Analysis of a catalytic acidic pair in the active center of cellulase from Aspergillus aculeatus

Four acidic amino acid residues, Asp97, Asp101, Glu118, and Glu202, were located in the cleft from the X-ray crystallographic analysis of FI-CMCase, endo-1,4-beta-glucanase (EC: 3.2.1.4) of Aspergillus aculeatus No. F-50. To identify the catalytic residues of the FI-CMCase, these residues were mutated to Glu or Ser from Asp97 and Asp101, and to Asp or Ser from Glu118 and Glu202 by site-directed mutagenesis, and totally 8 single mutant enzymes expressed in Escherichia coli were prepared: D97E, D97S, D101E, D101S, E118D, E118S, E202D, and E202S. Mutant enzymes E118S and E202S were not shown to have any detectable activity. Kinetic parameters of other mutant enzymes were measured after purification. The Km of mutant enzymes were not much different from that of wild type FI-CMCase, while the Vmax of mutant enzymes D97E, D97S, D101E, D101S, E118D, and D202E were much decreased to 1/50, 1/20, 1/4000, 1/2000, 1/800, and 1/1600 of the wild type FI-CMCase, respectively. From these results we concluded that Glu118 and Glu202 were most probable candidates for a catalytic pair of acidic amino acids in FI-CMCase.     

The role for an invariant aspartic acid in hypoxanthine phosphoribosyltransferases is examined using saturation mutagenesis, functional analysis, and X-ray crystallography

The role of an invariant aspartic acid (Asp137) in hypoxanthine phosphoribosyltransferases (HPRTs) was examined by site-directed and saturation mutagenesis, functional analysis, and X-ray crystallography using the HPRT from Trypanosoma cruzi. Alanine substitution (D137A) resulted in a 30-fold decrease of k(cat), suggesting that Asp137 participates in catalysis. Saturation mutagenesis was used to generate a library of mutant HPRTs with random substitutions at position 137, and active enzymes were identified by complementation of a bacterial purine auxotroph. Functional analyses of the mutants, including determination of steady-state kinetic parameters and pH-rate dependence, indicate that glutamic acid or glutamine can replace the wild-type aspartate. However, the catalytic efficiency and pH-rate profile for the structural isosteric mutant, D137N, were similar to the D137A mutant. Crystal structures of four of the mutant enzymes were determined in ternary complex with substrate ligands. Structures of the D137E and D137Q mutants reveal potential hydrogen bonds, utilizing several bound water molecules in addition to protein atoms, that position these side chains within hydrogen bond distance of the bound purine analogue, similar in position to the aspartate in the wild-type structure. The crystal structure of the D137N mutant demonstrates that the Asn137 side chain does not form interactions with the purine substrate but instead forms novel interactions that cause the side chain to adopt a nonfunctional rotamer. The results from these structural and functional analyses demonstrate that HPRTs do not require a general base at position 137 for catalysis. Instead, hydrogen bonding sufficiently stabilizes the developing partial positive charge at the N7-atom of the purine substrate in the transition-state to promote catalysis.     

Crystal structure of a catalytic site mutant of beta-amylase from Bacillus cereus var. mycoides cocrystallized with maltopentaose

The X-ray crystal structure of a catalytic site mutant of beta-amylase, E172A (Glu172 --> Ala), from Bacillus cereus var. mycoides complexed with a substrate, maltopentaose (G5), and the wild-type enzyme complexed with maltose were determined at 2.1 and 2.0 A resolution, respectively. Clear and continuous density corresponding to G5 was observed in the active site of E172A, and thus, the substrate, G5, was not hydrolyzed. All glucose residues adopted a relaxed (4)C(1) conformation, and the conformation of the maltose unit for Glc2 and Glc3 was much different from those of other maltose units, where each glucose residue of G5 is named Glc1-Glc5 (Glc1 is at the nonreducing end). A water molecule was observed 3.3 A from the C1 atom of Glc2, and 3.0 A apart from the OE1 atom of Glu367 which acts as a general base. In the wild-type enzyme-maltose complex, two maltose molecules bind at subsites -2 and -1 and at subsites +1 and +2 in tandem. The conformation of the maltose molecules was similar to that of the condensation product of soybean beta-amylase, but differed from that of G5 in E172A. When the substrate flips between Glc2 and Glc3, the conformational energy of the maltose unit was calculated to be 20 kcal/mol higher than that of the cis conformation by MM3. We suggest that beta-amylase destabilizes the bond that is to be broken in the ES complex, decreasing the activation energy, DeltaG(++), which is the difference in free energy between this state and the transition state.     

