Site-directed mutagenic replacement of glu-461 with gln in β-galactosidase (E. coli): Evidence that glu-461 is important for activity

Bader, Dorian; Ring, Mark; Huber, R.E.

doi:10.1016/s0006-291x(88)81222-1

Cited by 25 publications

(8 citation statements)

References 7 publications

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“…Mutagenesis studies of E. coli lacZ have demonstrated that a glutamine substitution at position E461 or E537 inactivates the protein (2,5,6). The analogous missense mutations were introduced into PHR1 at codon 169 or 270, and the mutated genes were tested for their ability to complement the morphological defects of a phr1⌬ mutant.…”

Section: Relationship Between Phr1p and Glycosidases A Basicmentioning

confidence: 99%

PHR1 and PHR2 of Candida albicans Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans

Fonzi

1999

J Bacteriol

181

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PHR1 and PHR2 encode putative glycosylphosphatidylinositol-anchored cell surface proteins of the opportunistic fungal pathogen Candida albicans. These proteins are functionally related, and their expression is modulated in relation to the pH of the ambient environment in vitro and in vivo. Deletion of either gene results in a pH-conditional defect in cell morphology and virulence. Multiple sequence alignments demonstrated a distant relationship between the Phr proteins and β-galactosidases. Based on this alignment, site-directed mutagenesis of the putative active-site residues of Phr1p and Phr2p was conducted and two conserved glutamate residues were shown to be essential for activity. By taking advantage of the pH-conditional expression of the genes, a temporal analysis of cell wall changes was performed following a shift of the mutants from permissive to nonpermissive pH. The mutations did not grossly affect the amount of polysaccharides in the wall but did alter their distribution. The most immediate alteration to occur was a fivefold increase in the rate of cross-linking between β-1,6-glycosylated mannoproteins and chitin. This increase was followed shortly thereafter by a decline in β-1,3-glucan-associated β-1,6-glucans and, within several generations, a fivefold increase in the chitin content of the walls. The increased accumulation of chitin-linked glucans was not due to a block in subsequent processing as determined by pulse-chase analysis. Rather, the results suggest that the glucans are diverted to chitin linkage due to the inability of the mutants to establish cross-links between β-1,6- and β-1,3-glucans. Based on these and previously published results, it is suggested that the Phr proteins process β-1,3-glucans and make available acceptor sites for the attachment of β-1,6-glucans.

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Section: Relationship Between Phr1p and Glycosidases A Basicmentioning

confidence: 99%

PHR1 and PHR2 of Candida albicans Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans

Fonzi

1999

J Bacteriol

181

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“…However, studies with Escherichia coli ␤-galactosidase and human glucocerebrosidase have shown that the mechanism of inactivation by related conduritol epoxide compounds differs from the catalytic mechanisms of these two enzymes. For both glucocerebrosidase and ␤-galactosidase, the amino acid residue identified as the catalytic nucleophile by labeling with conduritol epoxides was later demonstrated to be incorrect by site-directed mutagenesis experiments (6,7). The introduction of 2-deoxy-2-fluoro glycoside inhibitors by Withers and co-workers (8,9) provided true mechanism-based inhibition that correctly identified the catalytic nucleophiles in both E. coli ␤-galactosidase and glucocerebrosidase, as well as numerous other configuration-retaining glycosidases (reviewed in Ref.…”

mentioning

confidence: 99%

Catalytic Mechanism and Specificity for Hydrolysis and Transglycosylation Reactions of Cytosolic β-Glucosidase from Guinea Pig Liver

