Characterization of Streptococcus pneumoniae enoyl-(acyl-carrier protein) reductase (FabK).
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.
Marrakchi
Hedia
H
Protein Science Division, Department of Infectious Disease, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
Dewolf
Walter E
WE
Quinn
Chad
C
West
Joshua
J
Polizzi
Brian J
BJ
So
Chi Y
CY
Holmes
David J
DJ
Reed
Shannon L
SL
Heath
Richard J
RJ
Payne
David J
DJ
Rock
Charles O
CO
Wallis
Nicola G
NG
D000595:Amino Acid Sequence; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D009243:NAD / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016415:Sequence Alignment; D013296:Streptococcus pneumoniae / Q000201:enzymology* / Q000235:genetics
D009097:Multienzyme Complexes; D009243:NAD; D010088:Oxidoreductases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases