Search

[Display omitted] • Bacillus subtilis FabHA and FabHB vary in resistance to natural product inhibitors. • B. subtilis FabHA resembles Staphylococcal FabH. • B. subtilis FabHB resembles Escherichia coli FabH. Bacteria use the fatty acid composition of membrane lipids to maintain homeostasis of the bilayer. β-Ketoacyl-ACP synthase III (FabH) initiates fatty acid biosynthesis and is the primary determinant of the fatty acid composition. FabH condenses malonyl-acyl carrier protein with an acyl-Coenzyme A primer to form β -ketoacyl-acyl carrier protein which is used to make substrates for lipid synthesis. The acyl-Coenzyme A primer determines whether an acyl chain in the membrane has iso, anteiso, or no branching (straight chain) and biophysical properties of the membrane. The soil bacterium Bacillus subtilis encodes two copies of FabH (Bs FabHA and Bs FabHB), and here we solve their crystal structures. The substrate-free 1.85 Å and 2.40 Å structures of Bs FabHA and Bs FabHB show both enzymes have similar residues that line the active site but differ in the architecture surrounding the catalytic residues and oxyanion hole. Branching in the Bs FabHB active site may better accommodate the structure of an iso-branched acyl-Coenzyme A molecule and thus confer superior utilization to Bs FabHA for this primer type. The 2.02 Å structure of Bs FabHA•Coenzyme A shows how the active site architecture changes after binding the first substrate. The other notable difference is an amino acid insertion in Bs FabHB that extends a cap that covers the dimer interface. The cap topology is diverse across FabH structures and appears to be a distinguishing feature. FabH enzymes have variable sensitivity to natural product inhibitors and the availability of crystal structures help clarify how nature designs antimicrobials that differentially target FabH homologs. [ABSTRACT FROM AUTHOR]

Cyclolavandulyl diphosphate synthase (CLDS; estimated molecular weight 23.1 kDa) from the soil bacterium Streptomyces sp. CL190 is an enzyme that catalyzes both the condensation of two molecules of C5 dimethylallyl diphosphate (DMAPP) and the subsequent cyclization. CLDS was crystallized in the absence and the presence of the substrate DMAPP. Diffraction data were collected at a synchrotron source and the crystals diffracted to 2.00 and 1.73 Å resolution, respectively. The crystal obtained in the absence of DMAPP belonged to space group P212121, with unit-cell parameters a = 39.0, b = 87.5, c = 113.6 Å. The crystal obtained in the presence of DMAPP belonged to space group P1, with unit-cell parameters a = 46.9, b = 61.7, c = 82.2 Å, α = 74.0, β = 84.5, γ = 86.0°. [ABSTRACT FROM AUTHOR]

Highlights: [•] 20 Highly conserved residues in EloA were mutated. [•] Many mutants were inactive yet were expressed at levels comparable to the wild-type. [•] Mutation of the only conserved Cys did not abolish activity. [•] ELOs use a reaction mechanism distinct from that of the Claisen-like enzymes. [Copyright &y& Elsevier]

Autosomal-dominant Stargardt-like macular dystrophy [Stargardt3 (STGD3)] results from single allelic mutations in the elongation of very-long-chain fatty acids-like 4 (ELOVL4), whereas recessive mutations lead to skin and brain dysfunction. ELOVL4 protein localizes to the endoplasmic reticulum, where it mediates the condensation reaction catalyzing the formation of very-long-chain (VLC) (C-28 to C-40) fatty acids, saturated and polyunsaturated (PUFA). The defective gene product is truncated at the C terminus, leading to mislocalization and aggregation in other organelles. We hypothesized that the STGD3 truncated mutant may generate mislocalized, and therefore toxic, keto intermediates of fatty acid elongation, thereby contributing to the disease process. Using cell-based and cell-free microsome assays, we found that the truncated protein lacked innate condensation activity. Coexpression of different forms of wild-type and mutant ELOVL4 revealed a large dominant-negative effect of mutant protein on ELOVL4 localization and enzymatic activity, resulting in reduced VLC-PUFA synthesis. The reduction in VLC-PUFA levels in STGD3 and age-related macular degeneration may be a contributing factor to their retinal pathology. [ABSTRACT FROM AUTHOR]

Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine-aldimine complex to a substrate-aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

