Altering substrate specificity of phosphatidylcholine-preferring phospholipase C of Bacillus cereus by random mutagenesis of the headgroup binding site

PLC(Bc) is a 28.5 kDa monomeric enzyme that catalyzes the hydrolysis of the phosphodiester bond of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine to provide a diacylglycerol and the corresponding phosphorylated headgroup. Because single replacements of Glu4, Tyr56, and Phe66 in the headgroup binding pocket led to changes in substrate specificity [Martin et al. (2000) Biochemistry 39, 3410-3415], a combinatorial library of approximately 6000 maltose binding protein-PLC(Bc) fusion protein mutants containing random permutations of these three residues was generated to identify PLC(Bc) mutants with altered specificity profiles and high catalytic activities. Members of this library were screened for hydrolytic activity toward the water soluble substrates C6PC, C6PE, and C6PS using a novel protocol that was conducted in a 96-well format and featured the in situ cleavage of the fusion protein to release the mutant PLC(Bc)s. Ten mutant enzymes that exhibited significant preferences toward C6PE or C6PS were selected and analyzed by steady-state kinetics to determine their specificity constants, k(cat)/K(M). The C6PS selective clones E4G, E4Q/Y56T/F66Y, and E4K/Y56V exhibited higher specificity constants toward C6PS than wt, whereas Y56T, F66Y, and Y56T/F66Y were C6PE selective and had comparable or higher specificity constants than wt for C6PE. The corresponding wt residues were singly reinserted back into the E4Q/Y56T/F66Y and E4K/Y56V mutants via site-directed mutagenesis, and the E4Q/F66Y mutant thus obtained exhibited a 10-fold higher specificity constant toward C6PS than wt, a value significantly higher than other PLC(Bc) mutants. On the basis of available data, an aromatic residue at position 66 appears important for significant catalytic activity toward all three substrates, especially C6PC and C6PE. The charge of residue 4 also appears to be a determinant of enzyme specificity as a negatively charged residue at this position endows the enzyme with C6PC and C6PE preference, whereas a polar neutral or positively charged residue results in C6PS selectivity. Replacing Tyr56 with Val, Ala, Thr, or Ser greatly reduces activity toward C6PC. Thus, the substrate specificity of PLC(Bc) can be modulated by varying three of the amino acid residues that constitute the headgroup binding pocket, and it is now apparent that this enzyme is not evolutionarily optimized to hydrolyze phospholipids with ethanolamine or serine headgroups.     

Roles for cysteine residues in the regulatory CXXC motif of human mitochondrial branched chain aminotransferase enzyme

The redox-active dithiol/disulfide C315-Xaa-Xaa-C318 center has been implicated in the regulation of the human mitochondrial branched chain aminotransferase isozyme (hBCATm) [Conway, M. E., Yennawar, N., Wallin, R., Poole, L. B., and Hutson, S. M. (2002) Biochemistry 41, 9070-9078]. To explore further the mechanistic details of this CXXC center, mutants of the Cys residues at positions 315 and 318 of hBCATm were individually and in combination converted to alanine or serine by site-directed mutagenesis (C315A, C315S, C318A, C318S, C315/318A, and C315/318S). The effects of these mutations on cofactor absorbance, secondary structures, steady-state kinetics, and sensitivity toward hydrogen peroxide (H(2)O(2)) treatment were examined. Neither the UV-visible spectroscopic studies nor the circular dichroism data showed any major perturbations in the structure of the mutants. Kinetic analyses of the CXXC mutant proteins indicated primarily a modest reduction in k(cat) with no significant change in K(m). The largest effect on the steady-state kinetics was observed with the C315 single mutants, in which substitution of the thiol group resulted in a reduced k(cat) (to 26-33% of that of wild-type hBCATm). Moreover, the C315 single mutants lost their sensitivity to oxidation by H(2)O(2). The kinetic parameters of the C318 mutants were largely unaffected by the substitutions, and as with wild-type hBCATm, reaction of the C318A mutant protein with H(2)O(2) resulted in the complete loss of activity. In the case of oxidized C318A, this loss was largely irreversible on incubation with dithiothreitol. Mass spectrometry and dimedone modification results revealed overoxidation of the thiol group at position 315 to sulfonic acid occurring via a sulfenic acid intermediate in the H(2)O(2)-treated C318A enzyme. Thus, C315 appears to be the sensor for redox regulation of BCAT activity, whereas C318 acts as the "resolving cysteine", allowing for reversible formation of a disulfide bond.     

