of molecular mass 73 kDa on SDS-PAGE. Initial spectroscopic analysis of the protein in either detergent micelles or lipid bicelles revealed residual heme bound to the receptor, with a Soret maximum at 413 nm. Titration of the protein with exogenous heme gave a Soret peak at 437 nm in detergent micelles, and 402 nm in lipid bicelles. However, transfer of heme from hemoglobin yields a Soret maximum at 413 nm identical to that of the isolated protein. Further spectroscopic and kinetic analysis revealed that hemoglobin in the oxidized state is the most likely physiological substrate for ShuA. In addition, mutation of the conserved histidines, H86A or H420A, resulted in a loss of the ability of the receptor to efficiently extract heme from hemoglobin. In contrast the double mutant H86A/H420A was unable to extract heme from hemoglobin. These findings taken together confirm that both His-86 and His-420 are essential for substrate recognition, heme coordination, and transfer. Furthermore, the full-length TonB was shown to form a 1:1 complex with either apo-ShuA H86A/H420A or the wild-type ShuA. These observations provide a basis for future studies on the coordination and transport of heme by the TonB-dependent outer membrane receptors.     

Identification of heme and copper ligands in subunit I of the cytochrome bo complex in Escherichia coli.

The cytochrome bo complex is a terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli (Kita, K., Konishi, K., and Anraku, Y. (1984) J. Biol. Chem. 259, 3368-3374) and functions as a proton pump. It belongs to the heme-copper oxidase superfamily with the aa3-type cytochrome c oxidases in mitochondria and aerobic bacteria. In order to identify ligands of hemes and copper, we have substituted eight conserved histidines in subunit I by alanine and, in addition, His-106, -284, and -421 by glutamine and methionine. Western immunoblotting analysis showed that all the mutations do not affect the expression level of subunit I in the cytoplasmic membrane, indicating that these histidines are not crucial for its stability. A single copy expression vector carrying a single mutation at the invariant histidines, His-106, His-284, His-333, His-334, His-419, and His-421, of subunit I was unable to support the aerobic growth of a strain in which the chromosomal terminal oxidase genes (the cyo and cyd operons) have been deleted. The same mutations caused a complete loss of ubiquinol oxidase activity of the partially purified enzymes. Spectroscopic analysis of mutant oxidases in the cytoplasmic membrane revealed that substitutions of His-106 and -421 specifically eliminated a 563.5 nm peak of the low spin heme and that replacements of His-106, -284, and -419 reduced the extent of the CO-binding high spin heme. These spectroscopic properties of mutant oxidases were further confirmed with partially purified preparations. Atomic absorption analysis showed that substitutions of His-106, -333, -334, and -419 eliminated CuB almost completely. Based on these findings, we conclude that His-106 and -421 function as the axial ligands of the low spin heme and His-284 is a possible ligand of the high spin heme. His-333, -334, and -419 residues are attributed to the ligands of CuB. We present a helical wheel model of the redox center in subunit I, which consists of the membrane-spanning regions II, VI, VII, and X, and discuss the implications of the model.     

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.     

Use of Heme-Protein Complexes by the Yersinia enterocolitica HemR Receptor: Histidine Residues Are Essential for Receptor Function

The abilities of two bacterial active heme transporters, HmbR of Neisseria meningitidis and HemR of Yersinia enterocolitica, to use different heme sources were compared. While HmbR-expressing cells used only hemoglobin (Hb) and heme, HemR-expressing bacteria were able to grow on Hb, heme, myoglobin, hemopexin, catalase, human and bovine serum albumin-heme, and haptoglobin-hemoglobin complexes as sources of iron. Expression of functional HemR allowed Escherichia coli cells to respond to heme-containing peptides, microperoxidases MP-8, MP-9, and MP-11, suggesting the ability of HemR to transport heme covalently linked to other molecules. Comparison of HemR with other heme receptors identified several highly conserved histidine residues as well as two conserved amino acid motifs, the FRAP and NPNL boxes. A site-directed mutagenesis approach was used to investigate the roles of His128, His192, His352, and His461 residues in HemR function. The HemR receptor with histidine changed to lysine at position 128 (HemR(H128K)), HemR(H461L), HemR(H461A), and HemR(H128A,H461A) mutant receptors were unable to use Hb, human serum albumin-heme, and myoglobin as sources of porphyrin and iron. Utilization of free heme was also severely affected, with some residual heme uptake in cells expressing HemR(H128K), HemR(H461A), and HemR(H461L). Conversely, the HemR(H192T), HemR(H352A), HemR(H352K), and HemR(H192T,H352K) mutant receptors were fully functional. All mutant HemR proteins were expressed in the outer membrane at levels similar to that of the wild-type HemR receptor. Nonfunctional HemRs were able to bind heme- and Hb-agarose. A hypothetical model of the HemR function in which two conserved histidine residues, His128 and His461, participate in the transport of heme through the receptor pore is postulated.     

