Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens

摘要: 2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C-C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class reactions in chemistry and biochemistry. To investigate catalytic mechanism this enzyme, we determined three-dimensional structure at 2.0 A resolution using x-ray crystallography MAD methods. The overall includes small N-terminal domain mainly involved dimerization C-terminal Bet v1-like fold, which distinguishes classical alpha/beta-fold hydrolases. dumbbell-shaped substrate access tunnel was identified connect narrow interior amphiphilic pocket exterior solvent. is likely undergo significant conformational change upon binding active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, enzyme activity revealed that C-C proceeds via nucleophilic attack by water molecule, coordinated zinc ion. In addition, residues Tyr(121), Tyr(229), Asn(132), are predicted be hydrogen-bonded hydroxyl groups unhydrolyzed acetyl group, can finely tune position bound reactive orientation. Taken together, these results sites zinc-dependent explained its specificity as well.