Properties of Cys21-mutated muscle acylphosphatases

Cys21 is an invariant residue in muscle acylphosphatases, but is absent in the erythrocyte isozymes. To assess the importance of this residue in the muscle isozymes for catalytic, structural, and stability properties, two gene mutants have been prepared by oligonucleotide-directed mutagenesis and expressed inEscherichia coli cells; in these mutants, the codon for Cys21 was replaced by those for Ser and Ala, respectively. The two mutant enzymes, purified by immunoaffinity chromatography, showed kinetic and structural properties similar to those of the wild-type recombinant enzyme; however, the specific activity of the two mutants, especially that of the C21A mutant, was lower. The urea and thermal stabilities of the mutant enzymes were reduced with respect to those of the wild-type form, contrary to the susceptibility to inactivation by mercuric ions. The reported data support the possibility that Cys21 is involved in the stabilization of the enzyme active-site conformation.     

Stability of wild-type and mutant RTEM-1 beta-lactamases: effect of the disulfide bond

Uniquely among class A beta-lactamases, the RTEM-1 and RTEM-2 enzymes contain a single disulfide bond between Cys 77 and Cys 123. To study the possible role of this naturally occurring disulfide in stabilizing RTEM-1 beta-lactamase and its mutants at residue 71, this bond was removed by introducing a Cys 77----Ser mutation. Both the wild-type enzyme and the single mutant Cys 77----Ser confer the same high levels of resistance to ampicillin in vivo to Escherichia coli; at 30 degrees C the specific activity of purified Cys 77----Ser mutant is also the same as that of the wild-type enzyme. Also, neither wild-type enzyme nor the Cys 77----Ser mutant is inactivated by brief exposure to p-hydroxymercuribenzoate. However, above 40 degrees C the mutant enzyme is less stable than wild-type enzyme. After introduction of the Cys 77----Ser mutation, none of the double mutants (containing the second mutations at residue 71) confer resistance to ampicillin in vivo at 37 degrees C; proteins with Ala, Val, Leu, Ile, Met, Pro, His, Cys, and Ser at residue 71 confer low levels of resistance to ampicillin in vivo at 30 degrees C. The use of electrophoretic blots stained with antibodies against beta-lactamase to analyze the relative quantities of mutant proteins in whole-cell extracts of E. coli suggests that all 19 of the doubly mutant enzymes are proteolyzed much more readily than their singly mutant analogues (at Thr 71) that contain a disulfide bond.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutational analysis of residue roles in AraC function

The previously isolated hemiplegic, induction-negative, repression-positive mutants, H80R and Y82C, were found to be defective in the binding of arabinose. Randomization of other residues close to arabinose in the three-dimensional structure of AraC or that make strong interactions with arabinose yielded induction-negative, repression-positive mutants. The induction and repression properties of mutants obtained by randomizing individual residues of the N-terminal arm of AraC allowed identification of the domain with which that residue very likely makes its predominant interactions. Residues 8-14 of the arm appear to make their predominant interaction with the DNA-binding domain. Although the side-chain of residue 15 interacts directly with arabinose bound to the N-terminal dimerization domain, the properties of mutant F15L indicate that this mutation increases the affinity of the arm for the DNA-binding domain.     

Homology modeling of the structure of tobacco acetohydroxy acid synthase and examination of the active site by site-directed mutagenesis

