Purification and analysis of the structure of α-galactosidase from Escherichiacoli

Nagao, Yoshitaka; Nakada, Tetsuya; Imoto, Masanori; Shimamoto, Tadashi; Sakai, Shuzo; Tsuda, Masashi; Tsuchiya, Tomofusa

doi:10.1016/0006-291x(88)90584-0

Cited by 32 publications

(21 citation statements)

References 10 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The 13 C NMR data presented in Table V confirmed the findings from 1 H spectroscopy. In accord with previous analyses of various sugar phosphates (20,32,33), the most pronounced 13 C downfield shift was observed for the carbon carrying the phosphate ester. The marked downfield shift of 1.7-2.4 ppm recorded in Table V (column of data under "C-6") confirmed the terminal C-6 as the site of phosphorylation in all derivatives.…”

Section: Biosynthesis and Proof Of Structure Of The 6-p-␤-d-glucosidesupporting

confidence: 90%

“…2 Until recently, microbial phosphoglycosylhydrolases were found only in Family 1 of glycosylhydrolases. However, studies begun in our laboratory (9) and, subsequently, in other institutions, have identified a novel group of phosphoglucosylhydrolases in an increasing number of bacterial species, including Bacillus subtilis (10,11), Fusobacterium mortiferum (12), E. coli (13,14), Klebsiella pneumoniae (15), and Thermotoga maritima (16). By their inherent instability, oligomeric structure, sequence homology, and conserved signature pattern, these enzymes are assigned to Family 4 of glycosylhydrolases.…”

Section: His6mentioning

confidence: 99%

“…The differences found for the ␤-and ␥-carbons (C-5 and C-4, respectively) were comparatively small and have not been relied upon for structural assignments. Additional evidence for phosphorylation at O-6 in cellobiitol and the six ␤-D-glucosides is provided by the doublet splitting of the C-6 resonance with a coupling constant, 3 J(C,P), of about 4 -5 Hz that is, however, not detectable in the more complex 13 C data of the disaccharides.…”

Section: Biosynthesis and Proof Of Structure Of The 6-p-␤-d-glucosidementioning

confidence: 99%

See 2 more Smart Citations

β-Glucoside Kinase (BglK) from Klebsiella pneumoniae

Thompson

Lichtenthaler

Peters

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

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...

show abstract

Section: Biosynthesis and Proof Of Structure Of The 6-p-␤-d-glucosidesupporting

confidence: 90%

Section: His6mentioning

confidence: 99%

Section: Biosynthesis and Proof Of Structure Of The 6-p-␤-d-glucosidementioning

confidence: 99%

See 1 more Smart Citation

β-Glucoside Kinase (BglK) from Klebsiella pneumoniae

Thompson

Lichtenthaler

Peters

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…It has been reported that the E. coli ␣-galactosidase is active in its tetrameric form and it requires magnesium ions and NAD for activity (32). We observed that NADH can substitute NAD in this role (no activity was observed in the absence of NADH).…”

