Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens |
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Authors: | Yong-Xing He Liang Huang Yanyan Xue Xue Fei Yan-Bin Teng Sheryl B Rubin-Pitel Huimin Zhao and Cong-Zhao Zhou |
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Institution: | From the ‡Hefei National Laboratory for Physical Sciences at Microscale and School of Life Sciences, University of Science and Technology of China, Hefei, Anhui 230026, China and ;the Departments of §Chemical and Biomolecular Engineering and ;¶Chemistry and ;‖Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 |
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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 α/β-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 Tyr121, Tyr229, and Asn132, 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. |
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Keywords: | Enzymes/Hydrolases Enzymes/Metallo Enzymes/Structure Protein/Conformation 2 4-Diacetylphloroglucinol Biosynthesis C–C Bond Cleaving Hydrolase X-ray Diffraction Analysis Computational Modeling |
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