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.     

X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11?Å resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.     

The active site topology of Aspergillus niger endopolygalacturonase II as studied by site-directed mutagenesis

Strictly conserved charged residues among polygalacturonases (Asp-180, Asp-201, Asp-202, His-223, Arg-256, and Lys-258) were subjected to site-directed mutagenesis in Aspergillus niger endopolygalacturonase II. Specific activity, product progression, and kinetic parameters (K(m) and V(max)) were determined on polygalacturonic acid for the purified mutated enzymes, and bond cleavage frequencies on oligogalacturonates were calculated. Depending on their specific activity, the mutated endopolygalacturonases II were grouped into three classes. The mutant enzymes displayed bond cleavage frequencies on penta- and/or hexagalacturonate different from the wild type endopolygalacturonase II. Based on the biochemical characterization of endopolygalacturonase II mutants together with the three-dimensional structure of the wild type enzyme, we suggest that the mutated residues are involved in either primarily substrate binding (Arg-256 and Lys-258) or maintaining the proper ionization state of a catalytic residue (His-223). The individual roles of Asp-180, Asp-201, and Asp-202 in catalysis are discussed. The active site topology is different from the one commonly found in inverting glycosyl hydrolases.     

Identification of aspartic acid and histidine residues mediating the reaction mechanism and the substrate specificity of the human UDP-glucuronosyltransferases 1A

The human UDP-glucuronosyltransferase UGT1A6 is the primary phenol-metabolizing UDP-glucuronosyltransferase isoform. It catalyzes the nucleophilic attack of phenolic xenobiotics on UDP-glucuronic acid, leading to the formation of water-soluble glucuronides. The catalytic mechanism proposed for this reaction is an acid-base mechanism that involves an aspartic/glutamic acid and/or histidine residue. Here, we investigated the role of 14 highly conserved aspartic/glutamic acid residues over the entire sequence of human UGT1A6 by site-directed mutagenesis. We showed that except for aspartic residues Asp-150 and Asp-488, the substitution of carboxylic residues by alanine led to active mutants but with decreased enzyme activity and lower affinity for acceptor and/or donor substrate. Further analysis including mutation of the corresponding residue in other UGT1A isoforms suggests that Asp-150 plays a major catalytic role. In this report we also identified a single active site residue important for glucuronidation of phenols and carboxylic acid substrates by UGT1A enzyme family. Replacing Pro-40 of UGT1A4 by histidine expanded the glucuronidation activity of the enzyme to phenolic and carboxylic compounds, therefore, leading to UGT1A3-type isoform in terms of substrate specificity. Conversely, when His-40 residue of UGT1A3 was replaced with proline, the substrate specificity shifted toward that of UGT1A4 with loss of glucuronidation of phenolic substrates. Furthermore, mutation of His-39 residue of UGT1A1 (His-40 in UGT1A4) to proline led to loss of glucuronidation of phenols but not of estrogens. This study provides a step forward to better understand the glucuronidation mechanism and substrate recognition, which is invaluable for a better prediction of drug metabolism and toxicity in human.     

Functional mapping of charged residues of the 82-116 sequence in factor Xa: evidence that lysine 96 is a factor Va independent recognition site for prothrombin in the prothrombinase complex

It has been hypothesized that two antiparallel structures comprised of residues 82-91 and 102-116 in factor Xa (fXa) may harbor a factor Va- (fVa-) dependent prothrombin recognition site in the prothrombinase complex. There are 11 charged residues in the 82-116 loop of human fXa (Glu-84, Glu-86, Lys-90, Arg-93, Lys-96, Glu-97, Asp-100, Asp-102, Arg-107, Lys-109, and Arg-115). With the exception of Glu-84, which did not express, and Asp-102, which is a catalytic residue, we expressed the Ala substitution mutants of all other residues and evaluated their proteolytic and amidolytic activities in both the absence and presence of fVa. K96A and K109A activated prothrombin with 5-10-fold impaired catalytic efficiency in the absence of fVa. All mutants, however, exhibited normal activity toward the substrate in the presence of fVa. K109A also exhibited impaired amidolytic activity and affinity for Na(+); however, both fVa and higher Na(+) restored the catalytic defect caused by the mutation. Analysis of the X-ray crystal structure of fXa indicated that Glu-84 may interact by a salt bridge with Lys-109, explaining the lack of expression of E84A and the lower activity of K109A in the absence of fVa. These results suggest that none of the residues under study is a fVa-dependent recognition site for prothrombin in the prothrombinase complex; however, Lys-96 is a recognition site for the substrate independent of the cofactor. Moreover, the 82-116 loop is energetically linked to fVa and Na(+) binding sites of the protease.     

