Insights into the Functional Architecture of the Catalytic Center of a Maize β-Glucosidase Zm-p60.1

The maize (Zea mays) beta-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-A resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K(m) of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k(cat) was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.     

Identification of human parainfluenza virus type 2 (HPIV-2) V protein amino acid residues that reduce binding of V to MDA5 and attenuate HPIV-2 replication in nonhuman primates

Human parainfluenza virus type 2 (HPIV-2), an important pediatric respiratory pathogen, encodes a V protein that inhibits type I interferon (IFN) induction and signaling. Using reverse genetics, we attempted the recovery of a panel of V mutant viruses that individually contained one of six cysteine-to-serine (residues 193, 197, 209, 211, 214, and 218) substitutions, one of two paired charge-to-alanine (R175A/R176A and R205A/K206A) substitutions, or a histidine-to-phenylalanine (H174F) substitution. This mutagenesis was performed using a cDNA-derived HPIV-2 virus that expressed the V and P coding sequences from separate mRNAs. Of the cysteine substitutions, only C193S, C214S, and C218S yielded viable virus, and only the C214S mutant replicated well enough for further analysis. The H174F, R175A/R176A, and R205A/K206A mutants were viable and replicated well. The H174F and R205A/K206A mutants did not differ from the wild-type (WT) V in their ability to physically interact with MDA5, a cytoplasmic sensor of nonself RNA that induces type I IFN. Like WT HPIV-2, these mutants inhibited IFN-β induction and replicated efficiently in African green monkeys (AGMs). In contrast, the C214S and R175A/R176A mutants did not bind MDA5 efficiently, did not inhibit interferon regulatory factor 3 (IRF3) dimerization or IFN-β induction, and were attenuated in AGMs. These findings indicate that V binding to MDA5 is important for HPIV-2 virulence in nonhuman primates and that some V protein residues involved in MDA5 binding are not essential for efficient HPIV-2 growth in vitro. Using a transient expression system, 20 additional mutant V proteins were screened for MDA5 binding, and the region spanning residues 175 to 180 was found to be essential for this activity.     

Site-directed mutagenesis improves catalytic efficiency and thermostability of Escherichia coli pH 2.5 acid phosphatase/phytase expressed in Pichia pastoris

Escherichia coli pH 2.5 acid phosphatase gene (appA) and three mutants were expressed in Pichia pastoris to assess the effect of strategic mutations or deletion on the enzyme (EcAP) biochemical properties. Mutants A131N/ V134N/D207N/S211N, C200N/D207N/S211N, and A131N/ V134N/C200N/D207N/S211N had four, two, and four additional potential N-glycosylation sites, respectively. Extracellular phytase and acid phosphatase activities were produced by these mutants and the intact enzyme r-AppA. The N-glycosylation level was higher in mutants A131N/V134N/D207N/S211N (48%) and A131N/V134N/ C200N/D207N/S211N (89%) than that in r-AppA (14%). Despite no enhancement of glycosylation, mutant C200N/ D207N/S211N was different from r-AppA in the following properties. First, it was more active at pH 3.5-5.5. Second, it retained more (P < 0.01) phytase activity than that of r-AppA. Third, its specific activity of phytase was 54% higher. Lastly, its apparent catalytic efficiency kcat/Km for either p-nitrophenyl phosphate (5.8 x 10(5) vs 2.0 x 10(5) min(-1) M(-1)) or sodium phytate (6.9 x 10(6) vs 1.1 x 10(6) min(-1) M(-1)) was improved by factors of 1.9- and 5.3-fold, respectively. Based on the recently published E. coli phytase crystal structure, substitution of C200N in mutant C200N/D207N/S211N seems to eliminate the disulfide bond between the G helix and the GH loop in the alpha-domain of the protein. This change may modulate the domain flexibility and thereby the catalytic efficiency and thermostability of the enzyme.     