β-Ketoacyl-acyl carrier protein (ACP) synthase II (KASII) elongates 16:0-ACP to 18:0-ACP in the plastid, where it competes with three other enzymes at the first major branch point in fatty acid biosynthesis. Despite its key metabolic location, the influence of KASII in determining seed oil composition remains unclear, in part because the biochemical consequences of the fab1-1 mutation were unresolved. Thus, fab1-1, and a newly identified knockout allele, fab1-2, were analyzed in the context of the hypothesis that modulating KASII activity is sufficient to convert the composition of a temperate seed oil into that of a palm-like tropical oil. No homozygous fab1-2 individuals were identified in progeny of self-fertilized heterozygous fab1-2 plants, ≈1/4 of which aborted before the torpedo stage, suggesting that fab1-2 represents a complete loss of function and results in lethality when homozygous. Consistent with this hypothesis, homozygous fab1-2 plants were identified when a fab1-1 transgene was introduced, demonstrating that fab1-1 encodes an active KASII. Strong seed-specific hairpin-RNAi reductions in FAB1 expression resulted in abortion of ≈1/4 of the embryos in an apparent phenocopy of fab1-2 homozygosity. In less severe FAB1 hairpin-RNAi individuals, embryos developed normally and exhibited a 1:2:1 segregation ratio for palmitate accumulation. Thus, early embryo development appears sensitive to elevated 16:0, whereas at later stages, up to 53% of 16:0, i.e., a 7-fold increase over wild-type levels, is tolerated. These results resolve the role of KASII in seed metabolism and demonstrate that modulation of Arabidopsis KASII levels is sufficient to convert its temperate oilseed composition to that of a palm-like tropical oil. [ABSTRACT FROM AUTHOR]

Abstract: The very long chain fatty acids (VLCFA) incorporated into plant lipids are derived from the iterative addition of C2 units provided by malonyl-CoA to an acyl-CoA by the 3-ketoacyl-CoA synthase (KCS) component of a fatty acid elongase (FAE) complex. Mining of the Arabidopsis genome sequence database revealed 20 genes with homology to seed-specific FAE1 KCS. Eight of the 20 putative KCSs were cloned, expressed in yeast, and isolated as (His)6 fusion proteins. Five of the eight (At1g71160, At1g19440, At1g07720, At5g04530, and At4g34250) had little or no activity with C16 to C20 substrates while three demonstrated activity with C16, C18, and C20 saturated acyl-CoA substrates. At1g01120 KCS (KCS1) and At2g26640 KCS had broad substrate specificities when assayed with saturated and mono-unsaturated C16 to C24 acyl-CoAs while At4g34510 KCS was specific for saturated fatty acyl-CoA substrates. [Copyright &y& Elsevier]

Glucosinolates are a group of sulfur-rich thioglucoside natural products common in the Brassicaceae and related plant families. The first phase in the formation of many glucosinolates involves the chain extension of the amino acid methionine. Additional methylene groups are inserted into the side chain of methionine by a three-step elongation cycle involving 2-oxo acid intermediates. This investigation demonstrated the first step of this chain elongation cycle in a partially-purified preparation from arugula (Eruca sativa). The 2-oxo acid derived from methionine, 4-methylthio-2-oxobutanoic acid, was shown to condense with acetyl-CoA to form 2-(2′-methylthioethyl)malate. The catalyst, designated as a 2-(ω-methylthioalkyl)malate synthase, belongs to a family of enzymes that mediate the condensation of acyl-CoAs with 2-oxo acids, including citrate synthase of the citric acid cycle, and 2-isopropylmalate synthase of leucine biosynthesis. The 2-(ω-methylthioalkyl)malate synthase studied here shares properties with other enzymes of this class, but appears chromatographically distinct and is found only in extracts of plant species producing glucosinolates from chain-elongated methionine derivatives. Although the principal glucosinolates of arugula are formed from methionine that has undergone two rounds of chain elongation to form dihomomethionine, studies with substrates and substrate analogs of different chain lengths showed that the isolated enzyme is responsible only for the condensation step of the first round of elongation. [Copyright &y& Elsevier]

The major class of glucosinolates in Arabidopsis thaliana (L.) Heynh. are biosynthesized from methionine involving a three-step chain-elongation cycle. Each passage through the cycle results in the net addition of a single methylene group, with up to six cycles of elongation occurring in A. thaliana. The first reaction of the cycle is catalyzed by a methylthioalkylmalate synthase (MAMS), which condenses a ω-methylthio-2-oxoalkanoic acid with acetyl-CoA. Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. The Columbia ecotype is dominated by compounds that have undergone only two elongation cycles. The A. thaliana MAM1 protein exhibits basic sequence similarity to other previously described enzymes catalyzing the condensation of 2-oxo acids and acetyl-CoA, such as isopropylmalate synthase (EC 2.3.3.13), an enzyme of leucine biosynthesis, and homocitrate synthase (EC 2.3.3.14). It also shares similar properties with them, including the catalytic requirements for a divalent metal ion and an adenine nucleotide. However, the MAM1 protein does not show activity with the substrates of any of these other enzymes, and was chromatographically separable from isopropylmalate synthase in extracts of A. thaliana. Thus, MAM1 is exclusively an enzyme of secondary metabolism, distinct from primary metabolic enzymes catalyzing similar reactions. [ABSTRACT FROM AUTHOR]

