Limnanthes douglasii lysophosphatidic acid acyltransferases: immunological quantification, acyl selectivity and functional replacement of the Escherichia coli plsC gene.
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ABSTRACT:
SUBMITTER: Brown AP
PROVIDER: S-EPMC1222629 | biostudies-other | 2002 Jun
REPOSITORIES: biostudies-other
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Project description:The tetraploid Brassica napus possesses several seed-expressed microsomal lysophosphatidic acid acyltransferases (LPAAT ) including BAT1.5, which has been retained after genome duplication as a consequence of a subfunctionalisation of the gene encoding the ubiquitously expressed Kennedy pathway enzyme BAT1.13. Next, cDNA BAT1.3, encoding a LPAAT was subsequently isolated from an embryo library. The rapeseed LPAAT encoded by BAT1.3 is orthologous to the Arabidopsis thaliana At1g51260 gene product possibly associated with tapetum development and male fertility. However, BAT1.3 expression is predominant during the mid stages of embryo development in seeds of Brassica napus. Functional characterisation of BAT1.3 provides further support for a hypothesis of gene dosage sensitivity of LPAATs as does an analysis of the chromosomal localisation of LPAAT genes in Arabidopsis thaliana. The pattern of retention or loss of LPAAT genes after polyploidisation or segmental duplication is consistent with a model of balanced gene drive.
| S-EPMC3115043 | biostudies-literature
Project description:LpxD catalyzes the third step of lipid A biosynthesis, the R-3-hydroxyacyl-ACP-dependent N-acylation of UDP-3-O-(acyl)-alpha-D-glucosamine, and is a target for new antibiotic development. Here we report the 2.6 A crystal structure of the Escherichia coli LpxD homotrimer (EcLpxD). As is the case in Chlamydia trachomatis LpxD (CtLxpD), each EcLpxD chain consists of an N-terminal uridine-binding region, a left-handed parallel beta-helix (LbetaH), and a C-terminal alpha-helical domain. The backbones of the LbetaH domains of the two enzymes are similar, as are the positions of key active site residues. The N-terminal nucleotide binding domains are oriented differently relative to the LbetaH regions, but are similar when overlaid on each other. The orientation of the EcLpxD tripeptide (residues 303-305), connecting the distal end of the LbetaH and the proximal end of the C-terminal helical domains, differs from its counterpart in CtLpxD (residues 311-312); this results in a 120 degrees rotation of the C-terminal domain relative to the LbetaH region in EcLpxD versus CtLpxD. M290 of EcLpxD appears to cap the distal end of a hydrophobic cleft that binds the acyl chain of the R-3-hydroxyacyl-ACP donor substrate. Under standard assay conditions, wild-type EcLpxD prefers R,S-3-hydroxymyristoyl-ACP over R,S-3-hydroxypalmitoyl-ACP by a factor of 3, whereas the M290A mutant has the opposite selectivity. Both wild-type and M290A EcLpxD rescue the conditional lethality of E. coli RL25, a temperature-sensitive strain harboring point mutations in lpxD. Complementation with wild-type EcLpxD restores normal lipid A containing only N-linked hydroxymyristate to RL25 at 42 degrees C, as judged by mass spectrometry, whereas the M290A mutant generates multiple lipid A species containing one or two longer hydroxy fatty acids in place of the usual R-3-hydroxymyristate at positions 2 and 2'.
| S-EPMC2748855 | biostudies-literature
Project description:Portions of the intimin genes of Escherichia coli O157:H7 strain E319 and of the enteropathogenic E. coli O127:H6 strain E2348/69 were amplified by PCR and cloned into pET-28a+ expression vectors. The entire 934 amino acids (aa) of E. coli O157:H7 intimin, the C-terminal 306 aa of E. coli O157:H7 intimin, and the C-terminal 311 aa of E. coli O127:H6 intimin were expressed as proteins fused with a six-histidine residue tag (six-His tag) in pET-28a+. Rabbit antisera raised against the six-His tag-full-length E. coli O157:H7 intimin protein fusion cross-reacted in slot and Western blots with outer membrane protein preparations from the majority of enterohemorrhagic and enteropathogenic E. coli serotypes which have the intimin gene. The E. coli strains tested included isolates from humans and animals which produce intimin types alpha (O serogroups 86, 127, and 142), beta1 (O serogroups 5, 26, 46, 69, 111, 126, and 128), gamma 1 (O serogroups 55, 145, and 157), gamma 2 (O serogroups 111 and 103), and epsilon (O serogroup 103) and a nontypeable intimin (O serogroup 80), results based on intimin type-specific PCR assays. Rabbit antisera raised against the E. coli O157:H7 C-terminal fusion protein were much more intimin type-specific than those raised against the full-length intimin fusion protein, but some cross-reaction with other intimin types was also observed for these antisera. In contrast, the monoclonal antibody Intgamma1.C11, raised against the C-terminal E. coli O157 intimin, reacted only with preparations from intimin gamma 1-producing E. coli strains such as E. coli O157:H7.