Effects of heme ligand mutations including a pathogenic variant, H65R, on the properties of human cystathionine beta-synthase

Human cystathionine beta-synthase is a hemeprotein that catalyzes a pyridoxal phosphate (PLP)-dependent condensation of serine and homocysteine into cystathionine. Biophysical characterization of this enzyme has led to the assignment of the heme ligands as histidine and cysteinate, respectively, which has recently been confirmed by crystal structure determination of the catalytic core of the protein. Using site-directed mutagenesis, we confirm that C52 and H65 represent the thiolate and histidine ligands to the heme. Conversion of C52 to alanine or serine results in spectral properties of the resulting hemeprotein that are consistent with the loss of a thiolate ligand. Thus, the Soret peak blue-shifts from 428 to 415 and 417 nm in the ferric forms of the C52S and C52A mutants, respectively, and from 450 to 423 nm in the ferrous states of both mutants. Addition of CO to the dithionite-reduced ferrous C52 mutants results in spectra with Soret peaks at 420 nm. EPR spectroscopy of the ferric C52 variants reveals the predominance of a high-spin species. The H65R mutant, a variant described in a homocystinuric patient, has Soret peaks at 424, 421, and 420 nm in the ferric, ferrous, and ferrous CO states, respectively. EPR spectroscopy reveals predominance of the low-spin species. Both C52A and C52S mutations lead to protein with substoichiometric heme (19% with respect to wild type); however, the PLP content is comparable to that of wild-type enzyme. The heme and PLP contents of the H65R mutant are 40% and 75% that of wild-type enzyme. These results indicate that heme saturation does not dictate PLP saturation in these mutant enzymes. Both H65 and C52 variants display low catalytic activity, revealing that changes in the heme binding domain modulate activity, consistent with a regulatory role for this cofactor.     

Identification of essential residues in 2',3'-cyclic nucleotide 3'-phosphodiesterase. Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K(m) values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis.     

Roles of the heme proximal side residues tryptophan409 and tryptophan421 of neuronal nitric oxide synthase in the electron transfer reaction

Nitric oxide synthase (NOS) has an oxygenase domain with a thiol-coordinated heme active side similar to cytochrome P450. In contrast to cytochrome P450, however, conserved aromatic amino acids are situated in the heme proximal side of NOS. For example, in endothelial NOS (eNOS), the indole-ring nitrogen of Trp180 hydrogen-binds to the thiol of Cys186, the internal axial ligand to the heme. And, the aromatic side chain of Trp192 forms a bridge between this residue and the protein. Trp180 and Trp192 of eNOS correspond to Trp409 and Trp421 of neuronal NOS (nNOS), respectively. In order to understand the roles of the aromatic amino acids in catalysis, we generated Trp409His, Trp409Leu, Trp421His and Trp421Leu mutants of nNOS and determined their catalytic parameters. The Trp409Leu mutant was very poorly expressed in E. coli and was easily denatured during purification procedures. The NO formation activities of the Trp409His and Trp421Leu mutants were 11 and 25 micromol/min per micromol heme, respectively, and are lower than that (44 micromol/min per micromol heme) of the wild type. The activity (46 micromol/min per micromol heme) of the Trp421His mutant was comparable to that of the wild-type enzyme. However, NADPH oxidation rates of Trp421His (230 micromol/min per micromol heme) and Trp421Leu (104 micromol/min per microol heme) in the presence of L-Arg were much larger than those observed for the wild type (65 micromol/min per micromol heme) and the Trp409His mutant (43 micromol/min per micromol heme). The cytochrome c reduction rate of the Trp421His mutant was 6-fold larger than that of the wild type. The heme reduction rate with NADPH for the Trp421His mutant (0.09 min(-1)) was much lower than that (1.0 min(-1)) of the wild type. Taken together, it appears that Trp421 may be involved in inter-domain/inter-subunit electron transfer reactions.     

