The Crystal Structure of a Quercetin 2,3-Dioxygenase from Bacillus subtilis Suggests Modulation of Enzyme Activity by a Change in the Metal Ion at the Active Site(s)

Abstract

Common structural motifs, such as the cupin domains, are found in enzymes performing different biochemical functions while retaining a similar active site configuration and structural scaffold. The soil bacterium Bacillus subtilis has 20 cupin genes (0.5% of the total genome) with up to 14% of its genes in the form of doublets, thus making it an attractive system for studying the effects of gene duplication. There are four bicupins in B. subtilis encoded by the genes yvrK, yoaN, yxaG, and ywfC. The gene products of yvrK and yoaN function as oxalate decarboxylases with a manganese ion at the active site(s), whereas YwfC is a bacitracin synthetase. Here we present the crystal structure of YxaG, a novel iron-containing quercetin 2,3-dioxygenase with one active site in each cupin domain. Yxag is a dimer, both in solution and in the crystal. The crystal structure shows that the coordination geometry of the Fe ion is different in the two active sites of YxaG. Replacement of the iron at the active site with other metal ions suggests modulation of enzymatic activity in accordance with the Irving-Williams observation on the stability of metal ion complexes. This observation, along with a comparison with the crystal structure of YvrK determined recently, has allowed for a detailed structure-function analysis of the active site, providing clues to the diversification of function in the bicupin family of proteins.

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

“…designated strain FLA (flavonol utilization) was characterized with divalent Fe, Ni, and Co cofactors (43). The QDO from B. subtilis, has been characterized with divalent Fe, Mn, and Co cofactors (44) and tested for activity with divalent Mg, Ni, and Cd (45). It has been suggested that exchange of the active-site metal ion may allow biological modulation of enzyme activity (45).…”

Transcriptional response machineries of Bacillus subtilis conducive to plant growth promotion

“…23) Additionally, it was reported that qdoI encodes a quercetin 2,3-dioxygenase (EC 1.13.11.24) that converts quercetin to 2-protocatechuoyl-phloroglucinol carboxylic acid and carbon monoxide. 24) Further detail characterization revealed that this enzyme is also able to convert other flavonols, such as fisetin, tamarixetin, and galangin, to the corresponding depsides by a similar C-ring cleavage reaction, although its catalytic activity toward fisetin is relatively low. 25) In contrast to qdoI, there is little information about yxaH, except that it is predicted to encode a membrane protein by the sequence information.…”

“…3). A QdoR mutant capable of responding to The qdoI gene, one of the LmrA/QdoR regulon members, encodes quercetin 2,3-dioxygenase, which forms a dimer (two rotundate rectangles) 24) and catalyzes the C-ring cleavage of flavonols, as illustrated. 29) various flavonoids can be created by substituting the residues in the flavonoid-interacting domain probably including these three aromatic residues, and this mutant would be available as a novel biosensor to detect various flavonoids and related compounds in such a way as to introduce its gene into the B. subtilis strain carrying a reporter-fusion construct similar to that used in this study.…”

“…24,25) The resultant depsides can be hydrolyzed to a pair of the respective aromatic carboxylates by an endogenous esterase with broad substrate specificity. It was reported that B. subtilis also possesses the yfiE gene, whose expression is induced by catechol and which encodes a catechol-2,3-dioxygenase capable of converting catechol into 2-hydroxymuconic semialdehyde, 43) and the ywhB gene which encodes a 4-oxalocrotonate tautomerase capable of catalyzing the interconversion of 2-hydroxymuconate and 4-oxalocrotonate.…”

Excess Production ofBacillus subtilisQuercetin 2,3-Dioxygenase Affects Cell Viability in the Presence of Quercetin

“…5,9,10) The crystal structures of the enzymes from A. japonicus and B. subtilis revealed that both enzymes belong to the bicupin family, which accommodates metal ions in their catalytic active sites, formed by characteristic -barrel domains. 8,11) While the fungal enzyme contains Cu 2þ , 8,12) the enzyme from B. subtilis is assumed to prefer Mn 2þ as a cofactor, 13) but its recombinant protein has been purified as an iron enzyme from Escherichia coli. 5,11) On the other hand, the Streptomyces sp.…”