The same mechanism is proposed for KAS II, which also has a Cys-His-His active site triad. Comparison to the active site architectures of other thiolase fold enzymes carrying out a decarboxylation step suggests that chalcone synthase and KAS III with Cys-His-Asn triads use another mechanism in which both the histidine and the asparagine interact with the thioester oxo group. The acyl binding pockets of KAS I and KAS II are so similar that they alone cannot provide the basis for their differences in substrate specificity.

HighlightThe 101kb Cer-cqu gene cluster, encoding CER-C (a type III chalcone synthase-like diketone synthase), CER-Q (a lipase) and CER-U (a P450 enzyme), determines significant constituents of plant apoplasts.

The crystal structure of the fatty acid elongating enzyme L L-ketoacyl [acyl carrier protein] synthase I (KAS I) from Escherichia coli has been determined to 2.3 A î resolution by molecular replacement using the recently solved crystal structure of KAS II as a search model. The crystal contains two independent dimers in the asymmetric unit. KAS I assumes the thiolase K KL LK KL LK K fold. Electrostatic potential distribution reveals an acyl carrier protein docking site and a presumed substrate binding pocket was detected extending the active site. Both subunits contribute to each substrate binding site in the dimer.z 1999 Federation of European Biochemical Societies.

SUMMARYGlaucousness is described as the scattering effect of visible light from wax deposited on the cuticle of plant aerial organs. In wheat, two dominant genes lead to non-glaucous phenotypes: Inhibitor of wax 1 (Iw1) and Iw2. The molecular mechanisms and the exact extent (beyond visual assessment) by which these genes affect the composition and quantity of cuticular wax is unclear. To describe the Iw1 locus we used a genetic approach with detailed biochemical characterization of wax compounds. Using synteny and a large number of F 2 gametes, Iw1 was fine-mapped to a sub-cM genetic interval on wheat chromosome arm 2BS, which includes a single collinear gene from the corresponding Brachypodium and rice physical maps. The major components of flag leaf and peduncle cuticular waxes included primary alcohols, b-diketones and n-alkanes. Small amounts of C19-C27 alkyl and methylalkylresorcinols that have not previously been described in wheat waxes were identified. Using six pairs of BC 2 F 3 near-isogenic lines, we show that Iw1 inhibits the formation of b-and hydroxy-b-diketones in the peduncle and flag leaf blade cuticles. This inhibitory effect is independent of genetic background or tissue, and is accompanied by minor but consistent increases in n-alkanes and C 24 primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near-isogenic lines differing at Iw1.

beta-Ketoacyl-[acyl carrier protein (ACP)] synthase forms new carbon-carbon bonds in three steps: transfer of an acyl primer from ACP to the enzyme, decarboxylation of the elongating substrate and its condensation with the acyl primer substrate. Six residues of Escherichia coli beta-ketoacyl-ACP synthase I (KAS I) implicated in these reactions were subjected to site-directed mutagenesis. Analyses of the abilities of C163A, C163S, H298A, D306A, E309A, K328A, and H333A to carry out the three reactions lead to the following conclusions. The active site Cys-163 is not required for decarboxylation, whereas His-298 and His-333 are indispensable. Neither of the histidines is essential for increasing the nucleophilicity of Cys-163 to enable transfer of the acyl primer substrate. Maintenance of the structural integrity of the active site by Asp-306 and Glu-309 is required for decarboxylation but not for transfer. One function of Lys-328 occurs very early in catalysis, potentially before transfer. These results in conjunction with structural analyses of substrate complexes have led to a model for KAS I catalysis [Olsen, J. G., Kadziola, A., von Wettstein-Knowles, P., Siggaard-Andersen, M., and Larsen, S. (2001) Structure 9, 233-243]. Another facet of catalysis revealed by the mutant analyses is that the acyl primer transfer activity of beta-ketoacyl-ACP synthase I is inhibited by free ACP at physiological concentrations. Differences in the inhibitory response by individual mutant proteins indicate that interaction of free ACP with Cys-163, Asp-306, Glu-309, Lys-328, and His-333 might form a sensitive regulatory mechanism for the transfer of acyl primers.