Publications

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

  He Y.X., Huang L., Xue Y., Fei X., Teng Y.B., Rubin-Pitel S.B., Zhao H., Zhou C.Z.

  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 mechani ... >> More

  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. << Less

  J. Biol. Chem. 285:4603-4611(2010) [PubMed] [EuropePMC]

• Crystallization and preliminary X-ray diffraction analysis of a putative carbon-carbon bond hydrolase from Mycobacterium abscessus 103.

  Zhang Z., Jiang Y.L., Wu Y., He Y.X.

  The PhlG protein from Mycobacterium abscessus 103 (mPhlG), which shares 30% sequence identity with phloretin hydrolase from Eubacterium ramulus and 38% sequence identity with 2,4-diacetylphloroglucinol hydrolase from Pseudomonas fluorescens Pf-5, is a putative carbon-carbon bond hydrolase. Here, t ... >> More

  The PhlG protein from Mycobacterium abscessus 103 (mPhlG), which shares 30% sequence identity with phloretin hydrolase from Eubacterium ramulus and 38% sequence identity with 2,4-diacetylphloroglucinol hydrolase from Pseudomonas fluorescens Pf-5, is a putative carbon-carbon bond hydrolase. Here, the expression, purification and crystallization of mPhlG are reported. Crystals were obtained using a precipitant consisting of 100 mM citric acid pH 5.0, 1.0 M lithium chloride, 8%(w/v) polyethylene glycol 6000. The crystals diffracted to 1.87 Å resolution and belonged to space group P21, with unit-cell parameters a = 71.0, b = 63.4, c = 74.7 Å, α = 90.0, β = 103.2, γ = 90.0°. Assuming the presence of two mPhlG molecules in the asymmetric unit, VM was calculated to be 2.5 Å(3) Da(-1), which corresponds to a solvent content of 50%. << Less

  Acta Crystallogr. F Struct. Biol. Commun. 71:239-242(2015) [PubMed] [EuropePMC]

• Characterization of PhlG, a hydrolase that specifically degrades the antifungal compound 2,4-diacetylphloroglucinol in the biocontrol agent Pseudomonas fluorescens CHA0.

  Bottiglieri M., Keel C.

  The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been ... >> More

  The potent antimicrobial compound 2,4-diacetylphloroglucinol (DAPG) is a major determinant of biocontrol activity of plant-beneficial Pseudomonas fluorescens CHA0 against root diseases caused by fungal pathogens. The DAPG biosynthetic locus harbors the phlG gene, the function of which has not been elucidated thus far. The phlG gene is located upstream of the phlACBD biosynthetic operon, between the phlF and phlH genes which encode pathway-specific regulators. In this study, we assigned a function to PhlG as a hydrolase specifically degrades DAPG to equimolar amounts of mildly toxic monoacetylphloroglucinol (MAPG) and acetate. DAPG added to cultures of a DAPG-negative DeltaphlA mutant of strain CHA0 was completely degraded, and MAPG was temporarily accumulated. In contrast, DAPG was not degraded in cultures of a DeltaphlA DeltaphlG double mutant. To confirm the enzymatic nature of PhlG in vitro, the protein was histidine tagged, overexpressed in Escherichia coli, and purified by affinity chromatography. Purified PhlG had a molecular mass of about 40 kDa and catalyzed the degradation of DAPG to MAPG. The enzyme had a kcat of 33 s(-1) and a Km of 140 microM at 30 degrees C and pH 7. The PhlG enzyme did not degrade other compounds with structures similar to DAPG, such as MAPG and triacetylphloroglucinol, suggesting strict substrate specificity. Interestingly, PhlG activity was strongly reduced by pyoluteorin, a further antifungal compound produced by the bacterium. Expression of phlG was not influenced by the substrate DAPG or the degradation product MAPG but was subject to positive control by the GacS/GacA two-component system and to negative control by the pathway-specific regulators PhlF and PhlH. << Less

  Appl. Environ. Microbiol. 72:418-427(2006) [PubMed] [EuropePMC]

• Molecular and catalytic properties of 2,4-diacetylphloroglucinol hydrolase (PhlG) from Pseudomonas sp. YGJ3.

  Saitou H., Watanabe M., Maruyama K.

  Gene phlG encoding 2,4-diacetylphloroglucinol hydrolase was cloned from Pseudomonas sp. YGJ3 and expressed in Escherichia coli. Recombinant PhlG was purified homogeneously. It required 2-mercaptoethanol for stability. Km for 2,4-diacetylphloroglucinol and kcat were determined to be 24 µM and 5.8 s ... >> More

  Gene phlG encoding 2,4-diacetylphloroglucinol hydrolase was cloned from Pseudomonas sp. YGJ3 and expressed in Escherichia coli. Recombinant PhlG was purified homogeneously. It required 2-mercaptoethanol for stability. Km for 2,4-diacetylphloroglucinol and kcat were determined to be 24 µM and 5.8 s(-1) respectively. CoCl2 specifically and significantly activated PhlG. << Less

  Biosci. Biotechnol. Biochem. 76:1239-1241(2012) [PubMed] [EuropePMC]