The Structural Biologic Studies On Fungal Protein, LZ-8, From The Ganoderma Lucidium And On DAPG Hydrolase, PhlG, From Pseudomonas Fluorescens

1. The structure and function research on one fungal immunomodulatory protein, LZ-8, from the medicinal fungus Ganoderma lucidiumFungal immunomodulatory proteins is a family of fruiting body protein from mushrooms possessing immunomodulatory activity. They stimulate lymphocyte mitogenesis, suppress systemic anaphylaxis reactions and oedema, enhance transcription of IL-2, IFN-gamma and TNF-alpha, and haemagglutinate red blood cells.LZ-8 is a member of fungal immunomodulatory proteins (Fips) family, which was isolated from the Chinese traditional herb, Ling Zhi (Ganoderma lucidium). Despite sharing a very high level of sequence similarity to other members in this family, LZ-8 showed a unique anti-tumor activity on HL60 cells. To elucidate the structural basis of this uniqueness, we solved the crystal structure of LZ-8 at 2.10 ? resolution. Structural comparison with Fip-fve from Flammulina veltipes revealed that the conformations of the loops in the FNIII domain are unique. This finding provided us the structural basis for improving the medicinal activity of FNIII domain containing proteins via engineering the critical loops.2. The structure and proposed catalytic mechanism on 2, 4-diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescensThe PhlG enzyme is identified in certain strains of root-colonizing fluorescent Pseudomonas, well-known as biocontrol agents, catalyzes the conversion of the antibiotic diacetylphloroglucinol (DAPG) into less toxic monoacetylphloroglucinol (MAPG) by cleaving one of the carbon-carbon bonds linking the acetyl groups to the phenolic ring. Here, we report the 2.00 ? crystal structure of PhlG solved with the multiwavelength anomalous dispersion (MAD) method. The overall structure of PhlG folds into a Bet v1-like fold surrounded by several auxiliary regions, quite distinct from the classicα/βhydrolytic enzymes, making it the first identified hydrolytic enzyme with the Bet v1-like fold. Based on the computational docking results combined with enzymatic assay of PhlG mutants, we propose the solvent molecule coordinated by a zinc ion in a deep interior pocket functions as the attacking nucleophile activated by the Asp160 and/or Asp274 acting as the general base. Asn132 is determinant to anchor the unhydrolyzed acetyl so that the substrate is bound in a reacting pose while His214 and Tyr229 are also involved in positioning the substrate by forming hydrogen bonds with the DAPG hydroxyl near the reaction center. A putative dumbbell shaped substrate accessing tunnel was calculated, suggesting that conformational changes near the gorge of the substrate accessing tunnel must happen to let the substrate approach the active site. Furthermore, the PhlG activity is susceptible to excess amount of oxidized glutathione (GSSG), possibly due to the blocking of substrate accessing tunnel by glutathionylation of the cysteines nearby.