Structural And Functional Studies Of Hydrolase PhlG, Cytochrome B5 And Its Reductase

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C-C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol (DAPG) to form monoacetylphloroglucinol (MAPG), a rare class of reaction in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 (?) resolution using X-ray crystallography and multi-wavelength anomalous dispersion (MAD) methods. The overall structure comprises a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet vl-like fold, which distinguishes PhlG from the classicalα/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 enzymatic activity analysis revealed that cleavage of the DAPG 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.Cytochromes b5 are electron transfer hemoproteins ubiquitous in animals, plants, fungi and purple phototrophic bacteria. They are involved in a broad spectrum of reactions, including fatty acid desaturation, cholesterol biosynthesis, and cytochrome P450-dependent oxidation and hydroxylation reactions. Here, we report the crystal structures of cytochrome b5 and its reductase from the yeast Saccharomyces cerevisiae. Notably, Cyb5 monomers were assembled into dimers in the crystalline environment and the solvent-exposed edge of the haem group faces the interface between the two monomers, with one of the propionate groups pointing towards the helixα3 of the neighboring subunit. Also, the side-chains of Asp46 and Asp42, shown to be critical for electron transfer from cytochrome b5 to cytochrome c, sandwiched the propionate group of pyrrole II with Asp46 forming a direct hydrogen bond with it. Using computational docking method, we constructed the complex model of Cyb5 and Cbr1, which could shed light on the structural basis of the electron transfer between Cyb5 and Cbrl.Glutamine synthetase (GS, EC 6.3.1.2) plays an essential role in nitrogen assimilation by catalyzing the condensation of glutamate and ammonium to form glutamine, with concomitant hydrolysis of ATP. Following the recent publication of maize, human and canine GSII structures, we report the crystal structure of Glnl from the yeast Saccharomyces cerevisiae at the resolution of 2.95 A, with a citrate molecule binding to each glutamate binding site. Comparative structure analysis suggests that citrate binding could induce structural fluctuation of the segment Leu293-Ala300, which may serve the role of guarding the glutamate entrance to the active site. Besides a decameric quaternary structure and active sites similar to the published GSII structures, a novel back-to-back inter-ring interface was found. This additional interface enables Glnl to be assembled into a nanotube-like supramolecule, although the biological significance is still an open question.