Differential role of extracellular histidines in copper, zinc, magnesium and proton modulation of the P2X7 purinergic receptor

The P2X7 receptor is a non-selective cationic channel activated by extracellular ATP, belonging to the P2X receptor family. To assess the role of extracellular histidines on the allosteric modulation of the rat P2X7 receptor by divalent metals (copper, zinc and magnesium) and protons, these amino acid residues were singly substituted for corresponding alanines. Wild-type and mutated receptors were injected to Xenopus laevis oocytes; metal-related effects were evaluated by the two-electrode voltage-clamp technique. Copper inhibited the ATP-gated currents with a median inhibitory concentration of 4.4 +/- 1.0 micromol/L. The inhibition was non-competitive and time-dependent; copper was 60-fold more potent than zinc. The mutant H267A, resulted in a copper resistant receptor; mutants H201A and H130A were less sensitive to copper inhibition (p < 0.05). The rest of the mutants examined, H62A, H85A, and H219A, conserved the copper-induced inhibition. Only mutants H267A and H219A were less sensitive to the modulator action of zinc. Moreover, the magnesium-induced inhibition was abolished exclusively on the H130A and H201A mutants, suggesting that this metal may act at a novel cationic modulator site. Media acidification inhibited the ATP-gated current 87 +/- 3%; out of the six mutants examined, only H130A was significantly less sensitive to the change in pH, suggesting that His-130 could be involved as a pH sensor. In conclusion, while His-267 is critically involved in the copper or zinc allosteric modulation, the magnesium inhibitory effects is related to His-130 and His-201, His-130 is involved in proton sensing, highlighting the role of defined extracellular histidines in rat P2X7 receptor allosteric modulation.     

Histidine 140 plays a key role in the inhibitory modulation of the P2X4 nucleotide receptor by copper but not zinc

To elucidate the role of extracellular histidines in the modulation of the rat P2X4 receptor by trace metals, we generated single, double, and triple histidine mutants for residues 140, 241, and 286, replacing them with alanines. cDNAs for the wild-type and receptor mutants were expressed in Xenopus laevis oocytes and in human embryonic kidney 293 cells and examined by the two electrode and patch clamp techniques, respectively. Whereas copper inhibited concentration-dependently the ATP-gated currents in the wild-type and in the single or double H241A and H286A receptor mutants, all receptors containing H140A were insensitive to copper in both cell systems. The characteristic bell-shaped concentration-response curve of zinc observed in the wild-type receptor became sigmoid in both oocytes and human embryonic kidney cells expressing the H140A mutant; in these mutants, the zinc potentiation was 2.5-4-fold larger than in the wild-type. Results with the H140T and H140R mutants further support the importance of a histidine residue at this position. We conclude that His-140 is critical for the action of copper, indicating that this histidine residue, but not His-241 or His-286, forms part of the inhibitory allosteric metal-binding site of the P2X4 receptor, which is distinct from the putative zinc facilitator binding site.     

Regulation of the Escherichia coli antiterminator protein BglG by phosphorylation at multiple sites and evidence for transfer of phosphoryl groups between monomers

Activity of antiterminator protein BglG regulating the beta-glucoside operon in Escherichia coli is controlled by the phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) in a dual manner. It requires HPr phosphorylation to be active, whereas phosphorylation by the beta-glucoside-specific transport protein EIIBgl inhibits its activity. BglG and its relatives carry two PTS regulation domains (PRD1 and PRD2), each containing two conserved histidines. For BglG, histidine 208 in PRD2 was reported to be the negative phosphorylation site. In contrast, other antiterminators of this family are negatively regulated by phosphorylation of the first histidine in PRD1, and presumably activated by phosphorylation of the histidines in PRD2. In this work, a screen for mutant BglG proteins that escape repression by EIIBgl yielded exchanges of nine residues within PRD1, including conserved histidines His-101 and His-160, and C-terminally truncated proteins. Genetic and phosphorylation analyses indicate that His-101 in PRD1 is phosphorylated by EIIBgl and that His-160 contributes to negative regulation. His-208 in PRD2 is essential for BglG activity, suggesting that it is phosphorylated by HPr. Surprisingly, phosphorylation by HPr is not fully abolished by exchanges of His-208. However, phosphorylation by HPr is inhibited by exchanges in PRD1 and the phosphorylation of these mutants is restored in the presence of wild-type BglG. These results suggest that the activating phosphoryl group is transiently donated from HPr to PRD1 and subsequently transferred to His-208 of a second BglG monomer. The active His-208-phosphorylated BglG dimer can subsequently be inhibited in its activity by EIIBgl-catalyzed phosphorylation at His-101.     

