Molecular aspects of <i>β</i>-ketoacyl synthase (KAS) catalysis

Wettstein-Knowles, Penny von; Olsen, Johan G.; McGuire, Kirsten Arnvig; Larsen, S.

doi:10.1042/bst0280601

Cited by 25 publications

(14 citation statements)

References 0 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A previous study comparing decarboxylation activities of the E. coli enzymes revealed that, in contrast with KAS I, no acetyl‐ACP was recovered in KAS II assays even after 30 min at pH 6.8 [9,27]. That the KAS II enzyme was active in decarboxylation was confirmed in elongation assays that resulted in the synthesis of long‐chain acyl‐ACPs.…”

Section: Resultsmentioning

confidence: 84%

Fatty acid synthesis

Wettstein-Knowles¹,

Olsen

McGuire³

et al. 2006

The FEBS Journal

View full text Add to dashboard Cite

The formation of carbon-carbon bonds is a fundamental biochemical reaction. A number of enzymes involved in various biosynthetic pathways accomplish this by different means. Among these is a large family of enzymes involved in synthesis of fatty acids, waxes, flavins, natural drugs, and antibiotics making carboncarbon bonds by use of the Claisen condensation principle. Initially, an active site nucleophile induces a transesterification by nucleophilic attack on an acylthioester substrate. In the second step, a b-carbanion thioester is generated by either proton abstraction or decarboxylation. This strong nucleophile then attacks the carbonyl carbon of the first ester, resulting in a b-keto product (Scheme I). b-Ketoacyl-acyl carrier protein (ACP) synthase {3-oxoacyl-[acyl-carrier-protein] synthase (E.C. 2.3.1.41)} I (KAS I) and KAS II from Escherichia coli represent a set of decarboxylating condensing enzymes, which we refer to as the CHH b-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 b-ketoacyl-ACP synthases, including mitochondrial b-ketoacyl-ACP synthase, bacterial plus plastid b-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 coli b-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 N e , directed only towards the active site and malonyl-ACP binding area in the fatty acid complex.Abbreviations ACP, acyl carrier protein; KAS, b-ketoacyl-ACP synthase; WT-C8, KAS I-octanoyl complex.

show abstract

Section: Resultsmentioning

confidence: 84%

Fatty acid synthesis

Wettstein-Knowles¹,

Olsen

McGuire³

et al. 2006

The FEBS Journal

View full text Add to dashboard Cite

show abstract

“…This is inferred from the findings that fabF strains have no apparent growth phenotype and that fabB mutants require only unsaturated fatty acids for growth. In vitro tests with purified FabF and FabB proteins show that both enzymes are capable of catalyzing the condensation of malonyl-ACP and cis-3-decenoyl-ACP (24,55). The discrepancy between the genetic and biochemical observations indicates that the biochemical characterization of these enzymes does not accurately reflect their behavior in vivo.…”

Section: Discussionmentioning

confidence: 88%

“…Recently, it has been shown that FabF effectively titrates FabD (malonyl coenzyme A:ACP transacylase) and that overproduction of FabD offsets the toxicity of fabF plasmids (51). The exact role of FabB in unsaturated fatty acid biosynthesis has not been directly demonstrated in vitro and currently seems to be a puzzle (41,55). FabB is generally thought to catalyze elongation of the cis-3-decanoyl-ACP produced by FabA to 3-ketododecanoyl-ACP.…”

Section: Discussionmentioning

confidence: 99%

Escherichia coli FadR Positively Regulates Transcription of the fabB Fatty Acid Biosynthetic Gene

Campbell¹,

2001

View full text Add to dashboard Cite

In Escherichia coli expression of the genes of fatty acid degradation (fad) is negatively regulated at the transcriptional level by FadR protein. In contrast the unsaturated fatty acid biosynthetic gene, fabA, is positively regulated by FadR. We report that fabB, a second unsaturated fatty acid biosynthetic gene, is also positively regulated by FadR. Genomic array studies that compared global transcriptional differences between wild-type and fadR-null mutant strains, as well as in cultures of each strain grown in the presence of exogenous oleic acid, indicated that expression of fabB was regulated in a manner very similar to that of fabA expression. A series of genetic and biochemical tests confirmed these observations. Strains containing both fabB and fadR mutant alleles were constructed and shown to exhibit synthetic lethal phenotypes, similar to those observed in fabA fadR mutants. A fadR strain was hypersensitive to cerulenin, an antibiotic that at low concentrations specifically targets the FabB protein. A transcriptional fusion of chloramphenicol acetyltransferase (CAT) to the fabB promoter produces lower levels of CAT protein in a strain lacking functional FadR. The ability of a putative FadR binding site within the fabB promoter to form a complex with purified FadR protein was determined by a gel mobility shift assay. These experiments demonstrate that expression of fabB is positively regulated by FadR.

show abstract

“…The closest relatives to the FAE family by sequence homology and by function are the chalcone synthases/stilbene synthases and then different types of β‐ketoacyl‐ACP synthases (KAS), which are parts of fatty acid synthase (FAS) complexes (Plate ). Structure and biochemical mechanism of chalcone synthase as well as of several β‐keto fatty acid synthases have been elucidated by X‐ray crystallographic studies 35–42. Superimposition of the known three‐dimensional structures reveals an active site cysteine, a histidine and an asparagine (or another histidine) present in all crystal structures.…”

Section: Discussionmentioning

confidence: 99%

Flufenacet herbicide treatment phenocopies the fiddlehead mutant in Arabidopsis thaliana

Lechelt-Kunze

Meissner

Drewes

et al. 2003

Pest Management Science

View full text Add to dashboard Cite

In order to study the mode of action of herbicides we conducted a pilot study analysing phenotype and gene expression of flufenacet- and benfuresate-treated Arabidopsis thaliana (L) Heynhoe plants. Treatments with either herbicide caused phenocopies of the known Arabidopsis mutant fiddlehead, displaying fused organs and the typical fiddlehead-like inflorescence. Herbicide treatments of other plant species, including monocots, also gave rise to analogous organ fusions, indicating the presence of the target in a broad range of plants. Furthermore, many other herbicides with a proposed similar mode of action, eg chloroacetanilides, produced comparable fusion phenotypes in plants. The fiddlehead gene encodes a putative very-long-chain fatty acid elongase (VLCFAE), which corroborates earlier biochemical results pointing to the inhibition of VLCFA synthesis as mode of action of flufenacet. Gene expression profiles of herbicide-treated plants using the first 8247 gene Arabidopsis gene array of Affymetrix provided additional clues in support of inhibition of VLCFA synthesis. We discuss fiddlehead-like elongases as plant specific targets for flufenacet and many other herbicides.

show abstract

Molecular aspects of β-ketoacyl synthase (KAS) catalysis

Cited by 25 publications

References 0 publications

Fatty acid synthesis

Fatty acid synthesis

Escherichia coli FadR Positively Regulates Transcription of the fabB Fatty Acid Biosynthetic Gene

Flufenacet herbicide treatment phenocopies the fiddlehead mutant in Arabidopsis thaliana

Contact Info

Product

Resources

About