title: A unique mono- and diacylglycerol lipase from Penicillium cyclopium: heterologous expression, biochemical characterization and molecular basis for its substrate selectivity.
abstract: A cDNA gene encoding a mature peptide of the mono- and diacylglycerol lipase (abbreviated to PcMdl) from Penicillium cyclopium PG37 was cloned and expressed in Pichia pastoris GS115. The recombinant PcMdl (rePcMdl) with an apparent molecular weight of 39 kDa showed the highest activity (40.5 U/mL of culture supernatant) on 1,2-dibutyrin substrate at temperature 35°C and pH 7.5. The rePcMdl was stable at a pH range of 6.5-9.5 and temperatures below 35°C. The activity of rePcMdl was inhibited by Hg2+ and Fe3+, but not significantly affected by EDTA or the other metal ions such as Na+, K+, Li+, Mg2+, Zn2+, Ca2+, Mn2+, Cu2+, and Fe2+. PcMdl was identified to be strictly specific to mono- and diacylglycerol, but not triacylglycerol. Stereographic view of PcMdl docked with substrate (tri- or diacylglycerol) analogue indicated that the residue Phe256 plays an important role in conferring the substrate selectivity. Phe256 projects its side chain towards the substrate binding groove and makes the sn-1 moiety difficult to insert in. Furthermore, sn-1 moiety prevents the phosphorus atom (substitution of carboxyl carbon) from getting to the Oγ of Ser145, which results in the failure of triacylglycerol hydrolysis. These results should provide a basis for molecular engineering of PcMdl and expand its applications in industries.
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 2) Jiangsu Provincial Engineering Laboratory for Biomass Conversion and Process Integration, School of Life Science and Chemical Engineering, Huaiyin Institute of Technology, Huai'an China.
authors: Zhong-Biao
authors: ZB
authors: Tan
authors: School of Food Science and Technology, Jiangnan University, Wuxi, China.
authors: Jian-Fang
authors: JF
authors: Li
authors: Wuxi Medical School, Jiangnan University, Wuxi, China.
authors: Xue-Ting
authors: XT
authors: Li
authors: Wuxi Medical School, Jiangnan University, Wuxi, China.
authors: Ying
authors: Y
authors: Gu
authors: Wuxi Medical School, Jiangnan University, Wuxi, China.
authors: Min-Chen
authors: MC
authors: Wu
authors: Wuxi Medical School, Jiangnan University, Wuxi, China.
authors: Jing
authors: J
authors: Wu
authors: Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, China.
authors: Jun-Qing
authors: JQ
authors: Wang
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D002614:Chelating Agents / Q000737:chemistry; D004075:Diglycerides / Q000737:chemistry; D004492:Edetic Acid / Q000737:chemistry; D004795:Enzyme Stability; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007501:Iron / Q000737:chemistry; D008628:Mercury / Q000737:chemistry; D062105:Molecular Docking Simulation; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D008994:Monoacylglycerol Lipases / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D050178:Monoglycerides / Q000737:chemistry; D010407:Penicillium / Q000201:enzymology*; D010843:Pichia; D011485:Protein Binding; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D002614:Chelating Agents; D004075:Diglycerides; D050178:Monoglycerides; D004492:Edetic Acid; D007501:Iron; D008994:Monoacylglycerol Lipases; D008628:Mercury