Site-directed mutagenesis of Asp313, Glu315, and Asp391 residues in chitinase of Aeromonas caviae

Site-directed mutagenesis was used to explore the roles of amino acid residues involved in the activity of chitinase from Aeromonas caviae. Kinetic parameters for 4-methylumbelliferyl-N,N'-diacetyl-chitobiose or 4-methylumbelliferyl-N,N',N"-triacetylchitotriose hydrolysis were determined with wild-type and mutant chitinases. Chitinases with the mutations E315D (or Q) and D391E (or N) were severely impaired and had dramatically decreased kcat. However, the effect of the these mutations on the Km values were different. The function of the carboxyl group of Asp313 was partially replaced by the amide of Asn when the 4-methylumbelliferyl-N,N',N"-triacetylchitotriose substrate was used. Results indicated that Asp313, Glu315, and Asp391 might be the best candidates for the catalytic residues of chitinase A from Aeromonas caviae.     

Directed mutagenesis of apecific active site residues on Fibrobacter succinogenes 1,3-1,4-beta -D-glucanase significantly affects catalysis and enzyme structural stability

The functional and structural significance of amino acid residues Met(39), Glu(56), Asp(58), Glu(60), and Gly(63) of Fibrobacter succinogenes 1,3-1,4-beta-d-glucanase was explored by the approach of site-directed mutagenesis, initial rate kinetics, fluorescence spectroscopy, and CD spectrometry. Glu(56), Asp(58), Glu(60), and Gly(63) residues are conserved among known primary sequences of the bacterial and fungal enzymes. Kinetic analyses revealed that 240-, 540-, 570-, and 880-fold decreases in k(cat) were observed for the E56D, E60D, D58N, and D58E mutant enzymes, respectively, with a similar substrate affinity relative to the wild type enzyme. In contrast, no detectable enzymatic activity was observed for the E56A, E56Q, D58A, E60A, and E60Q mutants. These results indicated that the carboxyl side chain at positions 56 and 60 is mandatory for enzyme catalysis. M39F, unlike the other mutants, exhibited a 5-fold increase in K(m) value. Lower thermostability was found with the G63A mutant when compared with wild type or other mutant forms of F. succinogenes 1,3-1,4-beta-d-glucanase. Denatured wild type and mutant enzymes were, however, recoverable as active enzymes when 8 m urea was employed as the denaturant. Structural modeling and kinetic studies suggest that Glu(56), Asp(58), and Glu(60) residues apparently play important role(s) in the catalysis of F. succinogenes 1,3-1,4-beta-d-glucanase.     

Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase

To clarify the mechanism of carboxyl transfer from carboxylbiotin to pyruvate, the following conserved amino acid residues present in the carboxyl transferase domain of Bacillus thermodenitrificans pyruvate carboxylase were converted to homologous amino acids: Asp543, Glu576, Glu592, Asp649, Lys712, Asp713, and Asp762. The carboxylase activity of the resulting mutants, D543E, E576D, E576Q, E592Q, D649N, K712R, K712Q, D713E, D713N, D762E, and D762N, was generally less than that of the wild type from mutation, but it decreased the most to 5% or even less than that of the wild type with D543E, D576Q, D649N, K712R, and K712Q. The decrease in activity observed for Asp543, Asp649, and Lys712 mutants was not for structural reasons because their structures seemed to remain intact as assessed by gel filtration and circular dichroism. On the basis of these data, a mechanism is proposed where Lys712 and Asp543 serve as the key acid and base catalyst, respectively.     

