THE CRYSTAL STRUCTURE OF b-Ketoacyl-[ACP] synthase I FROM Escherichia coli

Johan Gotthardt Olsen and Anders Kadziola and Penny von Wettstein-Knowles¹ and Sine Larsen²

¹Center for Crystallographic Studies, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen O, University of Copenhagen,
² Institute for Molecular Biology, University of Copenhagen, Oster Farigmagsgade 2A, DK-1353 Copenhagen K\\

The b-Ketoacyl-[acyl carrier protein] synthases (KAS's) are the key enzymes in the fatty acid biosynthetic pathway. They function by transfering a fatty acyl moiety from acyl-acyl carrier protein (acyl-ACP) to its own active site cysteine residue where it is bound as a thioester. Subsequently, the enzyme catalyses a decarboxylation of an ACP bound malonyl moiety and the resulting carbanion attacks the bound fatty acyl residue at the thioester link, resulting in the product 3-oxoacyl-ACP. Apart from KAS III which exclusively catalyses the first condensation step, two distinctly different KAS enzymes exist, KAS I and KAS II, showing 38 \% sequence identity. In vitro, KAS I is capable of catalysing all the condensation reactions in the fatty acid biosynthetic pathway albeit with large variations in efficiency depending on substrate length and degree of saturation. In vivo the picture is different. Genetic studies have shown that the presence of KAS II is necessary for for the formation of cis-11-octadecenoic acid (C18:1) whereas KAS I is essential at the branching point of the pathway that leads to the synthesis of unsaturated fatty acids.

The crystallisation of KAS I has been reported earlier [Olsen et al. (1995) Prot. Pept. Lett. 1 (246-251)]. However, the numerous attempts to determine the structure by the multiple isomorphous replacement method were futile. The structure determination was finally accomplished by molecular replacement using the structure of KAS II as a search model.

The four independent molecules in the asymmetric unit are organised as two dimers. The overall fold is similar to that of KAS II [Huang et al. (1998) EMBO J 17 (1183-1191)] and the core a-b-a-b-a 'double whopper' structure is also found in yeast peroxisomal thiolase of the b-oxidation pathway, an enzyme which essentially carries out a reverse condensation reaction.

The active site is lined with residues which are conserved throughout all fatty acid condensing enzymes. The structural make-up of the active site crevice together with biochemical results and mutant studies provides us with enough information to suggest a possible mechanism of catalysis.