6-Phospho-β-galactosidases of Gram-positive and 6-phospho-β-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the <i>lac</i>G gene of <i>staphylococcus aureus</i>

Witt, Ellen; Frank, Rainer; Hengstenberg, Wolfgang

doi:10.1093/protein/6.8.913

Cited by 36 publications

(34 citation statements)

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“…A licH mutant grew on all substrates tested (data not shown). Thus, it is possible that other ␤-glucosidases with similar substrate specificities take over the function of the licH gene product (58). Strain BGT5/6 derived from integration of plasmid pST11 containing an insertion of the neo gene in the unique HpaI site of licR (Fig.…”

Section: Resultsmentioning

confidence: 99%

Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis

et al. 1997

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A new catabolic system in Bacillus subtilis involved in utilization of ␤-glucosidic compounds has been investigated. It consists of five genes encoding phosphotransferase system (PTS) enzyme II (licB and licC) and enzyme IIA (licA), a presumed 6-phospho-␤-glucosidase (licH), as well as a putative regulator protein (licR). The genes map around 334؇ of the B. subtilis chromosome, and their products are involved in the uptake and utilization of lichenan degradation products. These five genes are organized in two transcriptional units. A weak promoter precedes gene licR, and transcription is obviously terminated at a secondary structure immediately downstream of the reading frame, as shown by Northern RNA blot analysis. Genes licB, licC, licA, and licH constitute an operon. Initiation of transcription at the promoter in front of this operon presumably requires activation by the gene product of licR. The LicR protein shows an unusual domain structure, i.e., similarities to (i) the conserved transcriptional antiterminator BglG family signature and (ii) PTS enzyme II. Using RNA techniques and transcriptional lacZ fusions, we have shown that the expression of the licBCAH operon is inducible by products of lichenan hydrolysis, lichenan and cellobiose. The presence of excess glucose prevents the induction of this operon, indicating the control by carbon catabolite repression. Moreover, the expression of the operon requires the general PTS components and seems to be negatively controlled by the specific lic PTS enzymes.

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Section: Resultsmentioning

confidence: 99%

Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis

et al. 1997

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“…Glutamate 372 corresponds with the catalytically active nucleophile of the glycosyl hydrolases from Agrobacterium sp. and Staphylococcus aureus, while glutamate 207 has been identified as the acidbase catalyst in the staphylococcal enzyme (39,40). Purification and characterization of the P. furiosus ␤-glucosidase overproduced in E. coli.…”

Section: Vol 177 1995 Expression and Mutation Of The P Furiosus Cementioning

confidence: 99%

“…Conserved residues are indicated by an asterisk below the alignment, while dots represent amino acids with the same characteristics. The arrows above the sequence indicate the conserved glutamate residues that have been identified as acid-base catalyst and active site nucleophile (39,40). The highly charged region (residue 273 to 288) found within the CelB sequence is overlined.…”

Section: Vol 177 1995 Expression and Mutation Of The P Furiosus Cementioning

confidence: 99%

“…To investigate whether the conserved glutamate at position 372 in CelB is the catalytically active nucleophile, it was replaced by an aspartate or a glutamine residue analogous to the approach used for glycosyl hydrolases of mesophilic bacteria (39,40). The resulting P. furiosus CelB mutants E372D and E372Q were produced in E. coli in slightly lower amounts than the wild-type CelB (data not shown).…”

Section: Vol 177 1995 Expression and Mutation Of The P Furiosus Cementioning

confidence: 99%

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Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

et al. 1995

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The celB gene encoding the cellobiose-hydrolyzing enzyme ␤-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation initiation sites of the celB gene have been determined, and archaeal control sequences were identified. The celB gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of ␤-glucosidase that could be purified by a two-step purification procedure. The ␤-glucosidase produced by E. coli had kinetic and stability properties similar to those of the ␤-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of ␤-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus ␤-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200-or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100؇C.The most extensively studied representative of the hyperthermophilic organisms that have an optimum growth temperature above 85ЊC is Pyrococcus furiosus (12). P. furiosus is able to grow on a wide range of substrates, including complex polymers such as starch, glycogen, peptone, and casein or simple carbon compounds like cellobiose, maltose, and pyruvate (12,20,33). The main fermentation products are CO 2 and H 2 or alanine, the latter acting as an alternative electron sink (22). The disaccharides cellobiose and maltose are hydrolyzed by intracellular glucosidases (5, 20). The generated glucose was proposed to be further metabolized via a nonphosphorylated Entner-Doudoroff pathway (27, 33). However, recently it was discovered that sugars are fermented by P. furiosus via an Embden-Meyerhof pathway that involves two ADP-dependent kinases (19).Characterization of proteins from hyperthermophiles revealed that they are extremely thermostable and may have an optimum temperature of catalysis that exceeds the maximum growth temperature of their host (1,18). In addition to their remarkable thermostability, proteins from hyperthermophiles are often found to be highly resistant to chemical denaturation and to degradation by proteases (13). One of the most thermostable enzymes identified up to now is the ␤-glucosidase from P. furiosus, with a half-life of 85 h at 100ЊC (20). During growth on cellobiose, ␤-glucosidase can make up to 5% of the total cell protein of P. furiosus and is involved in the hydrolysis of the ␤-1,4-glycosidic bond between the two glucose moieties of the disaccharide (20). In addition, ␤-glucosidases constitute a group of well-studied enzymes among members of all three domains of life, Eucarya, Bacteria, and Archaea (16,17,35). Therefore, the pyrococcal ␤-glucosidase is a suitable model enzyme for the molecular characterization of structure-function relations of hyperthermostable enzymes. To study these relations by protein eng...