A reliable model of tobacco acetohydroxy acid synthase (AHAS) was obtained by homology modeling based on a yeast AHAS X-ray structure using the Swiss-Model server. Conserved residues at the dimer interface were identified, of which the functional roles of four residues, namely H142, E143, M489, and M542, were determined by site-directed mutagenesis. Eight mutants were successfully generated and purified, five of which (H142T, M489V, M542C, M542I, and M542V) were found to be inactive under various assay conditions. The H142K mutant was moderately altered in all kinetic parameters to a similar extent. In addition, the mutant was more thermo-labile than wild type enzyme. The E143A mutant increased the Km value more than 20-fold while other parameters were not significantly changed. All mutations carried out on residue M542 inactivated the enzyme. Though showing a single band on SDS-PAGE, the M542C mutant lost its native tertiary structure and was aggregated. Except M542C, each of the other mutants showed a secondary structure similar to that of wild type enzyme. Although all the inactive mutants were able to bind FAD, the mutants M489V and M542C showed a very low affinity for FAD. None of the active mutants constructed was strongly resistant to three tested herbicides. Taken together, the results suggest that the residues of H142, E143, M489, and M542 are essential for catalytic activity. Furthermore, it seems that H142 residue is involved in stabilizing the dimer interaction, while E143 residue may be involved in binding with substrate pyruvate. The data from the site-directed mutagenesis imply that the constructed homology model of tobacco AHAS is realistic.     

Cysteine-scanning analysis of the nucleobase-ascorbate transporter signature motif in YgfO permease of Escherichia coli: Gln-324 and Asn-325 are essential, and Ile-329-Val-339 form an alpha-helix

The nucleobase-ascorbate transporter (NAT) signature motif is a conserved sequence motif of the ubiquitous NAT/NCS2 family implicated in defining the function and selectivity of purine translocation pathway in the major fungal homolog UapA. To analyze the role of NAT motif more systematically, we employed Cys-scanning mutagenesis of the Escherichia coli xanthine-specific homolog YgfO. Using a functional mutant devoid of Cys residues (C-less), each amino acid residue in sequence (315)GSIPITTFAQNNGVIQMTGVASRYVG(340) (motif underlined) was replaced individually with Cys. Of the 26 single-Cys mutants, 16 accumulate xanthine to > or =50% of the steady state observed with C-less YgfO, 4 accumulate to low levels (10-25% of C-less), F322C, N325C, and N326C accumulate marginally (5-8% of C-less), and P318C, Q324C, and G340C are inactive. When transferred to wild type, F322C(wt) and N326C(wt) are highly active, but P318G(wt), Q324C(wt), N325C(wt), and G340C(wt) are inactive, and G340A(wt) displays low activity. Immunoblot analysis shows that replacements at Pro-318 or Gly-340 are associated with low or negligible expression in the membrane. More extensive mutagenesis reveals that Gln-324 is critical for high affinity uptake and ligand recognition, and Asn-325 is irreplaceable for active xanthine transport, whereas Thr-332 and Gly-333 are important determinants of ligand specificity. All single-Cys mutants react with N-ethylmaleimide, but regarding sensitivity to inactivation, they fall to three regions; positions 315-322 are insensitive to N-ethylmaleimide, with IC(50) values > or =0.4 mM, positions 323-329 are highly sensitive, with IC(50) values of 15-80 microM, and sensitivity of positions 330-340 follows a periodicity, with mutants sensitive to inactivation clustering on one face of an alpha-helix.     

Role of cysteine62 in DNA recognition by the P50 subunit of NF-kappa B.

A powerful chemical modification procedure has been developed to define determinants of DNA recognition by the p50 subunit of NF-kappa B. Differential labelling with [14C] iodoacetate has identified a conserved cysteine residue, Cys62, that was protected from modification by the presence of an oligonucleotide containing the specific recognition site of the protein. To determine the importance of this cysteine residue, each of the conserved cysteines in p50 was changed to serine and the DNA binding properties of the mutant proteins determined. Scatchard analysis indicated that the C62S mutant bound to its DNA recognition site with a 10-fold larger dissociation constant than the wild type protein, while the other two mutants bound with an intermediate affinity. Dissociation rate constant measurements correlated well with the dissociation constants for the wild type, C119S, and C273S p50 proteins, whereas the p50 C62S-DNA complex dissociated anomalously quickly. Competition analyses with oligonucleotide variants of the DNA recognition site and nonspecific E. coli DNA revealed that the C62S p50 mutant had an altered DNA binding site specificity and was impaired in its ability to discriminate between specific and non-specific DNA. Thus the sulphydryl group of Cys62 is an important determinant of DNA recognition by the p50 subunit of NF-kappa B.     

