Penny von Wettstein-Knowles

The X‐ray crystal structure of β‐ketoacyl [acyl carrier protein] synthase I

The crystal structure of the fatty acid elongating enzyme β‐ketoacyl [acyl carrier protein] synthase I (KAS I) from Escherichia coli has been determined to 2.3 Å 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 αβαβα 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.

Structures of β-Ketoacyl-Acyl Carrier Protein Synthase I Complexed with Fatty Acids Elucidate its Catalytic Machinery

BACKGROUND beta-ketoacyl-acyl carrier protein synthase (KAS) I is vital for the construction of the unsaturated fatty acid carbon skeletons characterizing E. coli membrane lipids. The new carbon-carbon bonds are created by KAS I in a Claisen condensation performed in a three-step enzymatic reaction. KAS I belongs to the thiolase fold enzymes, of which structures are known for five other enzymes. RESULTS Structures of the catalytic Cys-Ser KAS I mutant with covalently bound C10 and C12 acyl substrates have been determined to 2.40 and 1.85 A resolution, respectively. The KAS I dimer is not changed by the formation of the complexes but reveals an asymmetric binding of the two substrates bound to the dimer. A detailed model is proposed for the catalysis of KAS I. Of the two histidines required for decarboxylation, one donates a hydrogen bond to the malonyl thioester oxo group, and the other abstracts a proton from the leaving group. CONCLUSIONS 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.

Genetic control of β-diketone and hydroxy-β-diketone synthesis in epicuticular waxes of barley

Five eceriferum, (cer) mutants in barley which influence β-diketone and hydroxy-β-diketone synthesis in spike and internode epicuticular waxes have been characterized. The mutation cer-u (69) blocks the synthesis of hydroxy-β-diketones and leads to a compensatory increase in the amount of β-diketones, indicating that β-diketones are precursors of the hydroxy-β-diketones. Furthermore, highly lobed wax plates were observed for the first time on barley lemmas, in addition to the characteristic wax tubes. Both diketone classes are selectively and proportionally reduced in the spike wax of cer-i (16), which has shorter wax tubes. The three mutants cer-c (36), -q (42), and -c,u (108) synthesize neither diketone class and form no wax tubes. In contrast to the variable composition of most individual barley wax classes, only a single β-diketone was identified, namely hentriacontan-14,16-dione.SummaryFive eceriferum, (cer) mutants in barley which influence β-diketone and hydroxy-β-diketone synthesis in spike and internode epicuticular waxes have been characterized. The mutation cer-u69 blocks the synthesis of hydroxy-β-diketones and leads to a compensatory increase in the amount of β-diketones, indicating that β-diketones are precursors of the hydroxy-β-diketones. Furthermore, highly lobed wax plates were observed for the first time on barley lemmas, in addition to the characteristic wax tubes. Both diketone classes are selectively and proportionally reduced in the spike wax of cer-i16, which has shorter wax tubes. The three mutants cer-c36, -q42, and -c,u108 synthesize neither diketone class and form no wax tubes. In contrast to the variable composition of most individual barley wax classes, only a single β-diketone was identified, namely hentriacontan-14,16-dione.

Identification and Molecular Characterization of the β-Ketoacyl-[Acyl Carrier Protein] Synthase Component of theArabidopsisMitochondrial Fatty Acid Synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, β-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C4-C18 fatty acids exhibiting a bimodal distribution with peaks at C8 and C14-C16. Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.

Fatty acid synthesis. Role of active site histidines and lysine in Cys-His-His-type beta-ketoacyl-acyl carrier protein synthases.

β‐Ketoacyl‐acyl carrier protein (ACP) synthase enzymes join short carbon units to construct fatty acyl chains by a three‐step Claisen condensation reaction. The reaction starts with a trans thioesterification of the acyl primer substrate from ACP to the enzyme. Subsequently, the donor substrate malonyl‐ACP is decarboxylated to form a carbanion intermediate, which in the third step attacks C1 of the primer substrate giving rise to an elongated acyl chain. A subgroup of β‐ketoacyl‐ACP synthases, including mitochondrial β‐ketoacyl‐ACP synthase, bacterial plus plastid β‐ketoacyl‐ACP synthases I and II, and a domain of human fatty acid synthase, have a Cys‐His‐His triad and also a completely conserved Lys in the active site. To examine the role of these residues in catalysis, H298Q, H298E and six K328 mutants of Escherichia coliβ‐ketoacyl‐ACP synthase I were constructed and their ability to carry out the trans thioesterification, decarboxylation and/or condensation steps of the reaction was ascertained. The crystal structures of wild‐type and eight mutant enzymes with and/or without bound substrate were determined. The H298E enzyme shows residual decarboxylase activity in the pH range 6–8, whereas the H298Q enzyme appears to be completely decarboxylation deficient, showing that H298 serves as a catalytic base in the decarboxylation step. Lys328 has a dual role in catalysis: its charge influences acyl transfer to the active site Cys, and the steric restraint imposed on H333 is of critical importance for decarboxylation activity. This restraint makes H333 an obligate hydrogen bond donor at Nε, directed only towards the active site and malonyl‐ACP binding area in the fatty acid complex.

Structure of the human beta-ketoacyl [ACP] synthase from the mitochondrial type II fatty acid synthase.