Substitutions in the active site of chloramphenicol acetyltransferase: role of a conserved aspartate

The role of conserved Asp-199 in chloramphenicol acetyltransferase (CAT) has been investigated by site-directed mutagenesis. Substitution of Asp-199 by alanine results in a thermolabile mutant enzyme (Ala-199 CAT) with reduced kcat(13-fold) but similar Km values to wild type CAT. Replacement by asparagine gives rise to a thermostable mutant enzyme (Asn-199 CAT) with much reduced kcat(1500-fold). Furthermore, Asn-199 CAT shows anomalous inactivation kinetics with the affinity reagent 3-(bromo-acetyl)chloramphenicol. These results favor a structural role for Asp-199 rather than a catalytic one, in keeping with crystallographic evidence for involvement of Asp-199 in a tight salt bridge with Arg-18. Replacement of Arg-18 by valine results in a mutant enzyme (Val-18 CAT) with similar properties to Ala-199 CAT. The catalytic imidazole of His-19 appears to be conformationally constrained by hydrogen bonding between N1-H and the carbonyl oxygen of the same residue and by ring stacking with Tyr-25.     

Identification of a novel phosphatase sequence motif.

We have identified a novel, conserved phosphatase sequence motif, KXXXXXXRP-(X12-54)-PSGH-(X31-54)-SRXXXXX HXXXD, that is shared among several lipid phosphatases, the mammalian glucose-6-phosphatases, and a collection of bacterial nonspecific acid phosphatases. This sequence was also found in the vanadium-containing chloroperoxidase of Curvularia inaequalis. Several lines of evidence support this phosphatase motif identification. Crystal structure data on chloroperoxidase revealed that all three domains are in close proximity and several of the conserved residues are involved in the binding of the cofactor, vanadate, a compound structurally similar to phosphate. Structure-function analysis of the human glucose-6-phosphatase has shown that two of the conserved residues (the first domain arginine and the central domain histidine) are essential for enzyme activity. This conserved sequence motif was used to identify nine additional putative phosphatases from sequence databases, one of which has been determined to be a lipid phosphatase in yeast.     

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.     

Kinetic properties and structural characteristics of an unusual two-domain arginine kinase of the clam Corbicula japonica

Arginine kinase (AK) from the clam Corbicula japonica is a unique enzyme in that it has an unusual two-domain structure with molecular mass of 80 kDa. It lacks two functionally important amino acid residues, Asp-62 and Arg-193, which are conserved in other 40 kDa AKs and are assumed to be key residues for stabilizing the substrate-bound structure. K m arg and Vmax values for the recombinant two-domain AK were determined. These values were close to those of usual 40 kDa AKs, although Corbicula AK lacks the functionally important Asp-62 and Arg-193. Domain 2 of Corbicula AK was separated from the two-domain enzyme and was expressed in Escherichia coli. Domain 2 still exhibited activity. However, kinetic parameters for domain 2 appeared to be slightly, but significantly, different from those of two-domain AK. Thus, it is likely that the formation of the contiguous dimer alters the kinetic properties of its constituent domains significantly. Comparison of K d arg and K m arg for two-domain AK and its domain 2 showed that the affinity of the enzyme for arginine is greater in the presence of substrate ATP than in its absence. Presumably this difference is correlated with the large structural differences in the enzyme in the presence or absence of substrate, namely open and closed structures. We expressed three mutants of Corbicula AK domain 2 (His-60 to Gly or Arg, Asp-197 to Gly), and determined their K m arg and Vmax values. The affinity for the substrate arginine in mutant enzymes was reduced considerably, accompanied by a decrease in Vmax. These results suggest that His-60 and Asp-197 affect the substrate binding system, and are consistent with the hypothesis that a hydrogen bond is formed between His-60 and Asp-197 in Corbicula AK as a substitute for the Asp-62 and Arg-193 bond in normal AKs.     

