Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli I: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

Hilt, Wolfgang; Pfleiderer, Gerhard; Fortnagel, Peter

doi:10.1016/0167-4838(91)90281-4

Cited by 50 publications

(40 citation statements)

References 18 publications

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“…Also as a comparison, the K m value of WbpO Rf for UDPGalNAc at 7.79 mM is within the range of the reported values for UDP-Glc dehydrogenase (61,62) and glucose dehydrogenase (63,64). However, this value is relatively high when compared with those for UDP-GlcNAc dehydrogenase (0.28 mM, see Ref.…”

Section: Discussionmentioning

confidence: 70%

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Zhao¹,

Creuzenet²,

Bélanger³

et al. 2000

Journal of Biological Chemistry

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WbpO is associated with B-band lipopolysaccharide biosynthesis in Pseudomonas aeruginosa serotype O6. This protein is thought to catalyze the enzymatic conversion of UDP-N-acetyl-D-galactosamine (UDP-GalNAc) to UDP-N-acetyl-D-galactosaminuronic acid (UDP-GalNAcA). WbpO was overexpressed with a C-terminal hexahistidine tag. The soluble form of expressed WbpO (WbpO Sol ) exhibited a secondary structure with 29.2% ␣-helix and 20.1% ␤-strand. However, no enzymatic activity could be detected using either high performance anion exchange chromatography or capillary electrophoresis-mass spectrometry analysis. An insoluble form of expressed WbpO was purified in the presence of guanidine hydrochloride by immobilized metal ion affinity chromatography. After refolding, this preparation of WbpO (designated as WbpO Rf ) exhibited stable secondary structure at pH 7.5 to 8.2, and it was enzymatically active. Capillary electrophoresis-mass spectrometry and tandem mass spectrometry analysis showed that WbpO Rf catalyzed the conversion of UDP-GalNAc to UDP-GalNAcA. 26 and 22% of the substrate could be converted to UDP-GalNAcA in the presence of NAD ؉ and NADP ؉ as the cofactors, respectively. The K m values of WbpO Rf for UDP-GalNAc, NAD ؉ , and NADP ؉ were 7.79, 0.65, and 0.44 mM, respectively. WbpO Rf can also catalyze the conversion of UDP-GlcNAc to UDP-GlcNAcA. In conclusion, this is the first report of the overexpression, purification, and biochemical characterization of an NAD ؉ /NADP ؉ -dependent UDP-GalNAc dehydrogenase. Our results also complete the biosynthetic pathway for GalNAcA that is part of the O-antigen of P. aeruginosa serotype O6 lipopolysaccharide.

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Section: Discussionmentioning

confidence: 70%

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Zhao¹,

Creuzenet²,

Bélanger³

et al. 2000

Journal of Biological Chemistry

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“…1) was so-named because it encodes a putative D-glucose dehydrogenase that would be needed to make the D-glucuronic acid found in the ssGPLs of M. avium serovars 3 or 9 (Table 3). The deduced amino acid sequence demonstrated a high degree of similarity (69 and 65 %) to known D-glucose dehydrogenases of Bacillus subtilis (Hilt et al, 1991) and Pseudomonas aeruginosa (van Schie et al, 1985), respectively. The presence of this subcluster in the serovar 2 strains is anomalous because D-glucuronic acid is not found in the serovar-2-specific GPL.…”

Section: The Anomalous Ser3 § Gene Clustermentioning

confidence: 99%

Proposed pathway for the biosynthesis of serovar-specific glycopeptidolipids in Mycobacterium avium serovar 2

2003

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Members of the Mycobacterium avium complex are distinguished by the presence of highly antigenic surface molecules called glycopeptidolipids (GPLs) and the oligosaccharide portion of the serovar-specific GPL defines the 28 serovars. Previously, the genomic region (ser2) encoding the enzymes responsible for the glycosylation of the lipopeptide core to generate the serovar-2-specific GPLs has been described. In this work, the ser2 gene clusters of M. avium serovar 2 strains 2151 and TMC 724 were fully sequenced and compared to the homologous regions of M. avium serovar 1 strain 104, M. avium subsp. paratuberculosis and M. avium subsp. silvaticum. It was also determined that 104Rg, a mutant of strain 104 that produces truncated GPLs, lost several GPL biosynthesis genes by deletion. This comparison, together with analysis of protein similarities, supports a biosynthetic model in which serovar-2-specific GPLs are synthesized from a serovar-1-specific GPL intermediate that is derived from a non-specific GPL precursor. We also identified a gene encoding an enzyme that is necessary for the biosynthesis of serovar-3-and 9-specific GPLs, but not serovar-2-specific GPLs, suggesting that the different serovars may have evolved from the acquisition or loss of genetic information. In addition, a subcluster of genes for the biosynthesis and transfer of fucose, which are needed to make serovar-specific GPLs such as those of serovar 2, is found in the non-GPL-producing M. avium subspecies paratuberculosis and silvaticum.

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“…The purified enzymes from Bacillus cereus, B. megaterium, and B. subtilis have been characterized. [1][2][3] Amino acid sequence alignment has indicated that NAD(P) þ -dependent glucose dehydrogenases from these Bacillus species have more than 80% homology.…”

mentioning

confidence: 99%

Analysis of Bacterial Glucose Dehydrogenase Homologs from Thermoacidophilic ArchaeonThermoplasma acidophilum: Finding and Characterization of Aldohexose Dehydrogenase

Nishiya

Tamura

2004

Bioscience, Biotechnology, and Biochemistry

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Glucose dehydrogenase from Bacillus subtilis expressed in Escherichia coli I: purification, characterization and comparison with glucose dehydrogenase from Bacillus megaterium

Cited by 50 publications

References 18 publications

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

WbpO, a UDP-N-acetyl-d-galactosamine Dehydrogenase from Pseudomonas aeruginosa Serotype O6

Proposed pathway for the biosynthesis of serovar-specific glycopeptidolipids in Mycobacterium avium serovar 2

Analysis of Bacterial Glucose Dehydrogenase Homologs from Thermoacidophilic ArchaeonThermoplasma acidophilum: Finding and Characterization of Aldohexose Dehydrogenase

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