Catalytic mechanism of inulinase from Arthrobacter sp. S37

Detailed catalytic roles of the conserved Glu323, Asp460, and Glu519 of Arthrobacter sp. S37 inulinase (EnIA), a member of the glycoside hydrolase family 32, were investigated by site-directed mutagenesis and pH-dependence studies of the enzyme efficiency and homology modeling were carried out for EnIA and for D460E mutant. The enzyme efficiency (k_cat/K_m) of the E323A and E519A mutants was significantly lower than that of the wild-type due to a substantial decrease in k_cat, but not due to variations in K_m, consistent with their putative roles as nucleophile and acid/base catalyst, respectively. The D460A mutant was totally inactive, whereas the D460E and D460N mutants were active to some extent, revealing Asp460 as a catalytic residue and demonstrating that the presence of a carboxylate group in this position is a prerequisite for catalysis. The pH-dependence studies indicated that the pK_a of the acid/base catalyst decreased from 9.2 for the wild-type enzyme to 7.0 for the D460E mutant, implicating Asp460 as the residue that interacts with the acid/base catalyst Glu519 and elevates its pK_a. Homology modeling and molecular dynamics simulation of the wild-type enzyme and the D460E mutant shed light on the structural roles of Glu323, Asp460, and Glu519 in the catalytic activity of the enzyme.     

Substrate recognition mechanism of a glycosyltrehalose trehalohydrolase from Sulfolobus solfataricus KM1

Glycosyltrehalose trehalohydrolase (GTHase) is an α-amylase that cleaves the α-1,4 bond adjacent to the α-1,1 bond of maltooligosyltrehalose to release trehalose. To investigate the catalytic and substrate recognition mechanisms of GTHase, two residues, Asp252 (nucleophile) and Glu283 (general acid/base), located at the catalytic site of GTHase were mutated (Asp252→Ser (D252S), Glu (D252E) and Glu283→Gln (E283Q)), and the activity and structure of the enzyme were investigated. The E283Q, D252E, and D252S mutants showed only 0.04, 0.03, and 0.6% of enzymatic activity against the wild-type, respectively. The crystal structure of the E283Q mutant GTHase in complex with the substrate, maltotriosyltrehalose (G3-Tre), was determined to 2.6-Å resolution. The structure with G3-Tre indicated that GTHase has at least five substrate binding subsites and that Glu283 is the catalytic acid, and Asp252 is the nucleophile that attacks the C1 carbon in the glycosidic linkage of G3-Tre. The complex structure also revealed a scheme for substrate recognition by GTHase. Substrate recognition involves two unique interactions: stacking of Tyr325 with the terminal glucose ring of the trehalose moiety and perpendicularly placement of Trp215 to the pyranose rings at the subsites −1 and +1 glucose.     

Engineering of the pH-dependence of thermolysin activity as examined by site-directed mutagenesis of Asn112 located at the active site of thermolysin

Asn112 is located at the active site of thermolysin, 5-8 A from the catalytic Zn2+ and catalytic residues Glu143 and His231. When Asn112 was replaced with Ala, Asp, Glu, Lys, His, and Arg by site-directed mutagenesis, the mutant enzymes N112D and N112E, in which Asn112 is replaced with Asp and Glu, respectively, were secreted as an active form into Escherichia coli culture medium, while the other four were not. In the hydrolysis of a neutral substrate N-[3-(2-furyl)acryloyl]-Gly-L-Leu amide, the kcat/Km values of N112D and N112E exhibited bell-shaped pH-dependence, as did the wild-type thermolysin (WT). The acidic pKa of N112D was 5.7 +/- 0.1, higher by 0.4 +/- 0.2 units than that of WT, suggesting that the introduced negative charge suppressed the protonation of Glu143 or Zn2+-OH. In the hydrolysis of a negatively charged substrate, N-carbobenzoxy-l-Asp-l-Phe methyl ester (ZDFM), the pH-dependence of kcat/Km of the mutants decreased with increase in pH from 5.5 to 8.5, while that of WT was bell-shaped. This difference might be explained by the electrostatic repulsion between the introduced Asp/Glu and ZDFM, suggesting that introducing ionizing residues into the active site of thermolysin might be an effective means of modifying its pH-activity profile.     

