The α-galactosidase from Escherichia coli K12

Burstein, Claude; Képès, Adam

doi:10.1016/0304-4165(71)90053-5

Cited by 53 publications

(35 citation statements)

References 17 publications

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“…The a-galactosidase exhibited higher activity in phosphate than in Tris/HCl buffer, activity at pH 7-5 being 1.8 times greater in phosphate than Tris. In contrast to the E. coli a-galactosidase (Burstein & Kepes, 1971), there were no discernible differences in agalactosidase activity between extensively dialysed and non-dialysed cell-free enzyme preparations. Furthermore, addition of 10mM-EDTA had no effect.…”

Section: T a T G A T H T G T T T T C T T A C T T A A C~c~c A C G A T mentioning

confidence: 58%

Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Aduse-Opoku

Tao

Ferretti

et al. 1991

Journal of General Microbiology

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~ ~ ~~The aga gene coding for a-galactosidase in Streptococcus mutans was detected in a recombinant gene library constructed in phage A. The gene was subcloned into plasmid vectors and shown to specify a novel protein of M, 8OOOO. Characterization of a-galactosidase from S. mutans and from recombinant Escherichia coli expressing aga indicated that the enzyme functions as a tetramer. The amino acid composition of the a-galactosidase, deduced from nucleotide sequencing of aga, gave a predicted M, of 82022 and revealed regions of homology to agalactosidases encoded by the E. coli Raf plasmids and by Bacillus stearothermophilus. Inactivation of the aga gene in S. mutans resulted in loss of all a-galactosidase activity and abolished the ability to ferment melibiose; aglucosidase activity was also lost, due to an indirect effect on the dexB gene.

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Section: T a T G A T H T G T T T T C T T A C T T A A C~c~c A C G A T mentioning

confidence: 58%

Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Aduse-Opoku

Tao

Ferretti

et al. 1991

Journal of General Microbiology

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“…The suffices "ext" and "int" denote extracellular and intracellular niolecules, respectively. Suc, sucrose; Glc, glucose; Gal, galactose; Fru, fructose studies of the raffinose-induced a-galactosidase indicated properties different from the homologous E. coli enzyme specified by the me1 system [4,5]. These observations and the recent interest in the origin and nature of plasmid-borne metabolic functions [6-81 prompted a more extensive investigation of the new cc-galactosidase.…”

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confidence: 99%

Raffinose Metabolism in Escherichia coli K12

Schmid

Schmitt

1976

European Journal of Biochemistry

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The utilization by Escherichia coli K12 of raffinose as sole carbon source depends on a new raffinose transport system, an invertase and an a-galactosidase specified by the Raf-plasmid D 1021. The cc-galactosidase was purified to homogeneity from a mutant strain with constitutive synthesis of the enzyme. a-Galactosidase hydrolyzes p-nitrophenyl-a-D-galactoside ( K , 0.14 mM), methyl-a-11-galactoside ( K , 30 mM), melibiose ( K , 3.2 mM) and raffinose ( K , 60 mM). The enzymatic activity is strongly inhibited by Ag' , p-chloromercuriphenyl sulfonic acid and, to a lesser extent, by iodoacetamide. Isoelectric focusing indicates the existence of one form of a-galactosidase with an isoelectric point of 5.1. The purified enzyme has an sw,20 value of 11.7 k 0.3s and a molecular weight of 329000 k 4000; this value is not reduced at high dilutions. When examined by dodecylsulphate gel electrophoresis, purified a-galactosidase yields a single subunit band of molecular weight 82000 suggesting that the intact enzyme consists of four subunits. Amino acid analysis indicates the presence of approximately 712 amino acid residues per quarter molecule including 8 half-cystine residues. No carbohydrate moiety has been detected. High resolution electron micrographs and Markham rotation of a-galactosidase show enzyme molecules of approximately 11 x 11 nm containing foul-globular subunits in a tetragonal arrangement.The plasmid-coded a-galactosidase differs from the homologous E. coli enzyme by substrate affinities, cofactor requirements, stability and toluene resistance. It can, therefore, be used as a marker enzyme suitable for the detection in vivo of Raf-plasmids.In cells of Eschrrichia coli K12 the inducible functions of the me1 operon [l], namely melibiose permease and a-galactosidase, determine the utilization of a-galactosides such as melibiose (6-0-a-D-galactopyranosyl-D-ghcoside), melibiitol (6-0-a-Dgaiactopyranosyl-D-glucitol) and galactinol (6-0-a-D-galactopyranosyl-ueso-inositol). The trisaccharide raffinose (6-O-a-~-ga~actopyranosy~-~a-~-g~ucoside-2P-fructofuranoside) cannot be catabolized, since it is unable to induce specific transport or hydrolase activities.A recently discovered class of transmissible plasmids confers to E. coli the capacity to grow on raffinose as sole carbon source and to produce hydrogen sulphide Dedicated to Hans HerloK-lnhofen on the occasion of his 70th birthay.

