Characterization of an Arginine Decarboxylase from Streptococcus pneumoniae by Ultrahigh-Performance Liquid Chromatography-Tandem Mass Spectrometry

Abstract

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography-tandem mass spectrometry (LC-MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min^-1, and 63,218 min^-1 mM^-1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

Keywords: DFMA; DFMO; SP_0166; Streptococcus pneumoniae; arginine decarboxylase; polyamine.

Figure 1

Polyamine biosynthesis pathway substrates and products.

Figure 2

Expression and purification of recombinant SP_0166. The overexpressed and purified recombinant 43 kDa SP_0166 from S. pneumoniae TIGR4 was resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis.

Figure 3

UHPLC–MSMS chromatograms of (A) agmatine, (B) cadaverine, and (C) putrescine, which are the decarboxylation products of arginine, lysine, and ornithine, respectively, and (D) spermidine-d8 (internal standard). Four separate single monitoring (SRM) transitions are shown for synthesized polyamine standards within 10 min running time.

Figure 4

SP_0166 is an arginine decarboxylase. SP_0166 functions as an arginine decarboxylase, demonstrating the highest relative activity with arginine as a substrate. It exhibits relatively lower activity with lysine and negligible activity with ornithine.

Figure 5

Optimization of reaction conditions for polyamine analysis by LC–MS/MS. Panel (A) depicts the impact of pH on the activity of recombinant SP_0166, while panel (B) presents the time course of agmatine and cadaverine production from arginine and lysine substrates, respectively.

Figure 6

LC–MS/MS analysis of SP_0166 enzyme kinetics. (A) Comparison of the enzyme kinetics for the conversion of arginine to agmatine (●), lysine to cadaverine (○), and ornithine to putrescine (▼) at different substrate concentrations. The lines represent curve-fitting for each point. (B) Comparison of kinetic parameters. The results shown are the means of triplicate experiments, and data represent mean ± standard deviation.

Figure 7

Inhibition of recombinant SP_0166 decarboxylase activity by DFMA and DFMO. Inhibition potency of different concentrations of (A) DFMA and (B) DFMO on polyamine synthesis from arginine (●), lysine (○), and ornithine (▼) substrates. Inhibition potency of different concentrations of (C) DFMA and (D) DFMO on synthesis of N-carbamoyl putrescine (●), ornithine (○), and putrescine (▼) from arginine. The results shown are the means of triplicate experiments, and data represent mean ± standard deviation.