Investigating a catalytic mechanism of hyperthermophilic L-threonine dehydrogenase from Pyrococcus horikoshii

Based on our first structural data of L-threonine dehydrogenase (TDH) of Pyrococcus horikoshii (PhTDH), we examined its catalytic mechanism. The structural analysis indicated that a catalytic zinc atom at the active centre of PhTDH is coordinated by four residues (Cys42, His67, Glu68 and Glu152) with low affinity. These residues are highly conserved in alcohol dehydrogenases (ADHs) and TDHs. Several PhTDH mutants were prepared with respect to Glu152 and other residues, relating to the proton relay system that is substantially a rate-limiting step in ADH. It was found that the E152D mutant showed 3-fold higher turnover rate and reduced affinities toward L-threonine and NAD(+), compared to wild-type PhTDH. The kinetic analysis of Glu152 mutants indicated that the carboxyl group of Glu152 is important for expressing the catalytic activity. The results obtained from pH dependency of kinetic parameters suggested that Glu152 to Asp substitution causes the enhancement of deprotonation of His47 or ionization of zinc-bound water and threonine in the enzyme-NAD(+) complex. Furthermore, it was predicted that the access of threonine substrate to the enzyme-NAD(+) complex induces a large conformational change in the active domain of PhTDH. From these results, we propose here that the proton relay system works as a catalytic mechanism of PhTDH.     

Mechanism of the Ca2+-induced enhancement of the intrinsic factor VIIa activity

The intrinsic activity of coagulation factor VIIa (FVIIa) is dependent on Ca(2+) binding to a loop (residues 210-220) in the protease domain. Structural analysis revealed that Ca(2+) may enhance the activity by attenuating electrostatic repulsion of Glu(296) and/or by facilitating interactions between the loop and Lys(161) in the N-terminal tail. In support of the first mechanism, the mutations E296V and D212N resulted in similar, about 2-fold, enhancements of the amidolytic activity. Moreover, mutation of the Lys(161)-interactive residue Asp(217) or Asp(219) to Ala reduced the amidolytic activity by 40-50%, whereas the K161A mutation resulted in 80% reduction. Hence one of these Asp residues in the Ca(2+)-binding loop appears to suffice for some residual interaction with Lys(161), whereas the more severe effect upon replacement of Lys(161) is due to abrogation of the interaction with the N-terminal tail. However, Ca(2+) attenuation of the repulsion between Asp(212) and Glu(296) keeps the activity above that of apoFVIIa. Altogether, our data suggest that repulsion involving Asp(212) in the Ca(2+)-binding loop suppresses FVIIa activity and that optimal activity requires a favorable interaction between the Ca(2+)-binding loop and the N-terminal tail. Crystal structures of tissue factor-bound FVIIa(D212N) and FVIIa(V158D/E296V/M298Q) revealed altered hydrogen bond networks, resembling those in factor Xa and thrombin, after introduction of the D212N and E296V mutations plausibly responsible for tethering the N-terminal tail to the activation domain. The charge repulsion between the Ca(2+)-binding loop and the activation domain appeared to be either relieved by charge removal and new hydrogen bonds (D212N) or abolished (E296V). We propose that Ca(2+) stimulates the intrinsic FVIIa activity by a combination of charge neutralization and loop stabilization.     

Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism

Structures of the serine carboxypeptidase-like enzymes 1-O-sinapoyl-beta-glucose:L-malate sinapoyltransferase (SMT) and 1-O-sinapoyl-beta-glucose:choline sinapoyltransferase (SCT) were modeled to gain insight into determinants of specificity and substrate recognition. The structures reveal the alpha/beta-hydrolase fold as scaffold for the catalytic triad Ser-His-Asp. The recombinant mutants of SMT Ser173Ala and His411Ala were inactive, whereas Asp358Ala displayed residual activity of 20%. 1-O-sinapoyl-beta-glucose recognition is mediated by a network of hydrogen bonds. The glucose moiety is recognized by a hydrogen bond network including Trp71, Asn73, Glu87 and Asp172. The conserved Asp172 at the sequence position preceding the catalytic serine meets sterical requirements for the glucose moiety. The mutant Asn73Ala with a residual activity of 13% underscores the importance of the intact hydrogen bond network. Arg322 is of key importance by hydrogen bonding of 1-O-sinapoyl-beta-glucose and L-malate. By conformational change, Arg322 transfers L-malate to a position favoring its activation by His411. Accordingly, the mutant Arg322Glu showed 1% residual activity. Glu215 and Arg219 establish hydrogen bonds with the sinapoyl moiety. The backbone amide hydrogens of Gly75 and Tyr174 were shown to form the oxyanion hole, stabilizing the transition state. SCT reveals also the catalytic triad and a hydrogen bond network for 1-O-sinapoyl-beta-glucose recognition, but Glu274, Glu447, Thr445 and Cys281 are crucial for positioning of choline.     

Identification of essential amino acid residues of the NorM Na+/multidrug antiporter in Vibrio parahaemolyticus

NorM is a member of the multidrug and toxic compound extrusion (MATE) family and functions as a Na+/multidrug antiporter in Vibrio parahaemolyticus, although the underlying mechanism of the Na+/multidrug antiport is unknown. Acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM are conserved in one of the clusters of the MATE family. In this study, we investigated the role(s) of acidic amino acid residues Asp32, Glu251, and Asp367 in the transmembrane region of NorM by site-directed mutagenesis. Wild-type NorM and mutant proteins with amino acid replacements D32E (D32 to E), D32N, D32K, E251D, E251Q, D367A, D367E, D367N, and D367K were expressed and localized in the inner membrane of Escherichia coli KAM32 cells, while the mutant proteins with D32A, E251A, and E251K were not. Compared to cells with wild-type NorM, cells with the mutant NorM protein exhibited reduced resistance to kanamycin, norfloxacin, and ethidium bromide, but the NorM D367E mutant was more resistant to ethidium bromide. The NorM mutant D32E, D32N, D32K, D367A, and D367K cells lost the ability to extrude ethidium ions, which was Na+ dependent, and the ability to move Na+, which was evoked by ethidium bromide. Both E251D and D367N mutants decreased Na+-dependent extrusion of ethidium ions, but ethidium bromide-evoked movement of Na+ was retained. In contrast, D367E caused increased transport of ethidium ions and Na+. These results suggest that Asp32, Glu251, and Asp367 are involved in the Na+-dependent drug transport process.     

Biochemical analysis of point mutations in the 5'-3' exonuclease of DNA polymerase I of Streptococcus pneumoniae. Functional and structural implications

To define the active site of the 5'-3' exonucleolytic domain of the Streptococcus pneumoniae DNA polymerase I (Spn pol I), we have constructed His-tagged Spn pol I fusion protein and introduced mutations at residues Asp(10), Glu(88), and Glu(114), which are conserved among all prokaryotic and eukaryotic 5' nucleases. The mutations, but not the fusion to the C-terminal end of the wild-type, reduced the exonuclease activity. The residual exonuclease activity of the mutant proteins has been kinetically studied, together with potential alterations in metal binding at the active site. Comparison of the catalytic rate and dissociation constant of the D10G, E114G, and E88K mutants and the control fusion protein support: (i) a critical function of Asp(10) in the catalytic event, (ii) a role of Glu(114) in the exonucleolytic reaction, being secondarily involved in both catalysis and DNA binding, and (iii) a nonessential function of Glu(88) for the exonuclease activity of Spn pol I. Moreover, the pattern of metal activation of the mutant proteins indicates that none of the three residues is a metal-ligand at the active site. These findings and those previously obtained with D190A mutant of Spn pol I are discussed in relation to structural and mutational data for related 5' nucleases.     

