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Titolo:
GALACTOFURANOSE BIOSYNTHESIS IN ESCHERICHIA-COLI K-12 - IDENTIFICATION AND CLONING OF UDP-GALACTOPYRANOSE MUTASE
Autore:
NASSAU PM; MARTIN SL; BROWN RE; WESTON A; MONSEY D; MCNEIL MR; DUNCAN K;
Indirizzi:
GLAXO WELLCOME MED RES CTR,GUNNELS WOOD RD STEVENAGE SG1 2NY HERTS ENGLAND GLAXO WELLCOME MED RES CTR STEVENAGE SG1 2NY HERTS ENGLAND COLORADO STATE UNIV,DEPT MICROBIOL FT COLLINS CO 80523
Titolo Testata:
Journal of bacteriology
fascicolo: 4, volume: 178, anno: 1996,
pagine: 1047 - 1052
SICI:
0021-9193(1996)178:4<1047:GBIEK->2.0.ZU;2-8
Fonte:
ISI
Lingua:
ENG
Soggetto:
CELL-WALL ARABINOGALACTAN; RNA-POLYMERASE; CLONED GENES; EXPRESSION; RESIDUES; BINDING;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
25
Recensione:
Indirizzi per estratti:
Citazione:
P.M. Nassau et al., "GALACTOFURANOSE BIOSYNTHESIS IN ESCHERICHIA-COLI K-12 - IDENTIFICATION AND CLONING OF UDP-GALACTOPYRANOSE MUTASE", Journal of bacteriology, 178(4), 1996, pp. 1047-1052

Abstract

We have cloned two open reading frames (orf6 and orf8) from the Escherichia coli K-12 rfb region, The genes were expressed in E, coli undercontrol of the T7lac promoter, producing large quantities of recombinant protein, most of which accumulated in insoluble inclusion bodies, Sufficient soluble protein was obtained, however, for use in a radiometric assay designed to detect UDP-galactopyranose mutase activity (theconversion of UDP-galactopyranose to UDP-galactofuranose). The assay is based upon high-pressure liquid chromatography separation of sugar phosphates released from both forms of UDP-galactose by phosphodiesterase treatment, The crude orf6 gene product converted UDP-[alpha-D-U-C-14]-galactopyranose to a product which upon phosphodiesterase treatment gave a compound with a retention time identical to that of syntheticalpha-galactofuranose-1-phosphate. No mutase activity was detected inextracts from cells lacking the orf6 expression plasmid or from orf8-expressing cells, The orf6 gene product was purified by anion-exchangechromatography and hydrophobic interaction chromatography. Both the crude extract and the purified protein converted 6 to 9% of the UDP-galactopyranose to the furanose form, The enzyme was also shown to catalyze the reverse reaction; in this case an approximately 86% furanose-to-pyranose conversion was observed, These observations strongly suggestthat orf6 encodes UDP-galactopyranose mutase (EC 5.4.99.9), and we propose that the gene be designated glf accordingly, Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified UDP-galactopyranosemutase revealed one major band, and analysis by electrospray mass spectrometry indicated a single major species with a molecular weight of 42,960 +/- 8, in accordance with that calculated for the Glf protein, N-terminal sequencing revealed that the first 15 amino acids of the recombinant protein corresponded to those expected from the published sequence, UV-visible spectra of purified recombinant enzyme indicated that the protein contains a flavin cofactor, which we have identified asflavin adenine dinucleotide.

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Documento generato il 16/07/20 alle ore 19:56:21