Structure and catalytic mechanism of the evolutionarily unique bacterial chalcone isomerase

Abstract

Flavonoids represent a large class of secondary metabolites produced by plants. These polyphenolic compounds are well known for their antioxidative abilities, are antimicrobial phytoalexins responsible for flower pigmentation to attract pollinators and, in addition to other properties, are also specific bacterial regulators governing the expression of Rhizobium genes involved in root nodulation (Firmin et al., 1986). The bacterial chalcone isomerase (CHI) from Eubacterium ramulus catalyses the first step in a flavanone-degradation pathway by ring opening of (2S)-naringenin to form naringenin chalcone. The structural biology and enzymology of plant CHIs have been well documented, whereas the existence of bacterial CHIs has only recently been elucidated. This first determination of the structure of a bacterial CHI provides detailed structural insights into the key step of the flavonoid-degradation pathway. The active site could be confirmed by co-crystallization with the substrate (2S)-naringenin. The stereochemistry of the proposed mechanism of the isomerase reaction was verified by specific 1 H/ 2 H isotope exchange observed by 1 H NMR experiments and was further supported by mutagenesis studies. The active site is shielded by a flexible lid, the varying structure of which could be modelled in different states of the catalytic cycle using small-angle X-ray scattering data together with the crystallographic structures. Comparison of bacterial CHI with the plant enzyme from Medicago sativa reveals that they have unrelated folds, suggesting that the enzyme activity evolved convergently from different ancestor proteins. Despite the lack of any functional relationship, the tertiary structure of the bacterial CHI shows similarities to the ferredoxin-like fold of a chlorite dismutase and the stress-related protein SP1.

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

“…Based on these docking results, we assumed that His33 also functions as crucial residue for the flavanonol-isomerizing activity of CHI. This suggests a general acidbase catalysis as reported for the flavanone-converting activity of CHI (17). A transfer of the proton at position 3 of (ϩ)-taxifolin to His33 would facilitate the subsequent transformation to alphitonin, in accordance with the observed stereospecificity of taxifolin isomerization.…”

“…The X-ray crystal structures of CHI from E. ramulus (PDB ID no. 3ZPH and 4D06) (15,17) were obtained from the RCSB Protein Data Bank (www.rcsb.org). The structures were used as a template for flexible ligand docking with AutoDock 4.2 (25).…”

“…Based on the above-described docking results, the histidine at position 33 of CHI (position 34 referring to the amino acid sequence before posttranslational cleavage of the N-terminal methionine) (14,17) was replaced by alanine using site-directed mutagenesis. The mutated chi was heterologously expressed in E. coli at a similar level to the wild-type enzyme (see Fig.…”

“…S1 in the supplemental material). The structures of CHI and Phy have recently been solved, and catalytic amino acid residues have been identified by mutagenesis (15,17,18). CHI was crystallized in both ligand-free and ligand-bound forms, and the interaction of the active site His33 with naringenin was elucidated (15,17).…”

Assemblins as maturational proteases in herpesviruses

“…Compared to the triad, His-157A causes a 10-fold reduction in activity, but structural data indicate that a water molecule rescues a part of the activity. This effect was demonstrated for other enzymes as well [74]. The authors of the study assume a 150-fold reduction when excluding that effect [73].…”