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“…Hexose monophosphates may immediately enter the central energygenerating pathways, but prior to their dissimilation, accumulated disaccharide phosphates must first be hydrolyzed by sugar-specific disaccharide-phosphate hydrolases. Of the latter inducible enzymes, phospho-␤-galactosidase (EC 3.2.1.85) and phospho-␤-glucosidase (EC 3.2.1.86) have received considerable attention (6,7). By sequence-based alignment these enzymes are assigned to Family 1 of the 87-member superfamily of glycosylhydrolases (8).…”

Section: His6mentioning

confidence: 99%

β-Glucoside Kinase (BglK) from Klebsiella pneumoniae

Thompson

Lichtenthaler

Peters

et al. 2002

Journal of Biological Chemistry

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ATP-dependent ␤-glucoside kinase (BglK) has been purified from cellobiose-grown cells of Klebsiella pneumoniae. In solution, the enzyme (EC 2.7.1.85) exists as a homotetramer composed of non-covalently linked subunits of M r ϳ33,000. Determination of the first 28 residues from the N terminus of the protein allowed the identification and cloning of bglK from genomic DNA of K. pneumoniae. The open reading frame (ORF) of bglK encodes a 297-residue polypeptide of calculated M r 32,697. A motif of 7 amino acids (AFD 7 IG 9 GT) near the N terminus may comprise the ATP-binding site, and residue changes D7G and G9A yielded catalytically inactive proteins. BglK was progressively inactivated (t1 ⁄2 ϳ 19 min) by N-ethylmaleimide, but ATP afforded considerable protection against the inhibitor. By the presence of a centrally located signature sequence, BglK can be assigned to the ROK (Repressor, ORF, Kinase) family of proteins. Preparation of His6 BglK by nickel-nitrilotriacetic acid-agarose chromatography provided high purity enzyme in quantity sufficient for the preparative synthesis (200 -500 mg) of ten 6-phospho-␤-D-glucosides, including cellobiose-6-P, gentiobiose-6-P, cellobiitol-6-P, salicin-6-P, and arbutin-6-P. These (and other) derivatives are substrates for phospho-␤-glucosidase(s) belonging to Families 1 and 4 of the glycosylhydrolase superfamily. The structures, physicochemical properties, and phosphorylation site(s) of the 6-phospho-␤-D-glucosides have been determined by fast atom bombardmentnegative ion spectrometry, thin-layer chromatography, and 1 H and 13 C NMR spectroscopy. The recently sequenced genomes of two Listeria species, L. monocytogenes EGD-e and L. innocua CLIP 11262, contain homologous genes (lmo2764 and lin2907, respectively) that encode a 294-residue polypeptide (M r ϳ 32,200) that exhibits ϳ58% amino acid identity with BglK. The protein encoded by the two genes exhibits ␤-glucoside kinase activity and cross-reacts with polyclonal antibody to His6 BglK from K. pneumoniae. The location of lmo2764 and lin2907 within a ␤-glucoside (cellobiose):phosphotransferase system operon, may presage both enzymatic (kinase) and regulatory functions for the BglK homolog in Listeria species.The phosphoenolpyruvate-dependent sugar:phosphotransferase system (P-enolpyruvate:PTS), 1 was first described by Roseman and colleagues in Escherichia coli in 1964 (1). This multicomponent group translocation system is now recognized as the primary route for entry, and simultaneous phosphorylation, of carbohydrates by many bacterial species from both Gram-negative (2, 3) and Gram-positive genera (4, 5). Hexose monophosphates may immediately enter the central energygenerating pathways, but prior to their dissimilation, accumulated disaccharide phosphates must first be hydrolyzed by sugar-specific disaccharide-phosphate hydrolases. Of the latter inducible enzymes, phospho-␤-galactosidase (EC 3.2.1.85) and phospho-␤-glucosidase (EC 3.2.1.86) have received considerable attention (6, 7). By sequence-based alignment these enzymes...

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6-Phospho-β-galactosidases of Gram-positive and 6-phospho-β-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus

Cited by 36 publications

References 0 publications

Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis

Identification and characterization of a new beta-glucoside utilization system in Bacillus subtilis

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

β-Glucoside Kinase (BglK) from Klebsiella pneumoniae

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