Two distinct ways of organizing fatty acid biosynthesis exist: the multifunctional type I fatty acid synthase (FAS) of mammals, fungi, and lower eukaryotes with activities residing on one or two polypeptides; and the dissociated type II FAS of prokaryotes, plastids, and mitochondria with individual activities encoded by discrete genes. The β‐ketoacyl [ACP] synthase (KAS) moiety of the mitochondrial FAS (mtKAS) is targeted by the antibiotic cerulenin and possibly by the other antibiotics inhibiting prokaryotic KASes: thiolactomycin, platensimycin, and the α‐methylene butyrolactone, C75. The high degree of structural similarity between mitochondrial and prokaryotic KASes complicates development of novel antibiotics targeting prokaryotic KAS without affecting KAS domains of cytoplasmic FAS. KASes catalyze the C2 fatty acid elongation reaction using either a Cys‐His‐His or Cys‐His‐Asn catalytic triad. Three KASes with different substrate specificities participate in synthesis of the C16 and C18 products of prokaryotic FAS. By comparison, mtKAS carries out all elongation reactions in the mitochondria. We present the X‐ray crystal structures of the Cys‐His‐His‐containing human mtKAS and its hexanoyl complex plus the hexanoyl complex of the plant mtKAS from Arabidopsis thaliana. The structures explain (1) the bimodal (C6 and C10–C12) substrate preferences leading to the C8 lipoic acid precursor and long chains for the membranes, respectively, and (2) the low cerulenin sensitivity of the human enzyme; and (3) reveal two different potential acyl‐binding‐pocket extensions. Rearrangements taking place in the active site, including subtle changes in the water network, indicate a change in cooperativity of the active‐site histidines upon primer binding.

The Inhibitor of wax 1 locus (Iw1) prevents formation of β‐ and OH‐β‐diketones in wheat cuticular waxes and maps to a sub‐cM interval on chromosome arm 2BS

Glaucousness 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 F2 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, β-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 BC2 F3 near-isogenic lines, we show that Iw1 inhibits the formation of β- and hydroxy-β-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 C24 primary alcohols. No differences were found in cuticle thickness and carbon isotope discrimination in near-isogenic lines differing at Iw1.

Ultrastructure and origin of epicuticular wax tubes

Coiled wax ribbons have been found intermingled among the long, thin tubes characteristic of some barley and wheat cuticles. An analysis of the structure of these wax ribbons and their relationship to the tubes has led to the following hypothesis to explain the origin of the broad spectrum of observed epicuticular wax structures. Upon contact with air the wax extruded through pores onto the cuticle surface polymerizes. Fusion of wax from adjacent pores occurs before polymerization is complete. Continuous exudation of new wax pushes the initial wax away from the surface, thus leaving the most recent wax at the base of the structures. The particular morphologies produced depend on chemical composition of the exudate, as well as the number, closeness and arrangement of the pores, and the rate of exudation. These parameters are controlled by genes in interaction with the environment.

The physico-chemical basis of leaf wettability in wheat.

SummaryWild type wheat (Triticum aestivum L.) and three mutant lines that have reduced glaucousness on the flag leaf sheath have been examined for variations in glaucousness, contact angles, wax chemistry and wax morphology. On the sheath and culm, organs that are glaucous in the wild type, increasing glaucousness is correlated with increasing contact angles, an increasing proportion of β-diketones plus hydroxy-β-diketones in the was and an increasing proportion of wax tubes. Organs that were non-glaucous in all four lines, namely both surfaces of the vegetative leaves and the adaxial surface of the flag leaf, had high contact angles, a dense covering of wax plates and waxes rich in primary alcohols but devoid of β-diketones and hydroxy-β-diketones. The abaxial surface of the flag leaf was the most complex of the organ surfaces studied. In the wild type the glaucousness of the sheath continued onto this surface for 1–2 cm and this was correlated with the other characters studied as it was on the sheath. In the mutants, however, the tubes were absent. Flat ribbons of varying widths, a new wax structure in wheat, as well as various types of plates were found instead. These structures continued to the flag leaf tip and were also present on the abaxial surface of the wild type flag leaf. Changes in contact angle at the tip could not be correlated with the other measured parameters.

Biosynthesis of β-diketones and hydrocarbons in barley spike epicuticular wax☆

Abstract Aided by the analysis of induced, single gene mutants in barley, independent elongation systems were inferred for the synthesis of β-diketones (98% hentriacontan-14,16-dione) and hydrocarbons (primarily hentriacontane). This proposal has been substantiated by comparing the effects of preincubations of inhibitors on the ability of whole spikes to incorporate [2- 14 C]acetate into the various epicuticular wax lipids. Dithiothreitol and mercaptoethanol inhibited the incorporation of label into hydrocarbons, but not into β-diketones. Cyanide blocked the synthesis of β-diketones, while stimulating hydrocarbon formation more than twofold. β-diketone synthesis was far more sensitive to arsenite than was synthesis of hydrocarbons. Degradation of the asymmetric β-diketone molecules by base hydrolysis and determination of the amount of label in the resulting fragments revealed a specific inhibition by arsenite of label incorporation into the C-31 end, i.e., the end from which previous studies have shown elongation to proceed. Tissue slices prepared from spikes minus awns were able to incorporate into β-diketones 1 abel from [1- 14 C]palmitate and shorter evenchained fatty acids, but not from [1- 14 C]stearate. However, all fatty acids tested served equally well as hydrocarbon precursors. The elongation systems leading to the β-diketones and hydrocarbons are thought to diverge when the chain has 16 carbons. Thus, when a C 16 chain is elongated by the addition of a C 2 unit to form a C 18 β-keto acyl chain, the β-keto group is not reduced as in normal fatty acid synthesis, but both carbonyl groups are protected and retained during further elongation. After reaching a C 32 chain length, decarboxylation and release of the protected carbonyl groups yield the β-diketone.