Mutational,structural, and kinetic studies of the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase

Several residues lining the ATP-binding site of Methanobacterium thermoautotrophicum nicotinamide mononucleotide adenylyltransferase (NMNATase) were mutated in an effort to better characterize their roles in substrate binding and catalysis. Residues selected were Arg-11 and Arg-136, both of which had previously been implicated as substrate binding residues, as well as His-16 and His-19, part of the HXGH active site motif and postulated to be of importance in catalysis. Kinetic studies revealed that both Arg-11 and Arg-136 contributed to the binding of the substrate, ATP. When these amino acids were replaced by lysines, the apparent Km values of the respective mutants for ATP decreased by factors of 1.3 and 2.9 and by factors of 1.9 and 8.8 when the same residues were changed to alanines. All four Arg mutants displayed unaltered Km values for NMN. The apparent kcat values of the R11K and R136K mutants were the same as those of WT NMNATase but the apparent kcat values of the alanine mutants had decreased. Crystal structures of the Arg mutants revealed NAD+ and SO42- molecules trapped at their active sites. The binding interactions of NAD+ were unchanged but the binding of SO42- was altered in these mutants compared with wild type. The alanine mutants at positions His-16 and His-19 retained approximately 6 and 1.3%, respectively, of WT NMNATase activity indicating that His-19 is a key catalytic group. Surprisingly, this H19A mutant displayed a novel and distinct mode of NAD+ binding when co-crystallized in the presence of NAD+ and SO42-.     

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.     

Mutational analysis of the function of the a-subunit of the F0F1-APPase of Escherichia coli

In a model proposed for the structure of the a-subunit of the Escherichia coli F0F1-ATPase (Howitt, S.M., Gibson, F. and Cox, G.B. (1988) Biochim. Biophys. Acta 936, 74-80), a cluster of charged residues, including one arginine and four aspartic acid residues, lie on the periplasmic side of the membrane. On the cytoplasmic side, three pairs of lysine residues and an arginine residue are present. Site-directed mutagenesis was used to investigate the roles of these residues. It was found that none was directly involved in the proton pore. However, the substitutions of Asp-124 or Asp-44 by asparagine or Arg-140 by glutamine had similar effects in that the membranes from such mutants from which the F1-ATPase was removed were proton-impermeable. A combination of the Asp-44 mutation with either the Asp-124 or Arg-140 mutations in the same strain resulted in complete loss of oxidative phosphorylation. It was tentatively concluded that Asp-124 and Arg-140 form a salt bridge, as did Asp-44 with an unknown residue, and these salt bridges were concerned with the maintenance of correct a-subunit structure. Further support for this conclusion was obtained when second site revertants of a Glu-219 to histidine mutant were found to have either histidine or leucine replacing Arg-140. Thus, the lack of the Asp-124/Arg-140 salt bridge might enable repositioning of the helices of the a-subunit such that His-219 becomes a functional component of the proton pore.     

Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase

Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups. The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains. The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3. Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful. The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold. First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent. The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction. The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis. Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active. The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions. The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data. On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors. The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor. The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate. Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.     

Alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is a new member of the amidohydrolase superfamily