Site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase from Paenibacillus macerans to enhance substrate specificity towards maltodextrin for enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G)

In this work, the site-saturation engineering of lysine 47 in cyclodextrin glycosyltransferase (CGTase) from Paenibacillus macerans was conducted to improve the specificity of CGTase towards maltodextrin, which can be used as a cheap and easily soluble glycosyl donor for the enzymatic synthesis of 2-O-d-glucopyranosyl-l-ascorbic acid (AA-2G) by CGTase. When using maltodextrin as glycosyl donor, four mutants K47F (lysine→ phenylalanine), K47L (lysine→ leucine), K47V (lysine→ valine) and K47W (lysine→ tryptophan) showed higher AA-2G yield as compared with that produced by the wild-type CGTase. The transformation conditions (temperature, pH and the mass ratio of l-ascorbic acid to maltodextrin) were optimized and the highest titer of AA-2G produced by the mutant K47L could reach 1.97 g/l, which was 64.2 % higher than that (1.20 g/l) produced by the wild-type CGTase. The reaction kinetics analysis confirmed the enhanced maltodextrin specificity, and it was also found that compared with the wild-type CGTase, the four mutants had relatively lower cyclization activities and higher disproportionation activities, which was favorable for AA-2G synthesis. The mechanism responsible for the enhanced substrate specificity was further explored by structure modeling and it was indicated that the enhancement of maltodextrin specificity may be due to the short residue chain and the removal of hydrogen bonding interactions between the side chain of residue 47 and the sugar at ?3 subsite. Here the obtained mutant CGTases, especially the K47L, has a great potential in the production of AA-2G with maltodextrin as a cheap and easily soluble substrate.     

Key residues of loop 3 in the interaction with the interface residue at position 14 in triosephosphate isomerase from Trypanosoma brucei

Cysteine 14 is an interface residue that is fundamental for the catalysis and stability of homodimeric triosephosphate isomerase from Trypanosoma brucei (TbTIM). Its side chain is surrounded by a deep pocket of 11 residues that are part of loop 3 of the adjacent monomer. Mutation of this residue to serine (producing single mutant C14S) yields a wild-type-like enzyme that is resistant to the action of sulfhydryl reagents methylmethane thiosulfonate (MMTS) and 5,5-dithiobis(2-nitrobenzoate) (DTNB). This mutant enzyme was a starting point for probing by cysteine scanning the role of four residues of loop 3 in the catalysis and stability of the enzyme. Considering that the conservative substitution of either serine or alanine with cysteine would minimally alter the structure and properties of the environment of the residue in position 14, we made double mutants C14S/A69C, C14S/S71C, C14S/A73C, and C14S/S79C. Three of these double mutants were similar in their kinetic parameters to wild-type TbTIM and the single mutant C14S, but double mutant C14S/A73C showed a greatly reduced k cat. All enzymes had similar CD spectra, but all mutants had thermal stabilities lower than that of wild-type TbTIM. Intrinsic fluorescence was also similar for all enzymes, but the double mutants bound up to 50 times more 1-anilino-8-naphthalene sulfonate (ANS) and were susceptible to digestion with subtilisin. The double mutants were also susceptible to inactivation by sulfhydryl reagents. Double mutant C14S/S79C exhibited the highest sensitivity to MMTS and DTNB, bound a significant amount of ANS, and had the highest sensitivity to subtilisin. Thus, the residues at positions 73 and 79 are critical for the catalysis and stability of TbTIM, respectively.     

Site-directed mutagenesis studies on a putative fifth iron ligand of mouse 8S-lipoxygenase: retention of catalytic activity on mutation of serine-558 to asparagine, histidine, or alanine

