Sequence Conservation Does Not Always Signify a Functional Imperative as Observed in the Nitroreductase Superfamily

Abstract

Consensus sequences have the potential to help classify the structure and function of proteins and highlight key regions that may contribute to their biological properties. Often, the level of significance will track with the extent of sequence conservation, but this should not be considered universal. Arg and Lys dominate a position adjacent to the N1 and C2 carbonyl of flavin mononucleotide (FMN) bound in the proteins of the nitroreductase superfamily. Although this placement satisfies expectations for stabilizing the reduced form of FMN, the substitution of these residues in three subfamilies promoting distinct reactions demonstrates their importance to catalysis as only modest. Replacing Arg34 with Lys, Gln, or Glu enhances FMN binding to a flavin destructase (BluB) by twofold and diminishes FMN turnover by no more than 25%. Similarly, replacing Lys14 with Arg, Gln, or Glu in a nitroreductase (NfsB) does not perturb the binding of the substrate nitrofurazone. The catalytic efficiency does decrease by 21-fold for the K14Q variant, but no change in the midpoint potential of FMN was observed with any of the variants. Equivalent substitution at Arg38 in iodotyrosine deiodinase (IYD) affects catalysis even more modestly (<10-fold). While the Arg/Lys to Glu substitution inactivates NfsB and IYD, this change also stabilizes one-electron transfer in IYD contrary to predictions based on other classes of flavoproteins. Accordingly, functional correlations developed in certain structural superfamilies may not necessarily translate well to other superfamilies.

Scheme 1.

Three of the many diverse reactions catalyzed by enzymes within the NTR superfamily.

Scheme 2.

Reduction of oxidized flavin via hydride addition to the N5 position. The increase in electron density associated with reduction is thought to be stabilized by electrostatics and hydrogen bonding from the cationic amine and additional hydrogen bond donors (D) and acceptors (A).

Scheme 3.

FMN participates in hydride and single electron transfer

Scheme 4.

Dehalogenation via single electron transfer by IYD. (X = I⁻, Br⁻ and Cl⁻.

Figure 1.

Sequence conservation in subfamilies within the NTR superfamily. Weblogo plots of residues surrounding the cationic residue (R/K) interacting with the FMN N1 and C2 carbonyl (indicated with the blue circle). The R noted by the triangle anchors the 5’-phosphate of FMN. Conservation is based on 103, 731 and 211 sequences for BluB, NfsB and IYD, respectively and available from a structure function linkage database. The sequence logos were constructed by WebLogo.

Figure 2.

Representatives of the NTR superfamily and the polar residues proximal to their N1 and C2 carbonyl of FMN. The native α₂ homodimeric structures of (A) BluB (PDB: 2ISJ), (B) NfsB (PDB: 1YKI) and (C) IYD (PDB: 5KO8) are illustrated as cartoons along with their FMN. Distances between selected heteroatoms are listed in Å.

Figure 3.

Two electron reduction of flavoproteins in the presence of the reference dye AQDS. (A) NfsB K14R in 100 mM NaCl, 2 μM methyl viologen and 20 mM Tris pH 7.0 and (B) IYD R38K in 100 mM potassium phosphate pH 7.4 were reduced under anaerobic conditions from their FMN_ox to FMN_hq states by xanthine and xanthine oxidase in the presence of 20 μM of AQDS. Arrows indicate the increase and decrease of absorbances that were recorded at 30 s intervals for 2 h. Representative traces are shown for clarity. Reduction of FMN_ox and AQDS_ox was concurrently monitored at A₃₅₀ and A₃₂₅, respectively. Insert: the linear best fit of log(FMN_ox/FMN_hq) versus log(AQDS_ox/AQDS_red) was used to calculate the midpoint potential.^,

Figure 4.

Polar contacts and hydrogen bonding between I-Tyr, FMN and IYD. Lines indicate polar contacts with distances of less than 3 Å based on PDB 5KO8.

Figure 5.

Single electron reduction of FMN_ox to FMN_sq in IYD. (A) WT IYD and (B) its R38E variant in the presence of excess F-Tyr, 2 μM methyl viologen, 20 μM NB and 100 mM potassium phosphate pH 7.4 were reduced by xanthine and xanthine oxidase under anaerobic conditions. Arrows indicate the decrease of absorbances that were recorded in 30 s intervals for 3 h. Representative traces are shown for clarity. Reduction of FMN_ox and NB_ox was concurrently monitored at A₄₅₀ and A₆₂₀, respectively. Insert: the linear best fit of log(FMN_ox/FMN_sq) versus log(NB_ox/NB_red) was used to calculate the midpoint potential.^,

Figure 6.

Single electron reduction of FMN_sq to FMN_hq in IYD. (A) WT IYD and (B) its R38E variant in the presence of excess F-Tyr, 2 μM methyl viologen, 20 μM SFO and 100 mM potassium phosphate pH 7.4 were reduced by xanthine and xanthine oxidase under anaerobic conditions. Arrows indicate the decrease of absorbances that were recorded in 15 s intervals over 2 h. Representative traces are shown for clarity. The reduction of FMN_sq and SFO_ox was concurrently monitored at A₆₁₀ and A₅₂₀, respectively. Insert: the linear best fit of log(FMN_sq/FMN_hq) versus log(SFO_ox/SFO_red) was used to calculate the midpoint potential.^,