Histidine residues regulate the transition of photoexcited rhodopsin to its active conformation, metarhodopsin II.

The biologically active photoproduct of rhodopsin, metarhodopsin II (M II), exists in a pH-sensitive equilibrium with its precursor, metarhodopsin I (M I). Increasing acidity favors M II, with the midpoint of the pH titration curve at pH 6.4. To test the long-standing proposal that histidine protonation regulates this conformational transition, we characterized mutant rhodopsins in which each of the 6 histidines was replaced by phenylalanine or cysteine. Only mutants substituted at the 3 conserved histidines showed abnormal M I-M II equilibria. Those in which His-211 was replaced by phenylalanine or cysteine formed little or no M II at either extreme of pH, whereas mutants substituted at His-65 or at His-152 showed enhanced sensitivity to protons. The simplest interpretation of these results is that His-211 is the site where protonation strongly stabilizes the M II conformation and that His-65 and His-152 are sites where protonation modestly destabilizes the M II conformation.     

Histidine-607 and histidine-643 provide important interactions for metal support of catalysis in phosphodiesterase-5

Class I cyclic nucleotide phosphodiesterases (PDEs) share a catalytic domain containing 18 invariant residues. In cGMP-binding cGMP-specific PDE (PDE5), we showed previously that point mutation of nine of these profoundly decreases k(cat) when the assay is conducted in the presence of Mg(2+); seven of these are in the prototypical metal-binding motifs A and B (HX(3)HX(n)()E) that we identified earlier. Tandem arrangement of two of these metal-binding motifs in PDEs is novel, and whether residues within these motifs are involved in metal support of catalytic activity is a fundamental question in this field. This report shows that mutation of either His-607 (A motif) or His-643 (B motif) to alanine profoundly diminishes support of PDE catalysis by Mn(2+) or Mg(2+), but mutation of His-647 in B motif or of Glu in either motif does not. H607A and H643A mutants have much greater maximum catalytic rates supported by Mn(2+) than that by Mg(2+); catalytic activity of H603A mutant is supported weakly by either. In H607A and H643A, K(a)s for Mn(2+) and Mg(2+) are increased, but the effect of Mn(2+) is 2-fold greater than that of Mg(2+) in each. Mutation of any of the other conserved residues (Asn-604, Asp-644, His-675, Asp-714, and Asp-754) causes unremarkable changes in Mn(2+) or Mg(2+) support of catalysis. This study identifies specific residues in PDE5 that contribute to interactions with catalytically relevant metals. The combined data suggest that despite a high degree of sequence similarity between each HX(3)HX(n)()E motif in PDEs and certain metallo-endopeptidases, PDEs employ a distinct complement of residues for interacting with metals involved in catalysis.     

Conserved histidine residues in soybean lipoxygenase: functional consequences of their replacement.

