title: Crystal structure and computational analyses provide insights into the catalytic mechanism of 2,4-diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens.
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.
authors.affiliation: Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China.
authors.forename: Yong-Xing
authors.initials: YX
authors.lastname: He
authors.forename: Liang
authors.initials: L
authors.lastname: Huang
authors.forename: Yanyan
authors.initials: Y
authors.lastname: Xue
authors.forename: Xue
authors.initials: X
authors.lastname: Fei
authors.forename: Yan-Bin
authors.initials: YB
authors.lastname: Teng
authors.forename: Sheryl B
authors.initials: SB
authors.lastname: Rubin-Pitel
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
authors.forename: Cong-Zhao
authors.initials: CZ
authors.lastname: Zhou
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D002853:Chromatography, Liquid; D019295:Computational Biology; D018360:Crystallography, X-Ray; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010696:Phloroglucinol / Q000031:analogs & derivatives / Q000378:metabolism; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011551:Pseudomonas fluorescens / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; C059817:2,4-diacetylphloroglucinol; D010696:Phloroglucinol