Crystal structure of a triacylglycerol lipase from Penicillum expansum at 1.3 A determined by sulfur SAD

Fujian Institute of Research on the Structure of Matter, Chinese Academy of Sciences, Fuzhou, China.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.63). 05/2010; 78(6):1601-5. DOI: 10.1002/prot.22676
Source: PubMed
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Available from: Mingdong Huang, Sep 21, 2014
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    • "These three lipases have identical sequences. The crystal structure has been solved for one of the two P. expansum lipases discussed above (AAK07480; Bian et al. 2010) and this enabled determining the position of the residues, Ser (159)–Asp (215)–His (268), which constitute the catalytic triad of LipPE (Fig. 3). A multiple sequence alignment of LipPE and other four lipases with the highest identity demonstrated that the active-site sequence (GHSLG) was identical at the amino acid level (supplementary Fig. 1). "
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    ABSTRACT: Cold-active lipases are of significant interest as biocatalysts in industrial processes. We have identified a lipase that displayed activity towards long carbon-chain-p-nitrophenyl substrates (C12-C18) at 25 °C from the culture supernatant of an Antarctic Penicillium expansum strain assigned P. expansum SM3. Zymography revealed a protein band of around 30 kDa with activity towards olive oil. DNA fragments of a lipase gene designated as lipPE were isolated from the genomic DNA of P. expansum SM3 by genomic walking PCR. Subsequently, the complete genomic lipPE gene was amplified using gene-specific primers designed from the 5'- and 3'-regions. Reverse transcription PCR was used to amplify the lipPE cDNA. The deduced amino acid sequence consisted of 285 residues that included a predicted signal peptide. Three peptides identified by LC/MS/MS analysis of the proteins in the culture supernatant of P. expansum were also present in the deduced amino acid sequence of the lipPE gene suggesting that this gene encoded the lipase identified by initial zymogram activity analysis. Full analysis of the nucleotide and the deduced amino acid sequences indicated that the lipPE gene encodes a novel P. expansum lipase. The lipPE gene was expressed in E. coli for further characterization of the enzyme with a view of assessing its suitability for industrial applications.
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    ABSTRACT: Error-prone PCR was used to create more active or enantioselective variants of Penicillium expansum lipase (PEL). A variant with a valine to glycine substitution at residue 72 in the lid structure exhibited higher activity and enantioselectivity than those of wild-type PEL. Site-directed saturation mutagenesis was used to explore the sequence-function relationship and the substitution of Val72 of P. expansum lipase changed both catalytic activity and enantioselectivity greatly. The variant V72A, displayed a highest enantioselectivity enhanced to about twofold for the resolution of (R, S)-naproxen (E value increased from 104 to 200.7 for wild-type PEL and V72A variant, respectively). In comparison to PEL, the variant V72A showed a remarkable increase in specific activity towards p-nitrophenyl palmitate (11- and 4-fold increase at 25 and 35 °C, respectively) whereas it had a decreased thermostability. The results suggest that the enantioselective variant V72A could be used for the production of pharmaceutical drugs such as enantiomerically pure (S)-naproxen and the residue Val 72 of P. expansum lipase plays a significant role in the enantioselectivity and activity of this enantioselective lipase.
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