Hays¹,

VanderJagt²,

Bose³

et al. 1998

Journal of Biological Chemistry

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Cytosolic ␤-glucosidase (CBG) from mammalian liver is known for its broad substrate specificity and has been implicated in the transformation of xenobiotic glycosides. CBG also catalyzes a variety of transglycosylation reactions, which have been been shown with other glycosylhydrolases to function in synthetic and genetic regulatory pathways. We investigated the catalytic mechanism, substrate specificity, and transglycosylation acceptor specificity of guinea pig liver CBG by several methods. These studies indicate that CBG employs a two-step catalytic mechanism with the formation of a covalent enzyme-sugar intermediate and that CBG will transfer sugar residues to primary hydroxyls and equatorial but not axial C-4 hydroxyls of aldopyranosyl sugars. Kinetic studies revealed that correction for transglycosylation reactions is necessary to derive correct kinetic parameters for CBG. Further analyses revealed that for aldopyranosyl substrates, the activation energy barrier is affected most by the presence of a C-6 carbon and by the configuration of the C-2 hydroxyl, whereas the binding energy is affected modestly by the configuration and substituents at C-2, C-4, and C-5. These data indicate that the transglycosylation activity of CBG derives from the formation of a covalently linked enzymesugar intermediate and that the specificity of CBG for transglycosylation reactions is different from its specificity for hydrolysis reactions.A distinguishing feature of the cytosolic ␤-glucosidase (CBG) 1 of mammalian liver (EC 3.2.1.21) is its broad substrate specificity. The enzyme hydrolyzes ␤-D-galactopyranosides, ␤-D-fucopyranosides, ␤-D-xylopyranosides, and ␣-L-arabinopyranosides, in addition to ␤-D-glucopyranosides (1). The enzyme also catalyzes transglycosylation reactions in which a sugar residue is transferred from a substrate molecule to an acceptor to form a new glycoside (2). These properties are consistent with the fact that CBG is a configuration-retaining glycosidase (3). Collectively, these data suggest that the catalytic mechanism of CBG consists of a double-displacement reaction involving the formation of a stable enzyme-sugar intermediate, as originally proposed for configuration-retaining glycosidases by Koshland (4).Additional evidence of a two-step catalytic mechanism for CBG was derived from studies performed with the inhibitor, Br-conduritol-␤-epoxide, which is an irreversible, active sitedirected inactivator of the enzyme (5). However, studies with Escherichia coli ␤-galactosidase and human glucocerebrosidase have shown that the mechanism of inactivation by related conduritol epoxide compounds differs from the catalytic mechanisms of these two enzymes. For both glucocerebrosidase and ␤-galactosidase, the amino acid residue identified as the catalytic nucleophile by labeling with conduritol epoxides was later demonstrated to be incorrect by site-directed mutagenesis experiments (6, 7). The introduction of 2-deoxy-2-fluoro glycoside inhibitors by Withers and co-workers (8, 9) provided true mechanism-based i...

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“…␤-Galactosidases (␤-D-galactoside galactohydrolase or lactase; EC 3.2.1.23) are widely distributed in nature. With regard to reaction mechanism (2,16,18) and three-dimensional structure (9), the most thoroughly investigated ␤-galactosidase is obtained from E. coli (pH optimum, pH 7), whereas the most significant industrial ␤-galactosidases originate from yeasts and several Aspergillus species (pH optima, between pH 4 and 5). These fungal ␤-galactosidases are applied in food technology for hydrolyzing the ␤- (1,4) linkage between galactose and glucose in lactose, the worldwide production of which is approximately 5.75 million metric tons per year (6).…”

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confidence: 99%

Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus

Fischer

Scheckermann

Wagner

1995

Appl Environ Microbiol

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A new inducible intracellular ␤-galactosidase (EC 3.2.1.23) of the thermophilic fungus Thermomyces lanuginosus was purified by fractional salt precipitation, hydrophobic interaction, and anion exchange chromatography. The first 22 amino acid residues were determined by N-terminal sequencing. Electrophoretic investigations revealed a dimeric enzyme with a molecular mass of 75 to 80 kDa per identical subunit and an isoelectric point of 4.4 to 4.5. The native ␤-galactosidase was identified as a glycoprotein by the enzyme-linked immunosorbent assay technique. The ␤-galactosidase activity was optimal at pH 6.7 to 7.2, and the enzyme displayed stability between pH 6 and 9. It was completely stable at pH 6.8 and 47؇C for 2 h. After 191 h at 50؇C, the remaining ␤-galactosidase activity of an enzyme fraction after salt precipitation was 58%. The ␤-galactosidase hydrolyzed p-and o-NO 2-phenyl-␤-D-galactopyranoside, lactose, lactulose, MeOH-␤-D-galactopyranoside, phenyl-␤-D-galactopyranoside, and p-NO 2-phenyl-␣-L-arabinopyranoside. The kinetic constants (K m) measured for p-and o-NO 2-phenyl-␤-D-galactopyranoside and ␤-lactose were 4.8, 11.3, and 18.2 mM, respectively.

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Site-directed mutagenic replacement of glu-461 with gln in β-galactosidase (E. coli): Evidence that glu-461 is important for activity

Cited by 25 publications

References 7 publications

PHR1 and PHR2 of Candida albicans Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans

PHR1 and PHR2 of Candida albicans Encode Putative Glycosidases Required for Proper Cross-Linking of β-1,3- and β-1,6-Glucans

Catalytic Mechanism and Specificity for Hydrolysis and Transglycosylation Reactions of Cytosolic β-Glucosidase from Guinea Pig Liver

Purification and characterization of a thermotolerant beta-galactosidase from Thermomyces lanuginosus

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