The enzymatic activity of Pseudomonas fluorescens alpha-amino-beta-carboxymuconic-epsilon-semialdehyde decarboxylase (ACMSD) is critically dependent on a transition metal ion [Li, T., Walker, A. L., Iwaki, H., Hasegawa, Y., and Liu, A. (2005) J. Am. Chem. Soc. 127, 12282-12290]. Sequence analysis in this study further suggests that ACMSD belongs to the amidohydrolase superfamily, whose structurally characterized members comprise a catalytically essential metal cofactor. To identify ACMSD's metal ligands and assess their functions in catalysis, a site-directed mutagenesis analysis was conducted. Alteration of His-9, His-177, and Asp-294 resulted in a dramatic loss of enzyme activity, substantial reduction of the metal-binding ability, and an altered metallocenter electronic structure. Thus, these residues are confirmed to be the endogenous metal ligands. His-11 is implicated in metal binding because of the strictly conserved HxH motif with His-9. Mutations at the 228 site yielded nearly inactive enzyme variants H228A and H228E. The two His-228 mutant proteins, however, exhibited full metal-binding ability and a metal center similar to that of the wild-type enzyme as shown by EPR spectroscopy. Kinetic analysis on the mutants indicates that His-228 is a critical catalytic residue along with the metal cofactor. Since the identified metal ligands and His-228 are present in all known ACMSD sequences, it is likely that ACMSD proteins from other organisms contain the same cofactor and share similar catalytic mechanisms. ACMSD is therefore the first characterized member in the amidohydrolase superfamily that represents a C-C breaking activity.     

Cofactor and substrate binding to vanadium chloroperoxidase determined by UV-VIS spectroscopy and evidence for high affinity for pervanadate

The vanadate cofactor in vanadium chloroperoxidase has been studied using UV-VIS absorption spectroscopy. A band is present in the near-UV that is red-shifted as compared to free vanadate and shifts in both position and intensity upon change in pH. Mutation of vanadate binding residues has a clear effect on the spectrum. Substrate-induced spectral effects allow direct measurement of separate kinetics steps for the first time for vanadium haloperoxidases. A peroxo intermediate is formed upon addition of H(2)O(2), which causes a decrease in the absorption spectrum at 315 nm, as well as an increase at 384 nm. This peroxo form is very stable at pH 8.3, whereas it is less stable at pH 5.0, which is the optimal pH for activity. Upon addition of halides to the peroxo form, the native spectrum is re-formed as a result of halide oxidation. Stopped-flow experiments show that H(2)O(2) binding and Cl(-) oxidation occur on the millisecond to second time scale. These data suggest that the oxidation of Cl(-) to HOCl occurs in at least two steps. In the presence of H(2)O(2), the affinity for the vanadate cofactor was found to be much higher than previously reported for vanadate in the absence of H(2)O(2). This is attributed to the uptake of pervanadate by the apo-enzyme. Human glucose-6-phosphatase, which is evolutionarily related to vanadium chloroperoxidase, is also likely to have a higher affinity for pervanadate than vanadate. This could explain the enhanced insulin mimetic effect of pervanadate as compared to vanadate.     

Biochemical characterization of the chondroitinase B active site

Chondroitinase B from Flavobacterium heparinum is the only known lyase that cleaves the glycosaminoglycan, dermatan sulfate (DS), as its sole substrate. A recent co-crystal structure of chondroitinase B with a disaccharide product of DS depolymerization has provided some insight into the location of the active site and suggested potential roles of some active site residues in substrate binding and catalysis. However, this co-crystal structure was not representative of the actual enzyme-substrate complex, because the disaccharide product did not have the right length or the chemical structure of the minimal substrate (tetrasaccharide) involved in catalysis. Therefore, only a limited picture of the functional role of active site residues in DS depolymerization was presented in previous structural studies. In this study, by docking a DS tetrasaccharide into the proposed active site of the enzyme, we have identified novel roles of specific active site amino acids in the catalytic function of chondroitinase B. Our conformational analysis also revealed a unique, symmetrical arrangement of active site amino acids that may impinge on the catalytic mechanism of action of chondroitinase B. The catalytic residues Lys-250, Arg-271, His-272, and Glu-333 along with the substrate binding residues Arg-363 and Arg-364 were mutated using site-directed mutagenesis, and the kinetics and product profile of each mutant were compared with recombinant chondroitinase B. Mutating Lys-250 to alanine resulted in inactivation of the enzyme, potentially attributable to the role of the residue in stabilizing the carbanion intermediate formed during enzymatic catalysis. The His-272 and Glu-333 mutants showed diminished enzymatic activity that could be indicative of a possible role for one or both residues in the abstraction of the C-5 proton from the galactosamine. In addition, the Arg-364 mutant had an altered product profile after exhaustive digestion of DS, suggesting a role for this residue in defining the substrate specificity of chondroitinase B.     