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“…Requirement(s) for DTT and a Conserved Cysteine Residue for Enzyme Activity-In the presence of the requisite NAD ϩ and Mn 2ϩ cofactors, almost all GHF4 enzymes require a comparatively high concentration of DTT for optimum activity (17,20,21), but the role(s) of this reducing agent have yet to be defined (for discussion, see Ref. 19).…”

Section: Resultsmentioning

confidence: 99%

Genes malh and pagl of Clostridium acetobutylicum ATCC 824 Encode NAD+- and Mn2+-dependent Phospho-α-glucosidase(s)

Thompson

Hess

Pikis

2004

Journal of Biological Chemistry

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The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD ؉ , Mn 2؉ , and dithiothreitol-dependent phospho-␣-glucosidases that can be assigned to family 4 of the glycosylhydrolase superfamily. The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho-␣-glucosidase), respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system. C. acetobutylicum grows on a variety of ␣-linked glucosides, including maltose, methyl-␣-D-glucoside, and the five isomers of sucrose. In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl-␣-D-glucopyranoside 6-phosphate, but whether hydrolysis reflected expression of enzymes encoded by the malh or pagl genes was not discernible by spectrophotometric analysis or polyacrylamide gel electrophoresis. Resolution of this question required the cloning of the malh and pagl genes, and subsequent high expression, purification, and characterization of maltose-6 -phosphate hydrolase (MalH) and phospho-␣-glucosidase (PagL), respectively. MalH and PagL exhibit 50% residue identity, and in solution are tetramers comprising similar sized (ϳ50 kDa) subunits. The two proteins cross-react with polyclonal rabbit antibody against phospho-␣-glucosidase from Fusobacterium mortiferum. Purified MalH and PagL cleaved p-nitrophenyl-␣-D-glucopyranoside 6-phosphate with comparable efficiency, but only MalH catalyzed the hydrolysis of disaccharide 6 -phosphates formed via the phosphoenolpyruvate-dependent:sugar phosphotransferase system. Importantly, analysis of the proteome of C. acetobutylicum 824 by electrospray ionization-mass spectrometry confirmed expression of MalH during growth on many ␣-glucosides tested. Site-directed changes C169S and D170N yielded full-length, but catalytically inactive MalH. Of the two putative operons, our findings suggest that only proteins encoded by the mal operon participate in the dissimilation of maltose and related O-␣-linked glucosides by C. acetobutylicum 824.

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The α-galactosidase from Escherichia coli K12

Cited by 53 publications

References 17 publications

Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Biochemical and genetic analysis of Streptococcus mutans -galactosidase

Raffinose Metabolism in Escherichia coli K12

Genes malh and pagl of Clostridium acetobutylicum ATCC 824 Encode NAD+- and Mn2+-dependent Phospho-α-glucosidase(s)

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