Crystal Structure of ChrR—A Quinone Reductase with the Capacity to Reduce Chromate

Abstract

The Escherichia coli ChrR enzyme is an obligatory two-electron quinone reductase that has many applications, such as in chromate bioremediation. Its crystal structure, solved at 2.2 Å resolution, shows that it belongs to the flavodoxin superfamily in which flavin mononucleotide (FMN) is firmly anchored to the protein. ChrR crystallized as a tetramer, and size exclusion chromatography showed that this is the oligomeric form that catalyzes chromate reduction. Within the tetramer, the dimers interact by a pair of two hydrogen bond networks, each involving Tyr128 and Glu146 of one dimer and Arg125 and Tyr85 of the other; the latter extends to one of the redox FMN cofactors. Changes in each of these amino acids enhanced chromate reductase activity of the enzyme, showing that this network is centrally involved in chromate reduction.

H 2 O 2 Production in Species of the Lactobacillus acidophilus Group: a Central Role for a Novel NADH-Dependent Flavin Reductase

“…The ortholog in Pseudomonas putida has been shown to catalyze the transfer of electrons from NADH to the quinone pool (46). The crystal structure of the E. coli gene annotated as chromate reductase clearly demonstrates the amino acids that constitute the flavin binding site (47). A ClustalW multiple sequence alignment shows that 4 out of the 11 residues of this binding site are conserved in both LJ_0548 and LJ_0549: i.e., Ser18, Asn20, Glu82, and Ser117 (see Fig.…”

Insights into the mode of flavin mononucleotide binding and catalytic mechanism of bacterial chromate reductases: A molecular dynamics simulation study

“…9 In addition, the amino acids, Tyr128, Glu146, Arg125, and Tyr85, and associated hydrogen bond networks, were found to play a critical role in enhancing chromate reductase activity. 9 Henceforth, gene cloning and prediction of FMN_red protein with reductase activity through molecular biology techniques and structural bioinformatics are of immense importance for bioremediation of waste materials from a diverse spectrum of environment. The YcnD (1ZCH) forms a dimer with one FMN per subunit.…”

Mutations in FMN binding pocket diminish chromate reduction rates for Gh-ChrR isolated from <i>Gluconacetobacter hansenii</i>

“…pdb), the FMN cofactor is held within a pocket near the surface through noncovalent interactions within individual subunits involving 12 electrostatic and 5 hydrophobic contact interactions that primarily involve loops 1 (G ) [8]. The ribotyl phosphate group is positioned deep within the binding pocket, and is stabilized through a salt bridge with R 17 in loop 1 between the β1 sheet and α1 helix (Figure 1), which is highly conserved in other NAD(P)H-dependent FMN reductases [13,15,16]. The proximal S 15 within loop 1 appears to stabilize this interaction, acting to assist in positioning the ribotyl phosphate through the formation of a hydrogen bond.…”

“…Consistent with this latter mechanism, the crystal structure of ChrR (3s2y.pdb) suggests a likely ion binding site for chromate proximal to FMN within the NAD(P)H binding pocket [8]. As chromate reduction by ChrR involves an adventitious enzyme activity [9] with a low catalytic rate and binding affinity (k cat = 0.25 sec ; K m = 0.24 mM) [8,13], the precise role of the FMN cofactor in chromate reduction remains uncertain. To test the prediction that chromate binds proximal to FMN, and to better understand the possible role of FMN in promoting the binding and reduction of chromate, we have used site-directed mutagenesis to assess how modifications in the FMN binding pocket affect chromate binding and its NADH-dependent reduction.…”

“…ChrR acts on quinones and other organic substrates (e.g., cancer prodrugs) through a ping-pong bi-bi (double displacement) mechanism whereby NAD(P)H and organic substrates bind sequentially, such that NAD(P)H reduces FMN prior to substrate binding and reduction [3,[12][13][14]. In contrast, the reduction of chromate involves an ordered bireactant mechanism, whereby chromate must first bind prior to the association of NAD(P)H [8].…”