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 Å 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 ␣/␤-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.

Molecular and Catalytic Properties of 2,4-Diacetylphloroglucinol Hydrolase (PhlG) from <i>Pseudomonas</i> sp. YGJ3

“…The threedimensional structure of His 6 -PhlG indicates that these amino acid residues are located in different positions than the catalytic site of the enzyme. 7) PhlG was purified from E. coli XL1-Blue MRF 0 carrying pUHH1.3. Table 1 summarizes the purification procedure.…”

“…It lost about half of its activity when incubated at 47 C for 20 min, and was completely inactivated at 55 C. The kinetic constants, K m and k cat , were determined by double reciprocal plot to be 24 mM and 5. of PhlG-His 6 of P. fluorescens CHA0 indicated that Histagged PhlG had a markedly higher K m value than PhlG. 6,7) This might have been due to the presence of an extra peptide chain at the N-terminus in His-tagged PhlG. MAPG competitively inhibited the enzyme (K i ¼ 87 mM), while 2-hydroxy-and 4-hydroxyacetophenone were ineffective.…”

“…6) Gene phlG has been cloned from P. fluorescens CHA0 and P. fluorescens Pf-5 and expressed as PhlGHis 6 and His 6 -PhlG (the His-tags were attached to the Cand the N-terminus of PhlG respectively) in Escherichia coli. 6,7) The three-dimensional structure of His 6 -PhlG (PDB id 3HWP) revealed PhlG to be zinc hydrolase. 7) Characterization of PhlG is essential to understand the physiological basis of the biocontrol of pseudomonads, but little information is available on the biochemical properties of native PhlG without the His-tag.…”

“…6,7) The three-dimensional structure of His 6 -PhlG (PDB id 3HWP) revealed PhlG to be zinc hydrolase. 7) Characterization of PhlG is essential to understand the physiological basis of the biocontrol of pseudomonads, but little information is available on the biochemical properties of native PhlG without the His-tag. Although the biosynthesis of DAPG is significantly and intricately influenced by various metal compounds, including Cu 2 SO 4 , ZnSO 4 , and CoCl 2 , depending on culture conditions, 8) His-tagged PhlG is unsuitable for examination of the effect of metal ions on PhlG.…”

“…Its M r was determined to be 65 kDa by gel filtration, suggesting that it is composed of a homodimer like His 6 -PhlG of P. fluorescens Pf-5. 7) The enzyme bound Zn 2þ was qualitatively measured with 4-(2-pyridylazo)resorcinol by the method of Hunt et al 12) PhlG showed an absorption maximum at 279 nm, a shoulder at 290 nm, and an absorption minimum at 252 nm. The A 279 =A 252 ratio was 2.29.…”