Sequences of 13 lipoxygenases from various plant and mammalian species, thus far reported, display a motif of 38 amino acid residues which includes 5 conserved histidines and a 6th histidine about 160 residues downstream. These residues occur at positions 494, 499, 504, 522, 531, and 690 in soybean lipoxygenase isozyme L-1. Since the participation of iron in the lipoxygenase reaction has been established and existing evidence based on M?ssbauer and EXAFS spectroscopy suggests that histidines may be involved in iron binding, the effect of the above residues has been examined in soybean lipoxygenase L-1. Six singly mutated lipoxygenases have been produced in which each of the His residues has been replaced with glutamine. Two additional mutants have been constructed wherein the codons for His-494 and His-504 have been replaced by serine codons. All of the mutant lipoxygenases, which were obtained by expression in Escherichia coli, have mobilities identical to that of the wild-type enzyme on denaturing gel electrophoresis and respond to lipoxygenase antibodies. The mutated proteins H499Q, H504Q, H504S, and H690Q are virtually inactive, while H522Q has about 1% of the wild-type activity. H494Q, H494S, and H531Q are about 37%, 8%, and 20% as active as the wild type, respectively. His-517 is conserved in the several lipoxygenase isozymes but not in the animal isozymes. The mutant H517Q has about 33% of the wild-type activity. The inactive mutants, H499Q, H504Q, H504S, and H690Q, become insoluble when heated for 3 min at 65 degrees C, as does H522Q. The other mutants and the wild-type are stable under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemical modification and site-directed mutagenesis of conserved HXXH and PP-loop motif arginines and histidines in the murine bifunctional ATP sulfurylase/adenosine 5'-phosphosulfate kinase.

The sulfurylase domain of the mouse bifunctional enzyme ATP sulfurylase/adenosine 5'-phosphosulfate (APS) kinase contains HXXH and PP-loop motifs. To elucidate the functional importance of these motifs and of conserved arginines and histidines, chemical modification and site-directed mutagenesis studies were performed. Chemical modification of arginines and histidines with phenylglyoxal and diethyl pyrocarbonate, respectively, renders the enzyme inactive in sulfurylase, kinase, and overall assays. Data base searches and sequence comparison of bifunctional ATP sulfurylase/APS kinase and monofunctional ATP sulfurylases shows a limited number of highly conserved arginines and histidines within the sulfurylase domain. Of these conserved residues, His-425, His-428, and Arg-421 are present within or near the HXXH motif whereas His-506, Arg-510, and Arg-522 residues are present in and around the PP-loop. The functional role of these conserved residues was further studied by site-directed mutagenesis. In the HXXH motif, none of the alanine mutants (H425A, H428A, and R421A) had sulfurylase or overall activity, whereas they all exhibited normal kinase activity. A slight improvement in reverse sulfurylase activity (<10% residual activity) and complete restoration of forward sulfurylase was observed with R421K. Mutants designed to probe the PP-loop requirements included H506A, R510A, R522A, R522K, and D523A. Of these, R510A exhibited normal sulfurylase and kinase activity, R522A and R522K showed no sulfurylase activity, and H506A had normal sulfurylase activity but produced an effect on kinase activity (<10% residual activity). The single aspartate, D523A, which is part of the highly conserved GRD sequence of the PP-loop, affected both sulfurylase and kinase activity. This mutational analysis indicates that the HXXH motif plays a role only in the sulfurylase activity, whereas the PP-loop is involved in both sulfurylase and kinase activities. Residues specific for sulfurylase activity have also been distinguished from those involved in kinase activity.     

The roles of Glu-327 and His-446 in the bisphosphatase reaction of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase probed by NMR spectroscopic and mutational analyses of the enzyme in the transient phosphohistidine intermediate complex

