Conversion of NfsA, the major Escherichia coli nitroreductase, to a flavin reductase with an activity similar to that of Frp, a flavin reductase in Vibrio harveyi, by a single amino acid substitution

Abstract

NfsA is the major oxygen-insensitive nitroreductase of Escherichia coli, similar in amino acid sequence to Frp, a flavin reductase of Vibrio harveyi. Here, we show that a single amino acid substitution at position 99, which may destroy three hydrogen bonds in the putative active center, transforms NfsA from a nitroreductase into a flavin reductase that is as active as the authentic Frp and a tartrazine reductase that is 30-fold more active than wild-type NfsA.

FIG. 1

Amino acid changes in NfsA-97, a mutant showing a high level of FMN reductase activity. (A) Structures of wild-type NfsA, NfsA-97, and their chimeras (NfsA-97-52A and -99G). Filled boxes, sequences derived from wild-type NfsA; open boxes, sequences derived from NfsA-97. Cleavage sites for restriction endonucleases used for chimera formation are indicated by vertical arrows. Values for NADPH-FMN and nitrofurazone reductase activities in cell extracts are shown on the right. (B) Amino acid sequence homology between NfsA and Frp. Invariant amino acids are shown by white letters in black boxes. The locations of two amino acid substitutions in NfsA-97 are indicated by vertical lines labeled with the mutated amino acids. According to Tanner et al. (10), H-11, S-13, R-15, S-39, Q-67, G-131, K-167, and R-169 are involved in stabilizing the FMN cofactor. R-225 and R-133 may be required for properly steering substrates to the active center (FMN cofactor). Note that R-225 and R-133 have hydrogen bonds to E-99, which is replaced by a glycine residue in NfsA-97. Asterisks show invariant active center amino acids between Frp and NfsA (see Fig. 2).

FIG. 2

Disruption of presumed hydrogen bonds in the active center of NfsA by a Glu-to-Gly substitution at position 99. Sixteen of 17 amino acids surrounding the FMN cofactor are invariant between Frp and NfsA, strongly suggesting that the three-dimensional structure of NfsA is very similar to that of Frp. We presume the active center of NfsA to possess a hydrogen-bonding pattern virtually identical to that of Frp (10). Thus, this figure is adapted from Tanner et al. (10). As with Frp Glu-99, NfsA Glu-99 has hydrogen bonds with Arg-225 and Arg-133. These hydrogen bonds, which are labeled with three small arrows, are disrupted by the Glu-to-Gly substitution (see the thick vertical arrow) so that the structurally relaxed active center of the mutant NfsA can accommodate large molecules such as FMN as substrates. Invariant amino acids surrounding or forming the active center are His-11, Ser-13, Arg-15, Ser-39, Gln-67, Glu-99, Val-106, Asp-107, Gly-130, Gly-131, Arg-133, Asn-134, Phe-153, Lys-167, Arg-169, and Arg-225. Tyr-69 of Frp (in parentheses) is replaced by His-69 in NfsA. Residues marked with B are from the other subunit.

FIG. 3

Temperature dependence of nitrofurazone (A) and FMN (B) reductase activities. Filled circles, wild-type NfsA; open circles, NfsA-97-99G.