Figure 1.
A: ChrR monomer; B: ChrR FMN-binding site; and C: ChrR tetramer. Underlined residues in panel B indicate NAD(P)H-dependent FMN-reductase signature sequence. Side chain atoms of F19 have been removed for clarity. The residue R100 belongs to the symmetry related molecule. Abbreviations: E = Glu; F = Phe; G = Gly; N = Asn; R = Arg; S = Ser; Y = Tyr; Wat = Water molecule.
Table 1.
Data collection and refinement statistics.
Figure 2.
Oligomeric state of native ChrR and the mutants listed in Table 2 as determined by gel-filtration chromatography (Superdex 200 10/300).
Panel A shows absorbance at 280 nm as a function of the elution volume, and Panel B is the calibration curve. The active enzymes are essentially tetrameric (peak b), as the dimer fraction (peak c) and protein aggregates (peak a) are negligible.
Figure 3.
Hydrogen bonding network between FMN molecules from two different dimers of ChrR.
Residue Tyr128 is closer to the FMN belonging to the symmetry-related dimer (8.4 Å) than to the FMN of the original molecule (14.1 Å). Abbreviations: E = Glu; R = Arg; Y = Tyr.
Table 2.
Chromate reductase activity of ChrR mutant enzymes.
Figure 4.
Diagramatic representation of the possible ways by which Tyr128Asn substitution in ChrR may increase chromate reductase activity.
Fig. 4a and 4b show changes in hydrogen bonding between the dimers, and the surface diagrams (4c and 4d) show changes in the cavity around FMN. Both diagrams show the same location but for clarity the surface diagram view is rotated 90° on Z-axis and 45° on X-axis of the bonding diagram; Arg125 location is maintained at the top for direct comparison of the two views. See text for details.