Section: Figmentioning

confidence: 50%

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

Mandrich¹,

Caputo²,

Martin³

et al. 2002

Journal of Biological Chemistry

View full text Add to dashboard Cite

Aes, a 36-kDa acetylesterase from Escherichia coli, belongs to the hormone-sensitive lipase family, and it is involved in the regulation of MalT, the transcriptional activator of the maltose regulon. The activity of MalT is depressed through a direct protein-protein interaction with Aes. Although the effect is clear-cut, the meaning of this interaction and the conditions that trigger it still remain elusive. To perform a comparative thermodynamic study between the mesophilic Aes protein and two homologous thermostable enzymes, Aes was overexpressed in E. coli and purified. At the last step of the purification procedure the enzyme was eluted from a Mono Q HR 5/5 column as a major form migrating, anomalously, at 56 kDa on a calibrated Superdex 75 column. A minor peak that contains the Aes protein and a polypeptide of 50 kDa was also detected. By a combined analysis of size-exclusion chromatography and surface-enhanced laser desorption ionization-time of flight mass spectrometry, it was possible to demonstrate the presence in this peak of a stable 87-kDa complex, containing the Aes protein itself and the 50-kDa polypeptide in a 1:1 ratio. The homodimeric molecular species of Aes and of the 50-kDa polypeptide were also detected. The esterase activity associated with the 87-kDa complex, when assayed with p-nitrophenyl butanoate as substrate, proved 6-fold higher than the activity of the major Aes form of 56 kDa. Amino-terminal sequencing highlighted that the 50-kDa partner of Aes in the complex was the ␣-galactosidase from E. coli. The E. coli cells harboring plasmid pT7-SCII-aes and, therefore, expressing Aes were hampered in their growth on a minimal medium containing raffinose as a sole carbon source. Because ␣-galactosidase is involved in the metabolism of raffinose, the above findings suggest a potential role of Aes in the regulation of carbohydrate metabolism in E. coli.The Escherichia coli aes (or ybac) gene encodes the 36-kDa cytoplasmic protein Aes (1), a carboxylesterase belonging to the hormone-sensitive lipase family (HSL) 1 (2). Although the physiological role of this enzyme is still obscure, it has recently received greater attention due to the discovery that Aes is involved in the regulation of the maltose regulon in E. coli (reviewed in Ref. 3). The E. coli maltose system consists of 10 genes encoding proteins for the uptake and metabolism of maltodextrin and maltose (3). MalT is the transcriptional activator of the maltose regulon and the prototype of a new family of transcription factors (4, 5) and integrates several signals through its amino-terminal three domains (6). To activate mal genes transcription MalT acts together with the cyclic AMPcatabolite protein complex, the inducer maltotriose, and ATP (7). Several regulatory circuits modulate the activity of MalT, e.g. MalT expression is regulated by Mlc, a glucose-inducible repressor (8). In addition, MalT is negatively controlled by at least three proteins: MalY, MalK, and Aes. MalY is an enzyme with cystathionine ␤-lyase activity (9, 10), which ...

show abstract

“…As reported for other members of this unusual family, MalH is inherently unstable and Mn# + and NAD + are prerequisite cofactors for activity (Nagao et al, 1988 ;Thompson et al, 1998Thompson et al, , 1999Raasch et al, 2000). Whether the nucleotide and metal ion fulfil catalytic or structural functions has not been ascertained for any member of family 4.…”

Section: Substrate Specificity and Hydrolysis Of Chromogenic Substratmentioning

confidence: 99%

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum

et al. 2002

View full text Add to dashboard Cite

Fusobacterium mortiferum utilizes sucrose [glucose-fructose in α(1 2) linkage] and its five isomeric α-D-glucosyl-D-fructoses as energy sources for growth.Sucrose-grown cells are induced for both sucrose-6-phosphate hydrolase (S6PH) and fructokinase (FK), but the two enzymes are not expressed above constitutive levels during growth on the isomeric compounds. Extracts of cells grown previously on the sucrose isomers trehalulose α(1 1), turanose α(1 3), maltulose α(1 4), leucrose α(1 5) and palatinose α(1 6) contained high levels of an NAD M plus metal-dependent phospho-α-glucosidase (MalH). The latter enzyme was not induced during growth on sucrose. MalH catalysed the hydrolysis of the 6'-phosphorylated derivatives of the five isomers to yield glucose 6-phosphate and fructose, but sucrose 6-phosphate itself was not a substrate. Unexpectedly, MalH hydrolysed both α-and β-linked stereomers of the chromogenic analogue p-nitrophenyl glucoside 6-phosphate. The gene malH is adjacent to malB and malR, which encode an EII(CB) component of the phosphoenolpyruvate-dependent sugar :phosphotransferase system and a putative regulatory protein, respectively. The authors suggest that for F. mortiferum, the products of malB and malH catalyse the phosphorylative translocation and intracellular hydrolysis of the five isomers of sucrose and of related α-linked glucosides. Genes homologous to malB and malH are present in both Klebsiella pneumoniae and the enterohaemorrhagic strain Escherichia coli O157 :H7. Both these organisms grew well on sucrose, but only K. pneumoniae exhibited growth on the isomeric compounds.

show abstract

Purification and analysis of the structure of α-galactosidase from Escherichiacoli

Cited by 32 publications

References 10 publications

β-Glucoside Kinase (BglK) from Klebsiella pneumoniae

β-Glucoside Kinase (BglK) from Klebsiella pneumoniae

The Aes Protein and the Monomeric α-Galactosidase fromEscherichia coli Form a Non-covalent Complex

Metabolism of sucrose and its five isomers by Fusobacterium mortiferum

Contact Info

Product

Resources

About