Functional studies on wild-type and mutant Escherichia coli spermidine synthase by HPLC

Abstract

Escherichia coli (E. coli) is a Gram-negative, facultative anaerobic bacterium that is generally found in the intestine of warm-blooded organisms. Whole genome sequencing in E. coli strain K12 has been completed in 1997. The speE gene from E. coli strain K12, coded for spermidine synthase (EcSpdS), shares an operon with speD gene (coded for S-adenosyl-methionine decarboxylase). Spermidine synthase catalyzes the production of spermidine (SPD) from putrescine (PUT) and decarboxylated S-adenosylmethionine (dcSAM) in polyamine biosynthesis. The deduced amino acid sequence of the EcSpdS shares 30-36 % sequence identities with most spermidine synthases from mammalian cells, plants and bacteria. In this study, the speE gene (864 bases pair, coded for EcSpdS) was cloned into the pQE30 vector and over-expressed the 6x His-tagged recombinant EcSpdS in Escherichia coli strain SG13009. The over-expressed protein (33.8 kDa) was purified by Ni-NTA affinity chromatography at a yield of 30 mg/L of bacteria culture. Enzyme activities were determined through HPLC to detect whether product spermidine formed or reactant putrecine consumed after reaction. Optimal pH and temperature for the EcSpdS reaction were between pH 7 - 8 and 37 - 43℃. The apparent Km values for putrescine and dcSAM were 122.1 μM and 109.0 μM, respectively. Increasing concentrations of the first substrate, dcSAM, gave an array of parallel lines (in Lineweavre-Burk plot) for the second substrate, putrescine, kinetics and vice versa, suggesting a ping pong mechanism for EcSpdS reaction. The enzyme activities from EcSpdS mutants (D158A, C159A, C159S, T160A, P161D, P162A, I163A, P165Q), one residue different from wild type EcSpdS in gate-keeping loop region for each, were determined by both TLC and HPLC. The results showed that a complete loss of enzyme activity occurred in D158A and D161A mutants. Mutant protein, C159A, T160A and P165Q reduced enzyme catalytic efficiency to 77%, 61% or 57%, respectively. Up to present, the content of endogenous spermidine synthase was not reported in any SpdS species. Endogenous EcSpdS protein was estimated about 0.083% (1/1250) in total cell lysate after western blotting along with anti-EcSpdS serum. In contrast, less amount (0.031%) of endogenous spermidine synthase (incomplete gate keeping region in structure) in total cell lysate from Helicobacter pylori after western blotting along with anti-HpSpdS serum. After immunoprecipitation (IP), only one-third of endogenous EcSpdS or HpSpdS was recovered after the detection with western blotting.