Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis.
Enterococcus faecalis azoreductase (AzoA) is a very active enzyme with a broad spectrum of substrate specificity and is capable of degrading various azo dyes. The enzyme has an absolute requirement for reduced FMN, which delivers a total of four electrons from NADH to the substrate, resulting in the cleavage of the nitrogen double bond. In this study, we report the identification of amino acid residues critical for FMN binding in AzoA. FMN is stabilized by 22 amino acid residues, eight of which, Trp-105, Asn-106, Leu-107, Gly-150, Gly-151, Tyr-153, Asn-121 and Tyr-129, are involved in binding the FMN isoalloxazine ring. In silico analysis of the amino acid residues revealed that the Trp residue at position 105 of AzoA is the most likely significant contributor to the binding of FMN to the enzyme and is involved in FMN stabilization and destabilization. Site-directed mutagenesis analysis of Trp-105 was performed to determine the role of this amino acid residue in FMN binding and azo dye reductive activity. The mutant proteins were overexpressed in Escherichia coli and purified by anion-exchange and size-exclusion chromatography. The replacement of Trp-105 by the small side-chain amino acids Ala and Gly caused complete loss of both affinity for FMN and enzyme activity. Substitution of Tyr for Trp-105 did not significantly decrease the V(max) of the enzyme (22 % reduction). Substitutions with three bulky side-chain amino acids, Gln, Phe and His, produced enzymes with lower V(max) values (decreases of 68.2, 30.6 and 8.2-fold, respectively). However, these mutated enzymes maintained K(m) values similar to the wild-type enzyme. This study provides an insight into the catalytic properties of AzoA in FMN stabilization and enzyme activity.
Chen
Huizhong
H
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA.
Xu
Haiyan
H
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA.
Kweon
Ohgew
O
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA.
Chen
Siwei
S
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA.
Cerniglia
Carl E
CE
Division of Microbiology, National Center for Toxicological Research, US Food and Drug Administration, Jefferson, AR 72079-9502, USA.