Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK)

Abstract

The enoyl-(acyl-carrier protein) (ACP) reductase catalyses the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. An extensively characterized NADH-dependent reductase, FabI, is widely distributed in bacteria and plants, whereas the enoyl-ACP reductase, FabK, is a distinctly different member of this enzyme group discovered in Streptococcus pneumoniae. We were unable to delete the fabK gene from Strep. pneumoniae, suggesting that this is the only enoyl-ACP reductase in this organism. The FabK enzyme was purified and the biochemical properties of the reductase were examined. The visible absorption spectrum of the purified protein indicated the presence of a flavin cofactor that was identified as FMN by MS, and was present in a 1:1 molar ratio with protein. FabK specifically required NADH and the protein activity was stimulated by ammonium ions. FabK also exhibited NADH oxidase activity in the absence of substrate. Strep. pneumoniae belongs to the Bacillus / Lactobacillus / Streptococcus group that includes Staphylococcus aureus and Bacillus subtilis. These two organisms also contain FabK-related genes, suggesting that they may also express a FabK-like enoyl-ACP reductase. However, the genes did not complement a fabI (Ts) mutant and the purified flavoproteins were unable to reduce enoyl-ACP in vitro and did not exhibit NAD(P)H oxidase activity, indicating they were not enoyl-ACP reductases. The restricted occurrence of the FabK enoyl-ACP reductase may be related to the role of substrate-independent NADH oxidation in oxygen-dependent anaerobic energy metabolism.

Genome of the Opportunistic Pathogen Streptococcus sanguinis

“…Lipid biosynthesis apparently follows the classical bacterial type II fatty acid synthase complex pathway (34). As shown previously for S. pneumoniae (33,57), S. sanguinis encodes the enoyl-(acyl-carrier protein) reductase (EC 1.3.1.9) FabK instead of the widespread and conserved FabI type enzyme of other bacteria and plants. The FabK enzyme of S. pneumoniae is less sensitive to inhibition by the antimicrobial triclosan than FabI is (33,57).…”

“…As shown previously for S. pneumoniae (33,57), S. sanguinis encodes the enoyl-(acyl-carrier protein) reductase (EC 1.3.1.9) FabK instead of the widespread and conserved FabI type enzyme of other bacteria and plants. The FabK enzyme of S. pneumoniae is less sensitive to inhibition by the antimicrobial triclosan than FabI is (33,57). Therefore, S. sanguinis is probably more resistant than FabI-containing bacteria to inhibition of lipid biosynthesis by the triclosan used in some toothpastes.…”

Antibacterial targets in fatty acid biosynthesis

“…However, enthusiasm for development of inhibitors of FabI is somewhat tempered by the existence of a structurally and mechanistically unrelated enzyme, FabK, that can catalyze the same reaction as FabI in some important pathogens [22]. A recently developed inhibitor of FabI API-1252 (Fig.…”

Transcriptional Regulation of Fatty Acid Biosynthesis in Lactococcus lactis

“…This ␤-ketoacyl-ACP is dehydrated by FabZ (␤-hydroxyacyl-ACP dehydratase), resulting in a trans-2-enoyl-ACP (5, 6). The final step in lactococcal FAB elongation is a second reduction step executed by trans-2-enoyl-ACP reductase I FabI, giving an acyl-ACP (7,8). Further elongation rounds start by the condensation enzyme FabF ␤-ketoacyl-ACP synthase II through the addition of an acyl group from malonyl-ACP (9, 10).…”