Mutagenesis and mechanistic study of a glycoside hydrolase family 54 alpha-L-arabinofuranosidase from Trichoderma koningii

A GH (glycoside hydrolase) family 54 alpha-L-arabinofuranosidase from Trichoderma koningii G-39 (termed Abf) was successfully expressed in Pichia pastoris and purified to near homogeneity by cation-exchange chromatography. To determine the amino acid residues essential for the catalytic activity of Abf, extensive mutagenesis of 24 conserved glutamate and aspartate residues was performed. Among the mutants, D221N, E223Q and D299N were found to decrease catalytic activity significantly. The kcat values of the D221N and D299N mutants were 7000- and 1300-fold lower respectively, than that of the wild-type Abf. E223Q was nearly inactive. These results are consistent with observations obtained from the Aspergillus kawachii alpha-L-arabinofuranosidase three-dimensional structure. This structure indicates that Asp221 of T. koningii Abf is significant for substrate binding and that Glu223 as well as Asp299 function as a nucleophile and a general acid/base catalyst for the enzymatic reaction respectively. The catalytic mechanism of wild-type Abf was further investigated by NMR spectroscopy and kinetic analysis. The results showed that Abf is a retaining enzyme. It catalyses the hydrolysis of various substrates via the formation of a common intermediate that is probably an arabinosyl-enzyme intermediate. A two-step, double-displacement mechanism involving first the formation, and then the breakdown, of an arabinosyl-enzyme intermediate was proposed. Based on the kcat values of a series of aryl-alpha-L-arabinofuranosides catalytically hydrolysed by wild-type Abf, a relatively small Br?nsted constant, beta(lg)=-0.18, was obtained, suggesting that the rate-limiting step of the enzymatic reaction is the dearabinosylation step. Further kinetic studies with the D299G mutant revealed that the catalytic activity of this mutant depended largely on the pK(a) values (>6) of leaving phenols, with beta(lg)=-1.3, indicating that the rate-limiting step of the reaction becomes the arabinosylation step. This kinetic outcome supports the idea that Asp299 is the general acid/base residue. The pH activity profile of D299N provided further evidence strengthening this suggestion.     

Identification of a glutamic acid and an aspartic acid residue essential for catalytic activity of aspergillopepsin II, a non-pepsin type acid proteinase

Aspergillopepsin II from Aspergillus niger var. macrosporus is a non-pepsin type or pepstatin-insensitive acid proteinase. To identify the catalytic residues of the enzyme, all acidic residues that are conserved in the homologous proteinases of family A4 were replaced with Asn, Gln, or Ala using site-directed mutagenesis. The wild-type and mutant pro-enzymes were heterologously expressed in Escherichia coli and refolded in vitro. The wild-type pro-enzyme was shown to be processed into a two-chain active enzyme under acidic conditions. Most of the recombinant mutant pro-enzymes showed significant activity under acidic conditions because of autocatalytic activation except for the D123N, D123A, E219Q, and E219A mutants. The D123A, E219Q, and E219A mutants showed neither enzymatic activity nor autoprocessing activity under acidic conditions. The circular dichroism spectra of the mutant pro- and mature enzymes were essentially the same as those of the wild-type pro- and mature enzyme, respectively, indicating that the mutant pro-enzymes were correctly folded. In addition, two single and one double mutant pro-enzyme, D123E, E219D, and D123E/E219D, did not show enzymatic activity under acidic conditions. Taken together, Glu-219 and Asp-123 are deduced to be the catalytic residues of aspergillopepsin II.     