Kinetic and crystallographic analysis of active site mutants of Escherichia coli gamma-aminobutyrate aminotransferase

The E. coli isozyme of gamma-aminobutyrate aminotransferase (GABA-AT) is a tetrameric pyridoxal phosphate-dependent enzyme that catalyzes transamination between primary amines and alpha-keto acids. The roles of the active site residues V241, E211, and I50 in the GABA-AT mechanism have been probed by site-directed mutagenesis. The beta-branched side chain of V241 facilitates formation of external aldimine intermediates with primary amine substrates, while E211 provides charge compensation of R398 selectively in the primary amine half-reaction and I50 forms a hydrophobic lid at the top of the substrate binding site. The structures of the I50Q, V241A, and E211S mutants were solved by X-ray crystallography to resolutions of 2.1, 2.5, and 2.52 A, respectively. The structure of GABA-AT is similar in overall fold and active site structure to that of dialkylglycine decarboxylase, which catalyzes both transamination and decarboxylation half-reactions in its normal catalytic cycle. Therefore, an attempt was made to convert GABA-AT into a decarboxylation-dependent aminotransferase similar to dialkylglycine decarboxylase by systematic mutation of E. coli GABA-AT active site residues. Two of the twelve mutants presented, E211S/I50G/C77K and E211S/I50H/V80D, have approximately 10-fold higher decarboxylation activities than the wild-type enzyme, and the E211S/I50H/V80D has formally changed the reaction specificity to that of a decarboxylase.     

Complete reversal of coenzyme specificity of xylitol dehydrogenase and increase of thermostability by the introduction of structural zinc

Pichia stipitis NAD(+)-dependent xylitol dehydrogenase (XDH), a medium-chain dehydrogenase/reductase, is one of the key enzymes in ethanol fermentation from xylose. For the construction of an efficient biomass-ethanol conversion system, we focused on the two areas of XDH, 1) change of coenzyme specificity from NAD(+) to NADP(+) and 2) thermostabilization by introducing an additional zinc atom. Site-directed mutagenesis was used to examine the roles of Asp(207), Ile(208), Phe(209), and Asn(211) in the discrimination between NAD(+) and NADP(+). Single mutants (D207A, I208R, F209S, and N211R) improved 5 approximately 48-fold in catalytic efficiency (k(cat)/K(m)) with NADP(+) compared with the wild type but retained substantial activity with NAD(+). The double mutants (D207A/I208R and D207A/F209S) improved by 3 orders of magnitude in k(cat)/K(m) with NADP(+), but they still preferred NAD(+) to NADP(+). The triple mutant (D207A/I208R/F209S) and quadruple mutant (D207A/I208R/F209S/N211R) showed more than 4500-fold higher values in k(cat)/K(m) with NADP(+) than the wild-type enzyme, reaching values comparable with k(cat)/K(m) with NAD(+) of the wild-type enzyme. Because most NADP(+)-dependent XDH mutants constructed in this study decreased the thermostability compared with the wild-type enzyme, we attempted to improve the thermostability of XDH mutants by the introduction of an additional zinc atom. The introduction of three cysteine residues in wild-type XDH gave an additional zinc-binding site and improved the thermostability. The introduction of this mutation in D207A/I208R/F209S and D207A/I208R/F209S/N211R mutants increased the thermostability and further increased the catalytic activity with NADP(+).     

Dynamics of beta-glucosidase Zm-p60.1 ectopic expression during transgenic pollen development: a histochemical approach

Zm-p60.1 is maize cDNA coding cytokinin-glucoside specific beta-glucosidase. Indigogenic method was used for histochemical localization of Zm-p60.1 beta-glucosidase activity in various developmental stages of transgenic tobacco anthers. Expression of Zm-p60.1 cDNA in T7 tobacco plants is controlled by the CaMV 35S promoter. Another type of tobacco transformant expresses Zm-p60.1 under the control of LAT 52 promoter. Histochemical detection has proved different patterns of beta-glucosidase activity during tobacco pollen development in these two types of transformants. Zm-p60.1 beta-glucosidase activity had not direct influence on pollen germinability.     

