3HWP

Crystal structure and computational analyses provide insights into the catalytic mechanism of 2, 4-diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens

Summary for 3HWP

• Entry DOI: 10.2210/pdb3hwp/pdb
• Descriptor: PhlG, ZINC ION, CHLORIDE ION, ... (5 entities in total)
• Functional Keywords: beta-grip fold, hydrolase
• Biological source: Pseudomonas fluorescens
• Total number of polymer chains: 2
• Total formula weight: 71193.25

Authors

He, Y.-X.,Huang, L.,Xue, Y.,Fei, X.,Teng, Y.-B.,Zhou, C.-Z. (deposition date: 2009-06-18, release date: 2009-12-15, Last modification date: 2024-11-06)

Primary citation

He, Y.X.,Huang, L.,Xue, Y.,Fei, X.,Teng, Y.B.,Rubin-Pitel, S.B.,Zhao, H.,Zhou, C.Z.
Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens.
J.Biol.Chem., 285:4603-4611, 2010

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

PubMed: 20018877
DOI: 10.1074/jbc.M109.044180

Experimental method

X-RAY DIFFRACTION (2 Å)