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Author Xiao, X. ♦ Wang, F. ♦ Saito, A. ♦ Majka, J. ♦ Schlösser, A. ♦ Schrempf, H.
Source SpringerLink
Content type Text
Publisher Springer-Verlag
File Format PDF
Copyright Year ©2002
Language English
Subject Domain (in DDC) Social sciences ♦ Sociology & anthropology
Abstract During cultivation in the presence of N-acetylglucosamine or chitin, Streptomyces olivaceoviridis mycelium efficiently takes up [14C]-labelled N-acetylglucosamine. Uptake of the labelled compound can be completely inhibited by unlabelled N-acetylglucosamine and partially by chitobiose. After extraction of the membrane with Triton X-100, two forms of a protein that binds to N-acetylglucosamine and N,N'-diacetylchitobiose (chitobiose) were purified to homogeneity by two consecutive rounds of anionic exchange chromatography. The protein was named NgcE. Using surface plasmon resonance, its binding parameters were determined. It showed highest affinity for N-acetylglucosamine (KD=8.28×10–9 M) and for chitobiose (KD=2.87×10–8 M). Varying equilibrium dissociation constants in the micromolecular range were ascertained for chitotetraose (KD=4.5×10–6 M), chitopentaose (KD=1.03×10–6 M) and chitohexaose (KD=3.02×10–6 M); the lowest value was measured for chitotriose (KD=19.4×10–6 M). After having determined the sequences of several internal peptides from the binding protein by Edman degradation, the corresponding ngcE gene, which encodes a predicted lipid-anchored protein, was identified by reverse genetics. Using a genomic phage library of S. olivaceoviridis genes encoding two other membrane proteins (named NgcF and NgcG) were identified adjacent to ngcE. Each of these is predicted to have six membrane-spanning helices and a consensus motif for integral membrane proteins characteristic of ABC transporters. In addition, the gene for a predicted regulator protein (NgcR) was detected. The ngcEFG operon lacks a gene for an ATP-hydrolysing protein. NgcE is a new member of the CUT-1 family of ABC transporters for carbohydrates. Comparative studies of the wild-type and a mutant strain carrying an insertion within the ngc operon clearly demonstrate that the Ngc system mediates the uptake of N-acetylglucosamine and chitobiose in vivo.
ISSN 16174615
Age Range 18 to 22 years ♦ above 22 year
Educational Use Research
Education Level UG and PG
Learning Resource Type Article
Publisher Date 2002-04-09
Publisher Place Berlin/Heidelberg
e-ISSN 16174623
Journal Molecular and General Genetics MGG
Volume Number 267
Issue Number 4
Page Count 11
Starting Page 429
Ending Page 439


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Source: SpringerLink