Functional analysis of active site residues of Bacillus thuringiensis WB7 chitinase by site-directed mutagenesis

To investigate the roles of the active site residues in the catalysis of Bacillus thuringiensis WB7 chitinase, twelve mutants, F201L, F201Y, G203A, G203D, D205E, D205N, D207E, D207N, W208C, W208R, E209D and E209Q were constructed by site-directed mutagenesis. The results showed that the mutants F201L, G203D, D205N, D207E, D207N, W208C and E209D were devoid of activity, and the loss of the enzymatic activities for F201Y, G203A, D205E, W208R and E209Q were 72, 70, 48, 31 and 29%, respectively. The pH-activity profiles indicated that the optimum pH for the mutants as well as for the wildtype enzyme was 8.0. E209Q exhibited a broader active pH range while D205E, G203A and F201Y resulted in a narrower active pH range. The pH range of activity reduced 1 unit for D205E, and 2 units for G203A and F201Y. The temperature-activity profiles showed that the optimum temperature for other mutants as well as wildtype enzyme was 60°C, but 50°C for G203A, which suggested that G203A resulted in a reduction of thermostability. The study indicated that the six active site residues involving in mutagenesis played an important part in WB7 chitinase. In addition, the catalytic mechanisms of the six active site residues in WB7 chitinase were discussed.     

Site-directed mutagenesis of phosphate-contacting amino acids of bovine pancreatic deoxyribonuclease I

Bovine pancreatic deoxyribonuclease I (DNase I) is an endonuclease which cleaves double-stranded DNA. Cocrystal structures of DNase I with oligonucleotides have revealed interactions between the side chains of several amino acids (N74, R111, N170, S206, T207, and Y211) and the DNA phosphates. The effects these interactions have on enzyme catalysis and DNA hydrolysis selectivity have been investigated by site-directed mutagenesis. Mutations to R111, N170, T207, and Y211 severely compromised activity toward both DNA and a small chromophoric substrate. A hydrogen bond between R111 (which interacts with the phosphate immediately 5' to the cutting site) and the essential amino acid H134 is probably required to maintain this histidine in the correct orientation for efficient hydrolysis. Both T207 and Y211 bind to the phosphate immediately 3' to the cleavage site. Additionally, T207 is involved in binding an essential, structural, calcium ion, and Y211 is the nearest neighbor to D212, a critical catalytic residue. N170 interacts with the scissile phosphate and appears to play a direct role in the catalytic mechanism. The mutation N74D, which interacts with a phosphate twice removed from the scissile group, strongly reduced DNA hydrolysis. However, a comparison of DNase I variants from several species suggests that certain amino acids, which allow interaction with phosphates (positively charged or hydrogen bonding), are tolerated. S206, which binds to a DNA phosphate two positions away from the cleavage site, appears to play a relatively unimportant role. None of the enzyme variants, including a triple mutation in which N74, R111, and Y211 were altered, affected DNA hydrolysis selectivity. This suggests that phosphate binding residues play no role in the selection of DNA substrates.     

Factor XI homodimer structure is essential for normal proteolytic activation by factor XIIa, thrombin, and factor XIa