The reported crystal structures of plant and animal lipoxygenases (LOX) show that the nonheme iron in the catalytic domain is ligated by three histidines, the C-terminal isoleucine, and in certain structures also by a fifth iron ligand, an asparagine or histidine residue. Mouse 8-LOX and its homologues (e.g., human 15-LOX-2) are unique in having a serine in place of the usual Asn or His in this fifth position. To investigate the importance of the residue in mouse 8-LOX structure-function, the serine-558 was replaced by asparagine, histidine, or alanine using oligonucleotide-directed mutagenesis. Wild-type mouse 8-LOX and the mutant cDNAs were expressed in HeLa cells infected with vaccinia virus encoding T7 RNA polymerase and their relative lipoxygenase activities assessed by incubation with [14C]arachidonic acid or [14C]linoleic acid followed by HPLC analysis of the products. The Ser558Asn and Ser558His mutants had equivalent or greater activity than wild-type 8-LOX. They also exhibited some 15-LOX activity, indicating that small structural perturbations (in this case to a residue identical in mouse 8-LOX and its 15-LOX-2 homologues) can interchange the positional specificity of these closely related enzymes. Remarkably, the Ser558Ala mutant exhibited significant 8-LOX activity, indicating that this position is not an essential iron ligand in the enzyme. We conclude that mouse 8-LOX is catalytically competent with only four amino acid iron ligands, and that Ser-558 of the wild-type enzyme does not play an essential role in catalysis.     

Temperature-sensitive mutant of Bacillus subtilis glutamine synthetase obtained by random mutation

Random mutations were introduced into the B. subtilis glutamine synthetase gene by using nitrous acid, and a high temperature-sensitive mutant was selected. DNA sequencing of the restriction fragment containing the mutation revealed a single base-pair change resulting in the substitution of Leu 318 with Phe. The mutant enzyme was purified, and its kinetic and physical properties were characterized. The Mg2(+)-dependent activity and Mg2+ plus Mn2(+)-dependent activity of the mutant were less than 5% of those of the wild-type at 37 degrees C, and these activities decreased above 15 degrees C, whereas the Mn2(+)-dependent activity was nearly normal. Affinity of the mutant enzyme for glutamate was extremely decreased although the Km values for NH3 or ATP were almost the same as those of the wild-type. The mutant enzyme was more susceptible than the wild-type enzyme to digestion with chymotrypsin in the presence of glutamate, ATP, and Mg2+, although addition of glutamate, ATP, and Mn2+ completely protected both enzymes. These results and circular dichroism analyses suggested that Leu 318 is at the glutamate-binding site and that the substitution of Leu 318 for Phe reduces the ability of the enzyme to form the enzyme-substrate complex, probably supported by Mg2+.     

Highly conserved tyrosine 37 stabilizes desensitized states and restricts calcium permeability of ATP-gated P2X3 receptor

Tyrosine 37 in the first transmembrane (TM1) domain is highly conserved in ATP-gated P2X receptors suggesting its fundamental role. We tested whether Y37 contributes to the desensitization of P2X3 receptors, which is currently not well understood. By combining electrophysiological, imaging and modeling approaches, we studied desensitization of various Y37 P2X3 mutants and potential partners of Y37. Unlike the membrane current of the WT receptor, which desensitized in seconds, Y37A mutant current did not fully desensitize even after minutes-long applications of β,γ-meATP, α,β-meATP, ATP or 2MeS-ATP. The fractional calcium current was enhanced in the Y37A mutant. Y37F did not rescue the native P2X3 phenotype indicating a role for the hydroxyl group of Y37 for the WT receptor. Homology modeling indicated I318 or I319 in TM2 as potential partners for Y37 in the receptor closed state. We tested this hypothesis by creating a permanent interaction between the two residues via disulfide bond. Whereas single Y37C, I318C and I319C mutants were functional, the double mutants Y37C-I318C and Y37C-I319C were non-functional. Using a cyclic model of receptor operation, we suggest that the conserved tyrosine 37 links TM1 to TM2 of adjacent subunit to stabilize desensitized states and restricts calcium permeability through the ion channel.     

Lysine 183 and glutamic acid 157 of the TSH receptor: two interacting residues with a key role in determining specificity toward TSH and human CG

A naturally occurring mutation in the ectodomain of the TSH receptor (TSHr), K183R, has been described recently in a familial case of gestational hyperthyroidism. Hyperthyroidism was explained by the widening of the specificity of the mutant receptor toward human CG (hCG). In the present study, we attempted to understand in molecular terms the structure-phenotype relationships of this mutant in light of the available structural model of TSHr ectodomain established on the template of the atomic structure of the porcine ribonuclease inhibitor. To this aim, we studied by site-directed mutagenesis and functional assays in transfected COS cells the effects of substituting amino acids with different physicochemical properties for lysine 183. Unexpectedly, all TSHr mutants displayed widening of their specificity toward hCG. Molecular dynamics simulations suggested that the gain of function would be secondary to the release of a nearby glutamate residue (E157) from a salt bridge with K183. This hypothesis was supported by further site-directed mutagenesis experiments showing that the presence of an acidic residue in position 157, or in its vicinity, was required to observe the increase in sensitivity to hCG (an acidic residue in position 183 can partially fulfill the role of a free acidic residue in position 157 when tested on the background of a E157A mutant). Our results suggest also that additional natural mutations (especially K183M, N, or Q) in position 183 of TSHr are expected to be found in gestational hyperthyroidism.     

