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J. Biol. Chem., Vol. 276, Issue 4, 2816-2823, January 26, 2001
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From the The crystal structure of a major
oxygen-insensitive nitroreductase (NfsA) from Escherichia
coli has been solved by the molecular replacement method at
1.7-Å resolution. This enzyme is a homodimeric flavoprotein with one
FMN cofactor per monomer and catalyzes reduction of nitrocompounds
using NADPH. The structure exhibits an The atomic coordinates and the structure factors (code 1F5V, 1PGO, and 1BKJ) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
Structure and Site-directed Mutagenesis of a Flavoprotein from
Escherichia coli That Reduces Nitrocompounds
ALTERATION OF PYRIDINE NUCLEOTIDE BINDING BY A SINGLE AMINO ACID
SUBSTITUTION*
§,
,,§§
Departments of Applied Biological Chemistry
and Biotechnology, Graduate School of Agricultural and Life
Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the ¶ Biotechnology Research Center, University of Tokyo,
1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan, the ** Department of
Biophysics and Biochemistry, Graduate School of Science, University of
Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, and the
§§ Department of Microbiology and Immunology,
University of British Columbia, 6174 University Boulevard, Vancouver,
British Columbia V6T 1Z3, Canada
+ 
-fold, and is comprised
of a central domain and an excursion domain. The overall structure of
NfsA is similar to the NADPH-dependent flavin reductase of
Vibrio harveyi, despite definite difference in the spatial
arrangement of residues around the putative substrate-binding site. On
the basis of the crystal structure of NfsA and its alignment with the
V. harveyi flavin reductase and the
NADPH-dependent nitro/flavin reductase of Bacillus
subtilis, residues Arg203 and Arg208 of
the loop region between helices I and J in the vicinity of the
calalytic center FMN is predicted as a determinant for NADPH binding.
The R203A mutant results in a 33-fold increase in the Km value for NADPH indicating that the side chain
of Arg203 plays a key role in binding NADPH possibly to
interact with the 2'-phosphate group.
*
This work was supported in part by grants-in-aid for
Scientific Research from the Ministry of Education, Science and Culture of Japan, the Sakabe project of TARA, University of Tsukuba, performed in part using the Station BL6A at the Photon Factory, High Energy Accelerator Research Organization under the approval of the Photon Factory Program Advisory Committee Proposal number 97G091.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Dept. of Physiology, Medical University of
Oita, 1-1 Idaigaoka, Hasama, Oita 879-5503, Japan.
§§
To whom correspondence should be addressed: Dept. of Applied
Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan. Tel.: 81-3-5841-5165; Fax: 81-3-5841-8023; E-mail: amtanok@ mail.ecc.u-tokyo.ac.jp.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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