Coagulation factor XI (FXI) is a covalent homodimer consisting of two identical subunits of 80 kDa linked by a disulfide bond formed by Cys-321 within the Apple 4 domain of each subunit. Because FXI(C321S) is a noncovalent dimer, residues within the interface between the two subunits must mediate its homodimeric structure. The crystal structure of FXI demonstrates formation of salt bridges between Lys-331 of one subunit and Glu-287 of the other subunit and hydrophobic interactions at the interface of the Apple 4 domains involving Ile-290, Leu-284, and Tyr-329. FXI(C321S), FXI(C321S,K331A), FXI(C321S,E287A), FXI(C321S,I290A), FXI(C321S,Y329A), FXI(C321S,L284A), FXI(C321S,K331R), and FXI(C321S,H343A) were expressed in HEK293 cells and characterized using size exclusion chromatography, analytical ultracentrifugation, electron microscopy, and functional assays. Whereas FXI(C321S) and FXI(C321S,H343A) existed in monomer/dimer equilibrium (K(d) approximately 40 nm), all other mutants were predominantly monomers with impaired dimer formation by analytical ultracentrifugation (K(d)=3-38 microm). When converted to the active enzyme, FXIa, all the monomeric mutants activated FIX similarly to wild-type dimeric FXIa. In contrast, these monomeric mutants could not be activated efficiently by FXIIa, thrombin, or autoactivation in the presence of dextran sulfate. We conclude that salt bridges formed between Lys-331 of one subunit and Glu-287 of the other together with hydrophobic interactions at the interface, involving residues Ile-290, Leu-284, and Tyr-329, are essential for homodimer formation. The dimeric structure of FXI is essential for normal proteolytic activation of FXI by FXIIa, thrombin, or FXIa either in solution or on an anionic surface but not for FIX activation by FXIa in solution.     

Variation in relative substrate specificity of bifunctional beta-D-xylosidase/alpha-L-arabinofuranosidase by single-site mutations: roles of substrate distortion and recognition

To probe differential control of substrate specificities for 4-nitrophenyl-alpha-l-arabinofuranoside (4NPA) and 4-nitrophenyl-beta-d-xylopyranoside (4NPX), residues of the glycone binding pocket of GH43 beta-d-xylosidase/alpha-l-arabinofuranosidase from Selenomonas ruminantium were individually mutated to alanine. Although their individual substrate specificities (kcat/Km)(4NPX) and (kcat/Km)(4NPA) are lowered 330 to 280,000 fold, D14A, D127A, W73A, E186A, and H248A mutations maintain similar relative substrate specificities as wild-type enzyme. Relative substrate specificities (kcat/Km)(4NPX)/(kcat/Km)(4NPA) are lowered by R290A, F31A, and F508A mutations to 0.134, 0.407, and 4.51, respectively, from the wild type value of 12.3 with losses in (kcat/Km)(4NPX) and (kcat/Km)(4NPA) of 18 to 163000 fold. R290 and F31 reside above and below the C4 OH group of 4NPX and the C5 OH group of 4NPA, where they can serve as anchors for the two glycone moieties when their ring systems are distorted to transition-state geometries by raising the position of C1. Thus, whereas R290 and F31 provide catalytic power for hydrolysis of both substrates, the native residues are more important for 4NPX than 4NPA as the xylopyranose ring must undergo greater distortion than the arabinofuranose ring. F508 borders C4 and C5 of the two glycone moieties and can serve as a hydrophobic platform having more favorable interactions with xylose than arabinofuranose.     

Thrombin mutants with altered enzymatic activity have an impaired mitogenic effect on mouse fibroblasts and are inefficient modulators of stellation of rat cortical astrocytes.

We produced recombinant human thrombin mutants to investigate the correlation between the thrombin enzyme and mitogenic activity. Single amino acid substitutions were introduced in the catalytic triad (H43N, D99N, S205A, S205T), in the oxy-anion binding site (G203A) and in the anion binding exosite-1 region (R73E). Proteins were produced as prethrombin-2 mutants secreted in the culture medium of DXB11-derived cell lines. All mutants were activated by ecarin to the corresponding thrombin mutants; the enzymatic activity was assayed on a chromogenic substrate and on the procoagulant substrate fibrinogen. Mutations S205A and G203A completely abolished the enzyme activity. Mutations H43N, D99N and S205T dramatically impaired the enzyme activity toward both substrates. The R73E mutation dissociated the amidolytic activity and the clotting activity of the protein. The ability of thrombin mutants to induce proliferation was investigated in NIH3T3 mouse fibroblasts and rat cortical astrocytes. The ability of the thrombin mutants to revert astrocyte stellation was also studied. The mitogenic activity and the effect on the astrocyte stellation of the thrombin mutants correlated with their enzymatic activity. Furthermore the receptor occupancy by the inactive S205A mutant prevented the thrombin effects providing strong evidence that a proteolytically activated receptor is involved in cellular responses to thrombin.     

