Biochemical Characterization and Structural Insight into Interaction and Conformation Mechanisms of Serratia marcescens Lysine Decarboxylase (SmcadA)
| Autor: | Xian Zhang, Zhiming Rao, Tolbert Osire, Yang Taowei, Zhina Qiao, Meijuan Xu |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
structural conformation
Lysine Mutant Pharmaceutical Science lysine decarboxylase Serratia Cofactor Analytical Chemistry lcsh:QD241-441 03 medical and health sciences lcsh:Organic chemistry Drug Discovery Physical and Theoretical Chemistry Binding site 030304 developmental biology 0303 health sciences biology Lysine decarboxylase 030306 microbiology Chemistry Organic Chemistry cofactor interactions biology.organism_classification Enterobacteriaceae Biochemistry Chemistry (miscellaneous) Serratia marcescens biology.protein Molecular Medicine Serratia marcesenes |
| Zdroj: | Molecules, Vol 26, Iss 697, p 697 (2021) Molecules Volume 26 Issue 3 |
| ISSN: | 1420-3049 |
| Popis: | Inducible lysine decarboxylases (LDCs) are essential in various cellular processes of microorganisms and plants, especially under acid stress, which induces the expression of genes encoding LDCs. In this study, a novel Serratia marcesenes LDC (SmcadA) was successfully expressed in E. coli, purified and characterized. The protein had an optimal pH of 6 and a temperature of 40 ° C and phylogenetic analysis to determine the evolution of SmcadA, which revealed a close relation to Enterobacteriaceae, Klebsiella sp., among others. The molecular weight of SmcadA was approximately 75 kDa after observation on SDS-PAGE and structural modeling showed the protein as a decamer, comprised of five interlinked dimers. The biocatalytic activity of the purified wild-type SmcadA (WT) was improved through site directed mutations and the results showed that the Arg595Lys mutant had the highest specific activity of 286.55 U/mg, while the Ser512Ala variant and wild-type SmcadA had 215.72 and 179.01 U/mg, respectively. Furthermore, molecular dynamics simulations revealed that interactions through hydrogen bonds between the protein residues and cofactor pyridoxal-5-phosphate (PLP) are vital for biocatalysis. Molecular Dynamics (MD) simulations also indicated that mutations conferred structural changes on protein residues and PLP hence altered the interacting residues with the cofactor, subsequently influencing substrate bioconversion. Moreover, the temperature also induced changes in orientation of cofactor PLP and amino acid residues. This work therefore demonstrates the successful expression and characterization of the purified novel lysine decarboxylase from Serratia marcesenes and provided insight into the mechanism of protein&ndash cofactor interactions, highlighting the role of protein&ndash ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis. |
| Databáze: | OpenAIRE |
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