CYS5 AND CYS214 OF NAD(P)H-FLAVIN OXIDOREDUCTASE FROM ESCHERICHIA-COLI ARE LOCATED IN THE ACTIVE-SITE

Citation
F. Fieschi et al., CYS5 AND CYS214 OF NAD(P)H-FLAVIN OXIDOREDUCTASE FROM ESCHERICHIA-COLI ARE LOCATED IN THE ACTIVE-SITE, European journal of biochemistry, 237(3), 1996, pp. 870-875
Citations number
30
Categorie Soggetti
Biology
ISSN journal
00142956
Volume
237
Issue
3
Year of publication
1996
Pages
870 - 875
Database
ISI
SICI code
0014-2956(1996)237:3<870:CACONO>2.0.ZU;2-P
Abstract
The NAD(P)H:flavin oxidoreductase (NADPH:riboflavin oxidoreductase) fr om Escherichia coli, Fre, is a monomer of 26.1 kDa, which catalyzes th e reduction of free flavins by NADPH or NADH. A sequential ordered mec hanism, with NADPH binding first, operates. Fre is the prototype of a class of flavin reductases able to transfer electrons with no prosthet ic group. It has been previously reported that several members of this family, including Fre, were inactivated by thiol reagents such as N-e thylmaleimide (MalNEt). Amino acid sequence similarities among these e nzymes reveal that one of the three cysteines residues of Fre is highl y conserved. Altogether this suggested a crucial role of cysteine resi dues for catalysis. The three cysteine residues were mutated to serine residues. Single-mutant and double-mutant enzymes were as active as t he wild-type and K-m values for both substrates remained the same. Cys teine residues are thus not important for activity. Nevertheless, we s howed that cysteines 5 and 214, but not cysteine 149, were responsible for MalNEt inactivation. In addition, it has been found that riboflav in, but not NADPH, can protect Fre from MalNEt inactivation. This stro ngly suggested that Cys5 and Cys214 are located at the flavin-binding site of Fre and that flavin can bind to the enzyme in the absence of N ADPH.