Characterization of the beta-carbon processing reactions of the mammalian cytosolic fatty acid synthase: role of the central core.
The properties of the beta-ketoacyl reductase, dehydrase, and enoyl reductase components of the animal fatty acid synthase responsible for the reduction of the beta-ketoacyl moiety formed at each round of chain elongation have been studied by engineering and characterizing mutants defective in each of these three catalytic domains. These "beta-carbon processing" mutants leak the stalled four-carbon intermediates by direct transfer to CoA. However, enoyl reductase mutants leak beta-ketobutyryl, beta-hydroxybutyryl, and crotonyl moieties, a finding explained, at least in part, by the observation that the equilibrium and rate constant for the dehydrase reaction favor the formation of beta-hydroxy rather than enoyl moieties. In this regard, the type I animal fatty acid synthase resembles its type II counterpart in Escherichia coli in that both systems rely on the enoyl reductase to pull the beta-carbon processing reactions to completion. Kinetic and nucleotide binding measurements on fatty acid synthases mutated in either of the two nucleotide binding domains revealed that the NADPH binding sites are nonidentical, the enoyl reductase exhibiting higher affinity. Surprisingly, NADPH binding is also completely compromised by certain deletions and mutations in the central core region distant from the nucleotide binding sites. Comparable central core sequences are present in the structurally related modular polyketide synthases, except in those modules that lack all three beta-carbon processing enzymes. These findings suggest that the central core region of fatty acid and polyketide synthases plays an important role in facilitating the beta-carbon processing reactions.
Children's Hospital Oakland Research Institute, 5700 Martin Luther King, Jr., Way, Oakland, California 94609, USA.
Andrzej
A
Witkowski
Anil K
AK
Joshi
Stuart
S
Smith
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002244:Carbon / Q000378:metabolism*; D002384:Catalysis; D003600:Cytosol / Q000201:enzymology*; D019281:Dimerization; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D004926:Escherichia coli / Q000201:enzymology; D064429:Fatty Acid Synthases / Q000378:metabolism*; D005227:Fatty Acids / Q000096:biosynthesis*; D007700:Kinetics; D008322:Mammals / Q000378:metabolism*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D011487:Protein Conformation
D005227:Fatty Acids; D009249:NADP; D002244:Carbon; D010088:Oxidoreductases; D015094:3-Hydroxyacyl CoA Dehydrogenases; D050753:Enoyl-(Acyl-Carrier-Protein) Reductase (NADH); D064429:Fatty Acid Synthases