Engineering the cytokinin-glucoside specificity of the maize β-d-glucosidase Zm-p60.1 using site-directed random mutagenesis

The maize β-d-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-d-glucopyranoside versus the trans-zeatin-O-β-d-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-d-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.     

Developmental regulation of the maize Zm-p60.1 gene encoding a β-glucosidase located to plastids

A β-glucosidase that cleaves the biologically inactive hormone conjugates cytokinin-O- and kinetin-N3-glucosides is encoded by the maize Zm-p60.1 gene. The expression of the Zm-p60.1 gene was analyzed by Northern blot analysis and in-situ hybridization. It was found that the expression levels of the Zm-p60.1-specific mRNA changed after pollination of carpellate inflorescences. The Zm-p60.1 cDNA was expressed in E. coli and antibodies were raised against this protein. An antibody was used to determine the tissue-specific localization of this protein. By in situ immunolocalization experiments, this protein was found to be located in cell layers below the epidermis and around the vascular bundles of the coleoptile. In the primary leaf, the Zm-p60.1 protein was detected in cells of the outermost cell layer and around the vascular tissue. In floral tissue, Zm-p60.1 was present in the glumes, the carpels and in the outer cell layer of the style. In coleoptiles, as determined by immuno-electronmicroscopy, the Zm-p60.1 protein was located exclusively in the plastids. Received: 11 August 1998 / Accepted: 30 December 1998     

Residues at the active site of the esterase 2 from Alicyclobacillus acidocaldarius involved in substrate specificity and catalytic activity at high temperature.

The recently solved three-dimensional structure of the thermophilic esterase 2 from Alicyclobacillus acidocaldarius allowed us to have a snapshot of an enzyme-sulfonate complex, which mimics the second stage of the catalytic reaction, namely the covalent acyl-enzyme intermediate. The aim of this work was to design, by structure-aided analysis and to generate by site-directed and saturation mutagenesis, EST2 variants with changed substrate specificity in the direction of preference for monoacylesters whose acyl-chain length is greater than eight carbon atoms. Positions 211 and 215 of the polypeptide chain were chosen to introduce mutations. Among five variants with single and double amino acid substitutions, three were obtained, M211S, R215L, and M211S/R215L, that changed the catalytic efficiency profile in the desired direction. Kinetic characterization of mutants and wild type showed that this change was achieved by an increase in k(cat) and a decrease in K(m) values with respect to the parental enzyme. The M211S/R215L specificity constant for p-nitrophenyl decanoate substrate was 6-fold higher than the wild type. However, variants M211T, M211S, and M211V showed strikingly increased activity as well as maximal activity with monoacylesters with four carbon atoms in the acyl chain, compared with the wild type. In the case of mutant M211T, the k(cat) for p-nitrophenyl butanoate was 2.4-fold higher. Overall, depending on the variant and on the substrate, we observed improved catalytic activity at 70 degrees C with respect to the wild type, which was a somewhat unexpected result for an enzyme with already high k(cat) values at high temperature. In addition, variants with altered specificity toward the acyl-chain length were obtained. The results were interpreted in the context of the EST2 three-dimensional structure and a proposed catalytic mechanism in which k(cat), e.g. the limiting step of the reaction, was dependent on the acyl chain length of the ester substrate.     

Survey of catalytic residues and essential roles of glutamate-alpha170 and aspartate-alpha335 in coenzyme B12-dependent diol dehydratase

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K(+) was mutated to Ala or another amino acid residue. The Ealpha170A and Dalpha335A mutants were totally inactive, and the Halpha143A mutant showed only a trace of activity, indicating that Glu-alpha170, Asp-alpha335, and His-alpha143 are catalytic residues. The Qalpha141A, Qalpha296A, and Salpha362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-alpha296 is important for substrate binding and Gln-alpha296 and Gln-alpha141 for preventing the enzyme from mechanism-based inactivation. The Ealpha221A, Ealpha170H, and Dalpha335A did not form the (alphabetagamma)(2) complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-alpha170 and Asp-alpha335 mutants, Ealpha170D and Ealpha170Q were 2.2 +/- 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dalpha335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue alpha170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-alpha170 and Asp-alpha335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-alpha170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.     

Site-directed mutagenesis of the putative distal helix of peroxygenase cytochrome P450

CYP152A1 is an unusual, peroxygenase enzyme that catalyzes the beta- or alpha-hydroxylation of fatty acids by efficiently introducing an oxygen atom from H2O2 to the fatty acid. To clarify the mechanistic roles of amino acid residues in this enzyme, we have used site-directed mutagenesis of residues in the putative distal helix and measured the spectroscopic and enzymatic properties of the mutant proteins. Initially, we carried out Lys-scanning mutagenesis of amino acids in this region to determine residues of CYP152A1 that might have a mechanistic role. Among the Lys mutants, only P243K gave an absorption spectrum characteristic of a nitrogenous ligand-bound form of a ferric P450. Further investigation of the Pro243 site revealed that a P243H mutant also exhibited a nitrogen-bound form, but that the mutants P243A or P243S did not. On the hydroxylation of myristic acid by the Lys mutants, we observed a large decrease in activity for R242K and A246K. We therefore examined other mutants at amino acid positions 242 and 246. At position 246, an A246K mutant showed a roughly 19-fold lower affinity for myristic acid than the wild type. Replacing Ala246 with Ser decreased the catalytic activity, but did not affect affinity for the substrate. An A246V mutant showed slightly reduced activity and moderately reduced affinity. At position 242, an R242A showed about a fivefold lower affinity than the wild type for myristic acid. The Km values for H2O2 increased and Vmax values decreased in the order of wild type, R242K, and R242A when H2O2 was used; furthermore, Vmax/Km was greatly reduced in R242A compared with the wild type. If cumene hydroperoxide was used instead of H2O2, however, the Km values were not affected much by these substitutions. Together, our results suggest that in CYP152A1 the side chain of Pro243 is located close to the iron at the distal side of a heme molecule; the fatty acid substrate may be positioned near to Ala246 in the catalytic pocket, although Ala246 does not participate in hydrophobic interactions with the substrate; and that Arg242 is a critical residue for substrate binding and H2O2-specific catalysis.     

Influence of multiple cysteines on human 3-hydroxy-3-methylglutaryl-CoA lyase activity and formation of inter-subunit adducts

Human 3-hydroxy-3-methylglutaryl-CoA lyase catalyzes formation of acetyl-CoA and acetoacetate in a reaction that requires divalent cation and is stimulated by sulfhydryl protective reagents. The enzyme is a homodimer and inter-subunit adducts form in the absence of reducing agents or upon treatment with cysteine selective crosslinking agents. To address the influence of cysteines on enzyme activity and formation of inter-subunit and intra-subunit adducts, single serine substitutions have been engineered for each enzyme cysteine. Enzyme activity varies for each cysteine→serine mutant protein and different mutations have widely different effects on recovery of activity upon DTT treatment of non-reduced enzyme. These levels of enzyme activity do not strongly correlate with formation of inter-subunit adducts by these HMGCL mutants. C170S, C266S, and C323S proteins do not form inter-subunit disulfide adducts but such an adduct is restored in the C170S/C174S double mutant. Coexpression of HMGCL proteins encoded by C266S and C323S expression plasmids supports formation of a C266S/C323S heterodimer which does form a covalent inter-subunit adduct. These observations are interpreted in the context of competition between cysteines in formation of intra-subunit and inter-subunit heterodisulfide adducts.     