Identification of critical, conserved vicinal aspartate residues in mammalian and bacterial ADP-ribosylarginine hydrolases.

NAD:arginine ADP-ribosyltransferases and ADP-ribosylarginine hydrolases catalyze opposing arms of a putative ADP-ribosylation cycle. ADP-ribosylarginine hydrolases from mammalian tissues and Rhodospirillum rubrum exhibit three regions of similarity in deduced amino acid sequence. We postulated that amino acids in these consensus regions could be critical for hydrolase function. To test this hypothesis, hydrolase, cloned from rat brain, was expressed as a glutathione S-transferase fusion protein in Escherichia coli and purified by glutathione-Sepharose affinity chromatography. Conserved amino acids in each of these regions were altered by site-directed mutagenesis. Replacement of Asp-60 or Asp-61 with Ala, Gln, or Asn, but not Glu, significantly reduced enzyme activity. The double Asp-60 --> Glu/Asp-61 --> Glu mutant was inactive, as were Asp-60 --> Gln/Asp-61 --> Gln or Asp-60 --> Asn/Asp-61 --> Asn. The catalytically inactive single and double mutants appeared to retain conformation, since they bound ADP-ribose, a substrate analogue and an inhibitor of enzyme activity, with affinity similar to that of the wild-type hydrolase and with the expected stoichiometry of one. Replacing His-65, Arg-139, Asp-285, which are also located in the conserved regions, with alanine did not change specific activity. These data clearly show that the conserved vicinal aspartates 60 and 61 in rat ADP-ribosylarginine hydrolase are critical for catalytic activity, but not for high affinity binding of the substrate analogue, ADP-ribose.     

Heterologous expression of the vanadium-containing chloroperoxidase from Curvularia inaequalis in Saccharomyces cerevisiae and site-directed mutagenesis of the active site residues His(496), Lys(353), Arg(360), and Arg(490).

The vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis is heterologously expressed to high levels in the yeast Saccharomyces cerevisiae. Characterization of the recombinant enzyme reveals that this behaves very similar to the native chloroperoxidase. Site-directed mutagenesis is performed on four highly conserved active site residues to examine their role in catalysis. When the vanadate-binding residue His(496) is changed into an alanine, the mutant enzyme loses the ability to bind vanadate covalently resulting in an inactive enzyme. The negative charges on the vanadate oxygens are compensated by hydrogen bonds with the residues Arg(360), Arg(490), and Lys(353). When these residues are changed into alanines the mutant enzymes lose the ability to effectively oxidize chloride but can still function as bromoperoxidases. A general mechanism for haloperoxidase catalysis is proposed that also correlates the kinetic properties of the mutants with the charge and the hydrogen-bonding network in the vanadate-binding site.     

Mutational analysis of Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP)

Ca(2+)/calmodulin-dependent protein kinase phosphatase (CaMKP) is a member of the serine/threonine protein phosphatases and shares 29% sequence identity with protein phosphatase 2Calpha (PP2Calpha) in its catalytic domain. To investigate the functional domains of CaMKP, mutational analysis was carried out using various recombinant CaMKPs expressed in Escherichia coli. Analysis of N-terminal deletion mutants showed that the N-terminal region of CaMKP played important roles in the formation of the catalytically active structure of the enzyme, and a critical role in polycation stimulation. A chimera mutant, a fusion of the N-terminal domain of CaMKP and the catalytic domain of PP2Calpha, exhibited similar substrate specificity to CaMKP but not to PP2Calpha, suggesting that the N-terminal region of CaMKP is crucial for its unique substrate specificity. Point mutations at Arg-162, Asp-194, His-196, and Asp-400, highly conserved amino acid residues in the catalytic domain of PP2C family, resulted in a significant loss of phosphatase activity, indicating that these amino acid residues may play important roles in the catalytic activity of CaMKP. Although CaMKP(1-412), a C-terminal truncation mutant, retained phosphatase activity, it was found to be much less stable upon incubation at 37 degrees C than wild type CaMKP, indicating that the C-terminal region of CaMKP is important for the maintenance of the catalytically active conformation. The results suggested that the N- and C-terminal sequences of CaMKP are essential for the regulation and stability of CaMKP.     

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.