Isolation and Partial Characterization of Escherichia coli Mutants with Low Levels of Transfer Ribonucleic Acid Nucleotidyltransferase

To determine the function of the enzyme transfer ribonucleic acid (tRNA) nucleotidyltransferase in vivo, five mutants of Escherichia coli containing low levels of this enzyme were isolated. Since no selection procedure for such mutants existed, these strains were isolated by assay of large numbers of colonies from a heavily mutagenized stock. A procedure employing cells made permeable to tRNA and ATP was used to screen the large number of colonies required for the isolation. All the mutants contained less than 20% of the normal level of the AMP-incorporating activity of tRNA nucleotidyltransferase in extracts prepared by several methods, and the best mutant contained only about 2% of this activity. Three of the mutants also had equally low levels of the cytidine 5'-monophosphate-incorporating activity of the enzyme. Despite these low activities, the mutant strains displayed relatively normal growth characteristics at all temperatures examined. The enzyme in the mutant strains was not temperature sensitive, nor were any other abnormal biochemical properties detected. tRNA isolated from the mutant strains was missing significant amounts of its 3' terminal adenosine 5'-monophosphate residue, amounting to 10 to 15% in the best mutant. However, only small amounts of the terminal cytidine 5'-monophosphate residue were missing. The results indicate that tRNA nucleotidyltransferase is involved in some aspect of synthesis or repair of the 3' terminus of tRNA, and that the enzyme is present in large excess over its requirements for this function.     

Complex of an active mu-opioid receptor with a cyclic peptide agonist modeled from experimental constraints

Site-directed mutagenesis and design of Zn(2+)-binding centers have been used to determine a set of specific tertiary interactions between the mu-opioid receptor, a rhodopsin-like G protein-coupled receptor (GPCR), and its cyclic peptide agonist ligand, Tyr(1)-c(S-Et-S)[d-Cys(2)-Phe(3)-d-Pen(4)]NH(2) (JOM6). The binding affinity of the tetrapeptide is strongly dependent on the nature of its first and third residues and on substitutions at positions 213, 216, 237, 300, 315, and 318 of the mu-opioid receptor. His(1) and His(3) analogues of the ligand were able to form metal-binding complexes with the V300C and G213C/T315C receptor mutants, respectively. Direct contact of the Phe(3) residue of JOM6 with Gly(213), Asp(216), Thr(315), and Trp(318) of the receptor was suggested by the binding affinities of His(3)-, Nle(3)-, Leu(3)-, Aci(3)-, Delta(E)Phe(3)-, and Delta(Z)Phe(3)-substituted peptides with the G213C/T315C, D216V, T315C, and W318L mutants. The improved binding affinity of the free carboxylate analogue of JOM6 for binding to the E229D mutant revealed an interaction between the C-terminal group of the peptide and Glu(229) of the receptor. The experimental constraints that were obtained were applied for distance geometry modeling of the mu-receptor in complex with the tetrapeptide agonist ligand, JOM6. The active conformation of the opioid receptor was calculated using the crystal structure of "inactive" rhodopsin and published engineered and intrinsic metal-binding sites and disulfide bonds that allow or facilitate activation of GPCRs. Interhelical H-bonds existing in the mu-receptor were applied as additional distance constraints. The calculated model of the receptor-ligand complex can serve as a prototype of the active state for all rhodopsin-like GPCRs. It displays a strongly shifted transmembrane helix 6 (TM6) and reorientation of the conserved Trp(293) residue in TM6 upon its interaction with the agonist. Importantly, the binding pockets of the active and inactive states are not identical, which implies distinct interaction modes of agonists and antagonists. In the active state, the binding pocket of the mu-receptor is complementary to the previously proposed receptor-bound conformation of JOM6.     