基于结构B因子分析指导的头孢菌素C酰化酶动力学稳定性改造

【目的】筛选Pseudomonas sp.SE83 acy Ⅱ定点饱和突变库,获得动力学稳定性提高的头孢菌素C(CPC)酰化酶突变体,并对突变酶进行初步的结构-功能关系分析。【方法】靶标酶Pseudomonas sp.SE83 acy Ⅱ与Pseudomonas diminuta N176具有较高的同源性,通过分析N176的结构B因子,构建CPC酰化酶SE83定点饱和突变库;基于pH指示剂显色法,采用Biomek FX~P自动工作站建立CPC酰化酶高通量筛选方法,获得优良突变酶,对其活性、稳定性等酶学性质进行表征;利用SWISS-MODEL对突变体进行同源建模,探讨突变体结构与功能的关系。【结果】通过B因子分析和同源结构比对,共找出9个靶标位点;经过3轮筛选,发现R218及K226位点突变显著提高酶的热稳定性,其中最显著的R218Q和K226V在40°C的半衰期分别为野生型的3.77和2.77倍,催化效率k_(cat)/K_m分别为野生型的1.8和3.1倍。同源建模分析表明氢键作用和疏水相互作用的增加可能是突变体稳定性提高的原因。【结论】B因子指导的酶分子改造是一种高效可靠的动力学稳定性改造策略,突变体R218Q和K226V均可提高CPC酰化酶的稳定性和催化效率,对进一步的CPC酰化酶分子改造具有一定的参考价值和指导意义。     

Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant

Mammalian xanthine dehydrogenase can be converted to xanthine oxidase by modification of cysteine residues or by proteolysis of the enzyme polypeptide chain. Here we present evidence that the Cys(535) and Cys(992) residues of rat liver enzyme are indeed involved in the rapid conversion from the dehydrogenase to the oxidase. The purified mutants C535A and/or C992R were significantly resistant to conversion by incubation with 4,4'-dithiodipyridine, whereas the recombinant wild-type enzyme converted readily to the oxidase type, indicating that these residues are responsible for the rapid conversion. The C535A/C992R mutant, however, converted very slowly during prolonged incubation with 4,4'-dithiodipyridine, and this slow conversion was blocked by the addition of NADH, suggesting that another cysteine couple located near the NAD(+) binding site is responsible for the slower conversion. On the other hand, the C535A/C992R/C1316S and C535A/C992R/C1324S mutants were completely resistant to conversion, even on prolonged incubation with 4,4'-dithiodipyridine, indicating that Cys(1316) and Cys(1324) are responsible for the slow conversion. The crystal structure of the C535A/C992R/C1324S mutant was determined in its demolybdo form, confirming its dehydrogenase conformation.     

Amino acid residues 62 and 193 play the key role in regulating the synergism of substrate binding in oyster arginine kinase

The purpose of this study is to clarify the amino acid residues responsible for the synergism in substrate binding of arginine kinase (AK), a key enzyme in invertebrate energy metabolism. AKs contain a pair of highly conserved amino acids (D62 and R193) that form an ion pair, and replacement of these residues can cause a pronounced loss of activity. Interestingly, in the oyster Crassostrea AK, these residues are replaced by an N and a K, respectively. Despite this replacement, the enzyme retains high activity and moderate synergism in substrate binding (Kd/Km=2.3). We replaced the N62 by G or D and the K193 by G or R in Crassostrea AK, and also constructed the double mutants of N62G/K193G and N62D/K193R. All of the mutants retained 50-90% of the wild-type activity. In N62G and N62D mutants, the Kmarg for arginine binding was comparable to that of wild-type enzyme, but the Kdarg was increased 2-5-fold, resulting in a strong synergism (Kd/Km=4.9-11.3). On the other hand, in K193G and K193R mutants, the Kmarg was increased 4-fold, and synergism was lost almost completely (Kd/Km=1.0-1.4). The N62G/K193G double mutant showed similar characteristics to the K193G and K193R mutants. Another double mutant, N62D/K193R, similar to the amino acid pair in the wild-type enzyme, had characteristics similar to those of the wild-type enzyme. These results indicate that the amino acid residues 62 and 193 play the key role in mediating the synergism in substrate binding of oyster arginine kinase.