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.

FIGURE 1.

Conversion of DAPG to MAPG by PhlG.

FIGURE 2.

Overall structure of PhlG dimer. A, schematic representation of the PhlG dimer. The metal ions are denoted as gray spheres. B, multisequence alignment of PhlG homologs. The alignment was performed using ClustalW and ESPript. The residues involved in coordinating zinc ions are marked by black filled circles, and residues involved in catalysis are marked by black stars.

FIGURE 3.

Structure and topology of PhlG monomer. A, schematic representation of PhlG monomer, colored and labeled according to secondary structural elements. The metal ion is denoted as a gray sphere. B, topology diagram of the C-terminal Bet v1-like domain of PhlG. Compared with the prototypic Bet v1-like fold, the PhlG C-terminal domain has an additional β-strand (β3) in the β-barrel and several insertions of helices (helices α6, α7, α8, and α9).

FIGURE 4.

Active site, substrate docking model, and mutational analysis of PhlG. A, anomalous difference Fourier map confirms the zinc ion. The anomalous difference map density of the metal is comparable with that of selenium atoms of SeMet residues (Mse, SeMet). The anomalous difference Fourier map calculated with data collected at the wavelength of 0.9250 Å is shown in pink contoured at 5σ. At this wavelength, zinc has a modest anomalous signal (f″ = 2.24), whereas magnesium (f″ = 0.06) and calcium (f″ = 0.52) do not. The 2F_o − F_c σ_A-weighted map is shown in gray and contoured at 1.5σ. B, model of DAPG intermediate bound to the active site of PhlG. The docked DAPG intermediate is shown in ball and stick format, and the residues within 4 Å of DAPG are shown in stick format. The distance between the hydroxyl group of the acetyl group and the zinc ion is 2.2 Å. The water molecule coordinated to the zinc ion may serve as the attacking nucleophile because its distance to the carbon atom of the carbonyl group is 2.3 Å. Hydrogen bonds between the active site and docked DAPG are also shown in black dashes and the distances are labeled.

FIGURE 5.

Putative substrate access tunnel calculated by CAVER. A, front view of the substrate access tunnel denoted in mesh. B, close-up side view of the substrate access tunnel; residues in the vicinity are shown as sticks and labeled. C, overlay of HPLCs of glutathionylated PhlG enzyme and untreated PhlG wild-type (WT) enzyme: from top to bottom, PhlG wild-type enzyme treated with 10 mm GSSG, wild-type enzyme, DAPG, and MAPG. mAU, milliabsorbance unit.