Crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae

Abstract

Nitroreductases are flavoenzymes that catalyze nitrocompounds and are widely utilized in industrial applications due to their detoxification potential and activation of biomedicinal prodrugs. Type I nitroreductases are classified into subgroups depending on the use of NADPH or NADH as the electron donor. Here, we report the crystal structure of the fungal nitroreductase Frm2 from Saccharomyces cerevisiae, one of the uncharacterized subgroups of proteins, to reveal its minimal architecture previously observed in bacterial nitroreductases such as CinD and YdjA. The structure lacks protruding helical motifs that form part of the cofactor and substrate binding site, resulting in an open and wide active site geometry. Arg82 is uniquely conserved in proximity to the substrate binding site in Frm2 homologues and plays a crucial role in the activity of the active site. Frm2 primarily utilizes NADH to reduce 4-NQO. Because missing helical elements are involved in the direct binding to the NAD(P)H in group A or group B in Type I family, Frm2 and its homologues may represent a distinctive subgroup with an altered binding mode for the reducing compound. This result provides a structural basis for the rational design of novel prodrugs with the ability to reduce nitrogen-containing hazardous molecules.

Keywords: 4-nitroquinoline 1-oxide; NADH; Saccharomyces cerevisiae; crystal structure; nitroreductase.

Figure 1

Overall structure and active site of Frm2. (A) Homodimeric arrangement of Frm2 (Mol A: green, Mol B; blue) with labeled secondary structures. The α-helix 8 located at the C-terminal end is not visible in the structure. FMN binding pocket is located in dotted box. The key residues in the FMN binding groove and active site are shown as sticks in the enlarged surface diagram. (B) Comparison of the active site. Models of 4-NQO and FMN are superposed to the active site pocket of Frm2 (green) based on the substrate bound CinD complex structure (pdb 4bnb, gold). The positively conserved residue Arg82, corresponding to Lys88 in CinD, is positioned in close proximity to NO₂ moiety of 4-NQO. (C) Nitroreductase activities are shown by the reduction of NADH with the 4-NQO substrate for wild type Frm2 and for mutant proteins of the Arg82 residue (R82A and R82E). Specific activities are indicated in the table. Assays were performed in 1 mL of 50 mM Tris–Hcl (pH 7.5) containing 10 µM FMN, 0.2 mM NAD(P)H and 0.05 mM nitrocompounds. Reactions were started by adding 100 nM of enzyme, and changes in absorbance were measured for 20 min at room temperature. One unit is defined as µmoles NAD(P)H per min.

Figure 2

Protruding helical elements in the NAD(P)H interaction. (A) Frp structure from Vibrio harveyi in Group A has helices of I, J, and K (red) in the C-terminus for interaction to NADPH (pdb 2bkj). The unusually folded structure of nicotinamide adenine dinucleotide is located in proximity to the protruding helices. (B) NfnB structure from Mycobacterium smegmatis in Group B has helices of C and D (green) for the nondiscriminative interaction to NADPH (pdb 2wzw). (C) The minimal architecture of Frm2 without the protruding helices suggests an alternative binding mode for the reducing agent. (D) Frm2 utilizes mainly NADH, whereas NfsA in Group A utilizes the NADPH molecule. Kinetic parameters associated to substrate consumption in wild type are shown in a table.