The bisphosphatase domain derived from the rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase was studied by 1H-13C HMQC NMR spectroscopy of the histidine C2' and H2' nuclei. The bacterially expressed protein was specifically labeled with 13C at the ring C2' position of the histidines. Each of the seven histidine residues gave rise to a single cross-peak in the HMQC spectra, and these were assigned by use of a series of histidine-to-alanine point mutants. His-304, His-344, and His-469 exhibit 13C and 1H resonances that titrated with pH, while the remaining histidine-associated resonances did not. The 13C and 1H chemical shifts indicate that at neutral pH, His-304 and His-446 are deprotonated, while His-469 is protonated. The pKa of His-344 was determined to be 7.04. The 13C chemical shifts suggest that the deprotonated His-258 exists as the N1' tautomer, while His-392 and His-419 are protonated in the resting, wild-type enzyme. Mutation of the remaining member of the catalytic triad, Glu-327, to alanine in the resting enzyme caused an upfield shift of 1.58 and 1.30 ppm in the 1H and 13C dimensions, respectively, and significant narrowing of the His-258 cross-peak. Mutation of His-446 to alanine produced perturbations of the His-258 cross-peak that were similar to those detected in the E327A mutant. The His-392 resonances were also shifted by the E327A and H446A mutations. These observations strongly suggest that residues His-258, Glu-327, His-392, and His-446 exist within a network of interacting residues that encompasses the catalytic site of the bisphosphatase and includes specific contacts with the C-terminal regulatory region of the enzyme. The specifically 13C-labeled bisphosphatase was monitored during turnover by HMQC spectra acquired from the transient N3' phosphohistidine intermediate complex in the wild-type enzyme, the E327A mutant, and the H446A mutant. These complexes were formed during reaction with the physiological substrate fructose-2, 6-bisphosphate. Upon formation of the phosphohistidine at His-258, the 13C and 1H resonances of this residue were shifted downfield by 1.7 and 0.31 ppm, respectively, in the wild-type enzyme. The upfield shifts of the His-258 resonances in the E327A and H446A mutant resting enzymes were reversed when the phosphohistidine was formed, generating spectra very similar to that of the wild-type enzyme in the intermediate complex. In contrast, the binding of fructose-6-phosphate, the reaction product, to the resting enzyme did not promote significant changes in the histidine-associated resonances in either the wild-type or the mutant enzymes. The interpretation of these data within the context of the X-ray crystal structures of the enzyme is used to define the role of Glu-327 in the catalytic mechanism of the bisphosphatase and to identify His-446 as a putative link in the chain of molecular events that results in activation of the bisphosphatase site by cAMP-dependent phosphorylation of the hepatic bifunctional enzyme.     

The role of histidines in the acetate kinase from Methanosarcina thermophila

The role of histidine in the catalytic mechanism of acetate kinase from Methanosarcina thermophila was investigated by diethylpyrocarbonate inactivation and site-directed mutagenesis. Inactivation was accompanied by an increase in absorbance at 240 nm with no change in absorbance at 280 nm, and treatment of the inactivated enzyme with hydroxylamine restored 95% activity, results that indicated diethylpyrocarbonate inactivates the enzyme by the specific modification of histidine. The substrates ATP, ADP, acetate, and acetyl phosphate protected against inactivation suggesting at least one active site where histidine is modified. Correlation of residual activity with the number of histidines modified, as determined by absorbance at 240 nm, indicated that a maximum of three histidines are modified per subunit, two of which are essential for full inactivation. Comparison of the M. thermophila acetate kinase sequence with 56 putative acetate kinase sequences revealed eight highly conserved histidines, three of which (His-123, His-180, and His-208) are perfectly conserved. Diethylpyrocarbonate inactivation of the eight histidine --> alanine variants indicated that His-180 and His-123 are in the active site and that the modification of both is necessary for full inactivation. Kinetic analyses of the eight variants showed that no other histidines are important for activity. Analysis of additional His-180 variants indicated that phosphorylation of His-180 is not essential for catalysis. Possible functions of His-180 are discussed.     

Structural-based mutational analysis of D-aminoacylase from Alcaligenes faecalis DA1

D-Aminoacylase is an attractive candidate for commercial production of D-amino acids through its catalysis in the zinc-assistant hydrolysis of N-acyl-D-amino acids. We report here the cloning, expression, and structural-based mutation of the D-aminoacylase from Alcaligenes faecalis DA1. A 1,007-bp PCR product amplified with degenerate primers, was used to isolate a 4-kb genomic fragment, encoding a 484-residue D-aminoacylase. The enzyme amino-terminal segment shared significant homology within a variety of enzymes including urease. The structural fold was predicted by 3D-PSSM to be similar to urease and dihydroorotase, which have grouped into a novel alpha/beta-barrel amidohydrolase superfamily with a virtually indistinguishable binuclear metal centers containing six ligands, four histidines, one aspartate, and one carboxylated lysine. Three histidines, His-67, His-69, and His-250, putative metal ligands in D-aminoacylase, have been mutated previously, the remaining histidine (His-220) and aspartate (Asp-366) Asp-65, and four cysteines were then characterized. Substitution of Asp-65, Cys-96, His-220, and Asp-366 with alanine abolished the enzyme activity. The H220A mutant bound approximately half the normal complement of zinc ion as did H250N. However, the C96A mutant showed little zinc-binding ability, revealing that Cys-96 may replace the carboxylated lysine to serve as a bridging ligand. According to the urease structure, the conserved amino-terminal segment including Asp-65 may be responsible for structural stabilization.