Chloroacetamide herbicides inhibit very-long-chain fatty acid elongase, and it has been suggested that covalent binding to the active site cysteine of the condensing enzyme is responsible [Pest Manage Sci 56 (2000), 497], but direct evidence was not available. The proposal implied that other condensing enzymes might also be targets, and therefore we have investigated four purified recombinant type III plant polyketide synthases. Chalcone synthase (CHS) revealed a high sensitivity to the chloroacetamide metazachlor, with 50% inhibition after a 10 min pre-incubation with 1–2 molecules per enzyme subunit, and the inactivation was irreversible. Stilbene synthase (STS) inactivation required 20-fold higher amounts, and 4-coumaroyltriacetic acid synthase and pyrone synthase revealed no response at the highest metazachlor concentrations tested. A similar spectrum of differential responses was detected with other herbicides that also inhibit fatty acid elongase (metolachlor and cafenstrole). The data indicate that type III polyketide synthases are potential targets of these herbicides, but each combination has to be investigated individually. The interaction of metazachlor with CHS was investigated by mass spectrometric peptide mapping, after incubation of the enzymes with the herbicides followed by tryptic digestion. A characteristic mass shift and MS/MS sequencing of the respective peptide showed that metazachlor was covalently bound to the cysteine of the active site, and the same was found with STS. This is the first direct evidence that the active site cysteine in condensing enzymes is the primary common target of these herbicides. [Copyright &y& Elsevier]

With the aim of elucidating the mechanisms involved in the biosynthesis of medium-chain fatty acids in Cuphea lanceolata Ait., a crop accumulating up to 90% decanoic acid in seed triacylglycerols, cDNA clones of a β-ketoacyl-acyl carrier protein (ACP) synthase IV (clKAS IV, EC 2.3.1.41) were isolated from C. lanceolata seed embryos. The amino acid sequence deduced from clKAS IV cDNA showed 80% identity to other plant KAS II-type enzymes, 55% identity towards plant KAS I and over 90% towards other Cuphea KAS IV-type sequences. Recombinant clKAS IV was functionally overexpressed in Escherichia coli, and substrate specificity of purified enzyme showed strong preference for elongation of short-chain and medium-chain acyl-ACPs (C4- to C10-ACP) with nearly equal activity. Further elongation steps were catalysed with distinctly less activity. Moreover, short- and medium-chain acyl-ACPs exerted a chain-length-specific and concentration-dependent substrate inhibition of clKAS IV. Based on these findings a regulatory mechanism for medium-chain fatty acid synthesis in C. lanceolata is presented. [ABSTRACT FROM AUTHOR]

Chalcone (CHS) and stilbene (STS) synthases are related plant-specific polyketide synthases that are key enzymes in the biosynthesis of flavonoids and of stilbene phytoalexins, respectively. A phylogenetic tree constructed from 34 CHS and four STS sequences revealed that the STS formed no separate cluster but grouped with CHS from the same or related plants. This suggested that STS evolved from CHS several times independently. We attempted to simulate this by site-directed mutagenesis of an interfamily CHS/STS hybrid, which contained 107 amino acids of a CHS from Sinapis alba (N-terminal) and 287 amino acids of a STS from Arachis hypogaea. The hybrid had no enzyme activity. Three amino acid exchanges in the CHS part (Gln-100 to Glu, Val-103 to Met, Val-105 to Arg) were sufficient to obtain low STS activity, and one additional exchange (Gly-23 to Thr) resulted in 20-25% of the parent STS activity. A kinetic analysis indicated (1) that the hybrids had the same K for the substrate 4-coumaroyl-CoA but a lower V than the parent STS, and (2) that they had a different substrate preference than the parent STS and CHS. Most of the other mutations and their combinations led to enzymatically inactive protein aggregates, suggesting that the subunit folding and/or the dimerization was disturbed. We propose that STS evolved from CHS by a limited number of amino acid exchanges, and that the advantage gained by this enzyme function favored the selection of plants with improved STS activity. [ABSTRACT FROM AUTHOR]