Biochemical characterization and identification of the catalytic residues of a family 43 beta-D-xylosidase from Geobacillus stearothermophilus T-6

Beta-D-xylosidases are hemilcellulases that hydrolyze short xylooligosaccharides into xylose units. Here, we describe the characterization and kinetic analysis of a family 43 beta-xylosidase from Geobacillus stearothermophilus T-6 (XynB3). Enzymes in this family use an inverting single-displacement mechanism with two conserved carboxylic acids, a general acid, and a general base. XynB3 was most active at 65 degrees C and pH 6.5, with clear preference to xylose-based substrates. Products analysis indicated that XynB3 is an exoglycosidase that cleaves single xylose units from the nonreducing end of xylooligomers. On the basis of sequence homology, amino acids Asp15 and Glu187 were suggested to act as the general-base and general-acid catalytic residues, respectively. Kinetic analysis with substrates bearing different leaving groups showed that, for the wild-type enzyme, the k(cat) and k(cat)/K(m) values were only marginally affected by the leaving-group reactivity, whereas for the E187G mutant, both values exhibited significantly greater dependency on the pK(a) of the leaving group. The pH-dependence activity profile of the putative general-acid mutant (E187G) revealed that the protonated catalytic residue was removed. Addition of the exogenous nucleophile azide did not affect the activities of the wild type or the E187G mutant but rescued the activity of the D15G mutant. On the basis of thin-layer chromatography and (1)H NMR analyses, xylose and not xylose azide was the only product of the accelerated reaction, suggesting that the azide ion does not attack the anomeric carbon directly but presumably activates a water molecule. Together, these results confirm the suggested catalytic role of Glu187 and Asp15 in XynB3 and provide the first unequivocal evidence regarding the exact roles of the catalytic residues in an inverting GH43 glycosidase.     

Conserved Asp327 of walker B motif in the N-terminal nucleotide binding domain (NBD-1) of Cdr1p of Candida albicans has acquired a new role in ATP hydrolysis

The Walker A and B motifs of nucleotide binding domains (NBDs) of Cdr1p though almost identical to all ABC transporters, has unique substitutions. We have shown in the past that Trp326 of Walker B and Cys193 of Walker A motifs of N-terminal NBD of Cdr1p have distinct roles in ATP binding and hydrolysis, respectively. In the present study, we have examined the role of a well conserved Asp327 in the Walker B motif of the N-terminal NBD, which is preceded (Trp326) and followed (Asn328) by atypical amino acid substitutions and compared it with its equivalent well conserved Asp1026 of the C-terminal NBD of Cdr1p. We observed that the removal of the negative charge by D327N, D327A, D1026N, D1026A, and D327N/D1026N substitutions, resulted in Cdr1p mutant variants that were severely impaired in ATPase activity and drug efflux. Importantly, all of the mutant variants showed characteristics similar to those of the wild type with respect to cell surface expression and photoaffinity drug analogue [125I] IAAP and [3H] azidopine labeling. Although the Cdr1p D327N mutant variant showed comparable binding with [alpha-32P] 8-azido ATP, Cdr1p D1026N and Cdr1p D327N/D1026N mutant variants were crippled in nucleotide binding. That the two conserved carboxylate residues Asp327 and Asp1026 are functionally different was further evident from the pH profile of ATPase activity. The Cdr1p D327N mutant variant showed approximately 40% enhancement of its residual ATPase activity at acidic pH, whereas no such pH effect was seen with the Cdr1p D1026N mutant variant. Our experimental data suggest that Asp327 of N-terminal NBD has acquired a new role to act as a catalytic base in ATP hydrolysis, a role normally conserved for Glu present adjacent to the conserved Asp in the Walker B motif of all the non-fungal transporters.     

Probing the catalytically essential residues of the alpha-L-fucosidase from the hyperthermophilic archaeon Sulfolobus solfataricus

Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.