Abstract
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–cofactor interactions, highlighting the role of protein–ligand interactions in altering cofactor and binding site residue conformations, thus contributing to improved biocatalysis.
Highlights
Inducible amino acid decarboxylases have been implicated in various cellular processes in microorganisms and plants [1], for example, they regulate acid induced stress in bacterium existing in the stomach and urinary tract of microorganisms
Microbial lysine decarboxylases (LDCs) have been extensively studied and have been categorized into two distinct groups: the constitutive LDCs, which are essential in cellular metabolic processes and pH sensitive inducible LDCs involved in the conversion of lysine to cadaverine [7], a polyamine which modulates cellular pH, and is associated with the adaptation of microorganisms like Escherichia coli, Vibrio cholerae, and Salmonella enterica to acidic conditions, forming part of outer cell membranes of Gram-negative bacteria by interacting with peptidoglycan
The Phylogenetic tree revealed that the Serratia marcesenes lysine decarboxylase (SmcadA) had a high similarity to LDCs from Enterobacteriaceae, Klebsiella sp., pantoea deleyi, Kluyvera ascorbate, Salmonella sp., Raoultella sp., Escherichia coli sp., and Metakosakonia massiliensis (Figure 1A). this means that LDCs from the above strains evolved from the same ancestry and could be involved in related cellular processes
Summary
Inducible amino acid decarboxylases have been implicated in various cellular processes in microorganisms and plants [1], for example, they regulate acid induced stress in bacterium existing in the stomach and urinary tract of microorganisms. Microbial lysine decarboxylases (LDCs) have been extensively studied and have been categorized into two distinct groups: the constitutive LDCs, which are essential in cellular metabolic processes and pH sensitive inducible LDCs involved in the conversion of lysine to cadaverine [7], a polyamine which modulates cellular pH, and is associated with the adaptation of microorganisms like Escherichia coli, Vibrio cholerae, and Salmonella enterica to acidic conditions, forming part of outer cell membranes of Gram-negative bacteria by interacting with peptidoglycan It is necessary for membrane integrity [8,9] and is required for biofilm formation [10]. Coupled with increased interest in understanding the mechanisms of protein–protein, protein–ligand interactions, and allosteric orientations, we applied molecular dynamics simulation to gain insight into the structure–functional mechanism of protein–cofactor interactions for the wild-type and two site-directed mutations (Ser512Ala and Arg595Lys) constructed and characterized in our related study [18]
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