Since the early twentieth century, with the isolation of penicillin and streptomycin in the 1940s, the modern era of anti-infective drug development has gained momentum. Due to the enormous success of early drug discovery, many infectious diseases were successfully prevented and eradicated. However, this initial hope was wrongheaded, and pathogens evolved as a significant threat to human health. Drug resistance develops as a result of natural selection's relentless pressure, necessitating the identification of new drug targets and the creation of chemotherapeutics that bypass existing drug resistance mechanisms. Fatty acid biosynthesis (FAS) is a crucial metabolic mechanism for bacteria during their growth and development. Several crucial enzymes involved in this biosynthetic pathway have been identified as potential targets for new antibacterial agents. In Escherichia coli (E. coli), this pathway has been extensively investigated. The present review focuses on progress in the development of Kas A, Kas B, and Fab H inhibitors as mono-therapeutic antibiotics., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Condensing enzymes catalyze the committed reaction of fatty acid elongation and determine the chain length of fatty acids accepted and produced by the elongation complex. While necessary for the elongation of very-long-chain fatty acids (VLCFAs), identified plant condensing enzymes cannot efficiently produce VLCFAs longer than 28 carbons, which are precursors for the most abundant cuticular waxes of most plant species that have been surveyed. The eceriferum2 (cer2) mutant of Arabidopsis thaliana has a severe wax-deficient phenotype and specifically lacks waxes longer than 28 carbons, but the CER2 protein does not share sequence similarity with condensing enzymes. Instead, CER2 is homologous to BAHD acyltransferases. Heterologous expression in yeast previously demonstrated that CER2, and a small clade of BAHD acyltransferases with high sequence identity to CER2, can extend the chain-length specificity of the condensing enzyme CER6. This biochemical function is distinct from that of the broader BAHD acyltransferase family. The product specificity and physiological functions of individual CER2-LIKE proteins are unique. Here, we demonstrate that CER2 physically interacts with the fatty acid elongase. We cloned chimeric CER2-LIKE proteins and expressed these in yeast cells to identify the features that define the substrate specificities of CER2-LIKEs. We generated homology-based structural models to compare CER2-LIKEs and BAHD acyltransferases. In addition, based on the current phylogenetic analysis of the CER2-LIKE clade, we describe two further Arabidopsis CER2-LIKE genes, CER2-LIKE3 and CER2-LIKE4. We used yeast expression and mutant analysis to characterize these genes. Collectively, these results expand our knowledge of the functions of CER2-LIKEs, the BAHD acyltransferase family and cuticular wax metabolism., (� The Author(s) 2020. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Pseudomonas aeruginosaproduces a number of alkylquinolone-type secondary metabolites best known for their antimicrobial effects and involvement in cell-cell communication. In the alkylquinolone biosynthetic pathway, the β-ketoacyl-(acyl carrier protein) synthase III (FabH)-like enzyme PqsBC catalyzes the condensation of octanoyl-coenzyme A and 2-aminobenzoylacetate (2-ABA) to form the signal molecule 2-heptyl-4(1H)-quinolone. PqsBC, a potential drug target, is unique for its heterodimeric arrangement and an active site different from that of canonical FabH-like enzymes. Considering the sequence dissimilarity between the subunits, a key question was how the two subunits are organized with respect to the active site. In this study, the PqsBC structure was determined to a 2 Å resolution, revealing that PqsB and PqsC have a pseudo-2-fold symmetry that unexpectedly mimics the FabH homodimer. PqsC has an active site composed of Cys-129 and His-269, and the surrounding active site cleft is hydrophobic in character and approximately twice the volume of related FabH enzymes that may be a requirement to accommodate the aromatic substrate 2-ABA. From physiological and kinetic studies, we identified 2-aminoacetophenone as a pathway-inherent competitive inhibitor of PqsBC, whose fluorescence properties could be used forin vitrobinding studies. In a time-resolved setup, we demonstrated that the catalytic histidine is not involved in acyl-enzyme formation, but contributes to an acylation-dependent increase in affinity for the second substrate 2-ABA. Introduction of Asn into the PqsC active site led to significant activity toward the desamino substrate analog benzoylacetate, suggesting that the substrate 2-ABA itself supplies the asparagine-equivalent amino function that assists in catalysis., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

The survival of Mycobacterium tuberculosis depends on mycolic acids, very long α-alkyl-β-hydroxy fatty acids comprising 60-90 carbon atoms. However, despite considerable efforts, little is known about how enzymes involved in mycolic acid biosynthesis recognize and bind their hydrophobic fatty acyl substrates. The condensing enzyme KasA is pivotal for the synthesis of very long (C38-42) fatty acids, the precursors of mycolic acids. To probe the mechanism of substrate and inhibitor recognition by KasA, we determined the structure of this protein in complex with a mycobacterial phospholipid and with several thiolactomycin derivatives that were designed as substrate analogs. Our structures provide consecutive snapshots along the reaction coordinate for the enzyme-catalyzed reaction and support an induced fit mechanism in which a wide cavity is established through the concerted opening of three gatekeeping residues and several α-helices. The stepwise characterization of the binding process provides mechanistic insights into the induced fit recognition in this system and serves as an excellent foundation for the development of high affinity KasA inhibitors.