Versicolorin B synthase:

مرکز و کتابخانه مطالعات اسلامی به زبان های اروپایی

منو

" Versicolorin B synthase: "
J. C. Silva

Document Type	:	Latin Dissertation
Language of Document	:	English
Record Number	:	1113421
Doc. No	:	TLpq304242433
Main Entry	:	J. C. Silva
Title & Author	:	Versicolorin B synthase:\ J. C. Silva
College	:	The Johns Hopkins University
Date	:	1996
student score	:	1996
Degree	:	Ph.D.
Page No	:	305
Abstract	:	Versicolorin B synthase (VBS) catalyzes the dehydrative cyclization of ()-versiconal hemiacetal (24) to the bisfuran ring system of ()-versicolorin B (25), an essential transformation in the aflatoxin biosynthesis by Aspergillus parasiticus owing to the mutagenic nature of aflatoxin B (1). The purification of native VBS facilitated the isolation of both cDNA and genomic clones of VBS to give a 1985 base genomic vbs DNA sequence interrupted by one intron of 53 nucleotides. Southern blotting, nucleotide sequencing and detailed restriction mapping of these clones supported the theory that the aflatoxin B biosynthetic genes in A. parasiticus are clustered. VBS, which shows 58% similarity and 38% identity with glucose oxidase from A. niger, possesses significant homology to the signature sequence of the GMC oxidoreductase family. The enzyme behaved as a dimer under non-denaturing conditions and migrated with usdM\sb{r}usd 78,000 under SDS-PAGE conditions. Comparisons to wild-type VBS by SDS-PAGE after PNGase F treatment revealed that extensive glycosylation accounted for the mass discrepency (7000 1500) between the native and inactive, bacterially-expressed VBS. Several expression systems in Saccharomyces cerevisiae were surveyed; one that incorporated a secretion signal (suc2) was found to be most successful. Yeast-derived VBS, of indistinguishable mass and kinetic properties to the wild-type enzyme, could be obtained in 50-100 fold greater amounts. Analysis of its pH-rate and stability profile suggested catalytic roles for one or two acidic residues (pKa = 3.7 0.1) and a basic residue (pKa = 7.3 0.1), a finding supported by chemical modification experiments suggesting the involvement of both aspartate and/or glutamate and histidine. Over-production of VBS in yeast marks the first aflatoxin biosynthetic enzyme to be so obtained and opens the way to direct study of the enzymology of this complex pathway.
Subject	:	aflatoxin B
	:	Biochemistry
	:	Biological sciences
	:	Molecular biology
	:	Pure sciences
	:	sb1