Involvement in K+ access of Leu318 at the extracellular domain flanking M3 and M4 of the Na+,K+-ATPase alpha-subunit

The effect of point mutation in the sequence 316TWLE319, which occurs in the extracellular loop flanking the third (M3) and the fourth (M4) transmembrane segment (L3/4) of the Na+,K+-ATPase alpha-subunit, was examined. Mutation of Glu319 to Asp yielded an enzyme with full activity, whereas substituting Glu319 to Ala resulted in a severe loss of activity. A negative charge was introduced along the sequence, one residue at a time, from Thr316 to Leu318 (by E-scanning) in the mutant construct with Glu319 already mutated to Gln. The activity that had been reduced to 60% by the mutation of Glu319 to Gln was restored upon the introduction of a negative charge by E-scanning. When Leu318 was replaced by Glu in a series of scanning experiments, the K+ sensitivity of the ATPase activity was lowered. The lowering of K+ sensitivity was further demonstrated when a mutation of Leu318 to Glu was introduced into the wild-type enzyme. Furthermore, mutants with Leu318 to Gln, Arg, and Phe displayed lower K+ sensitivity similar to that of Leu318 to Glu mutant. Leu318 may be in access path for K+, and any substitution at this position may interfere with access of K+ from outside the cell.     

Mutation of fungal endoglucanases into glycosynthases and characterization of their acceptor substrate specificity

Humicola insolens mutant Cel7B E197A is a powerful endo-glycosynthase displaying an acceptor substrate specificity restricted to β-d-glucosyl, β-d-xylosyl, β-d-mannosyl and β-d-glucosaminyl in +1 subsite. Our aim was to extend this substrate specificity to β-d-N-acetylglucosaminyl, in order to get access to a wider array of oligosaccharidic structures obtained through glycosynthase assisted synthesis. In a first approach a trisaccharide bearing a β-d-N-acetylglucosaminyl residue was docked at the +1 subsite of H. insolens Cel7B, indicating that the mutation of only one residue, His209, could lead to the expected wider acceptor specificity. Three H. insolens Cel7B glycosynthase mutants (H209A, H209G and H209A/A211T) were produced and expressed in Aspergillus oryzae. In parallel, sequence alignment investigations showed that several cellulases from family GH7 display an alanine residue instead of histidine at position 209. Amongst them, Trichoderma reesei Cel7B, an endoglucanase sharing the highest degree of sequence identity with Humicola Cel7B, was found to naturally accept a β-d-N-acetylglucosaminyl residue at +1 subsite. The T. reesei Cel7B mutant nucleophile E196A was produced and expressed in Saccharomyces cerevisiae, and its activity as glycosynthase, together with the H. insolens glycosynthase mutants, was evaluated toward various glycosidic acceptors.     

重组具有改良特性的D-氨基酸氧化酶

在大肠杆菌细胞中表达三角酵母D-氨基酸氧化酶, 并对重组酶的性质进行了研究。制备的单一突变体与野生型酶相比, 具有2.4倍的热稳定性或底物特异性变化光谱。结果显示突变的TvDAAO在氧化头孢菌素中催化效果优于野生型酶。并将一个突变的重组TvDAAO制备成结晶, 并解析了2.8 ?分辨率下的晶体结构。     

Cysteine-scanning mutagenesis around transmembrane segment VI of Tn10-encoded metal-tetracycline/H(+) antiporter

Each amino acid in putative transmembrane helix VI and its flanking regions, from Ser-156 to Thr-185, of a Cys-free mutant of the Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) was individually replaced by Cys. All of the cysteine-scanning mutants showed a normal level of tetracycline resistance except for the S156C mutant, which showed moderate resistance, indicating that there is no essential residue located in this region. All 20 mutants from S159C to W178C showed no reactivity with N-ethylmaleimide (NEM), whereas the mutants of the flanking regions from S156C to H158C and F179C to T185C were highly or moderately reactive with NEM. These results indicate that like transmembrane helices III and IX, the transmembrane helix VI comprising residues Ser-159-Trp-178 is totally embedded in the hydrophobic environment.