| S-EPMC119882 | biostudies-literature
Project description:SlyD is a Ni(II)-binding protein that contributes to nickel homeostasis in Escherichia coli. The C-terminal domain of SlyD contains a rich variety of metal-binding amino acids, suggesting broader metal binding capabilities, and previous work demonstrated that the protein can coordinate several types of first-row transition metals. However, the binding of SlyD to metals other than Ni(II) has not been previously characterized. To improve our understanding of the in vitro metal-binding activity of SlyD and how it correlates with the in vivo function of this protein, the interactions between SlyD and the series of biologically relevant transition metals [Mn(II), Fe(II), Co(II), Cu(I), and Zn(II)] were examined by using a combination of optical spectroscopy and mass spectrometry. Binding of SlyD to Mn(II) or Fe(II) ions was not detected, but the protein coordinates multiple ions of Co(II), Zn(II), and Cu(I) with appreciable affinity (K(D) values in or below the nanomolar range), highlighting the promiscuous nature of this protein. The order of affinities of SlyD for the metals examined is as follows: Mn(II) and Fe(II) < Co(II) < Ni(II) ~ Zn(II) ? Cu(I). Although the purified protein is unable to overcome the large thermodynamic preference for Cu(I) and exclude Zn(II) chelation in the presence of Ni(II), in vivo studies reveal a Ni(II)-specific function for the protein. Furthermore, these latter experiments support a specific role for SlyD as a [NiFe]-hydrogenase enzyme maturation factor. The implications of the divergence between the metal selectivity of SlyD in vitro and the specific activity in vivo are discussed.
| S-EPMC3253375 | biostudies-literature
Project description:We recently used a synthetic/bottom-up approach to establish the identity of the four enzymes composing an engineered functional reversal of the -oxidation cycle for fuel and chemical production in Escherichia coli (J. M. Clomburg, J. E. Vick, M. D. Blankschien, M. Rodriguez-Moya, and R. Gonzalez, ACS Synth Biol 1:541–554, 2012, http://dx.doi.org/10.1021/sb3000782).While native enzymes that catalyze the first three steps of the pathway were identified, the identity of the native enzyme(s) acting as the trans-enoyl coenzyme A (CoA) reductase(s) remained unknown, limiting the amount of product that could be synthesized (e.g., 0.34 g/liter butyrate) and requiring the overexpression of a foreign enzyme (the Euglena gracilis trans-enoyl-CoA reductase [EgTER]) to achieve high titers (e.g., 3.4 g/liter butyrate). Here, we examine several native E. coli enzymes hypothesized to catalyze the reduction of enoyl-CoAs to acyl-CoAs. Our results indicate that FabI, the native enoyl-acyl carrier protein (enoyl-ACP) reductase (ENR) from type II fatty acid biosynthesis, possesses sufficient NADH-dependent TER activity to support the efficient operation of a -oxidation reversal. Overexpression of FabI proved as effective as EgTER for the production of butyrate and longer-chain carboxylic acids. Given the essential nature of fabI, we investigated whether bacterial ENRs from other families were able to complement a fabI deletion without promiscuous reduction of crotonyl-CoA. These characteristics from Bacillus subtilis FabL enabled deltaffabI complementation experiments that conclusively established that FabI encodes a native enoyl-CoA reductase activity that supports the ?-oxidation reversal in E. coli.
| S-EPMC4309689 | biostudies-literature
Project description:Acyl carrier protein (ACP) is an essential co-factor protein in fatty acid biosynthesis that shuttles covalently bound fatty acyl intermediates in its hydrophobic pocket to various enzyme partners. To characterize acyl chain-ACP interactions and their influence on enzyme interactions, we performed 19 molecular dynamics (MD) simulations of Escherichia coli apo-, holo-, and acyl-ACPs. The simulations were started with the acyl chain in either a solvent-exposed or a buried conformation. All four short-chain (< or = C10) and one long-chain (C16) unbiased acyl-ACP MD simulation show the transition of the solvent-exposed acyl chain into the hydrophobic pocket of ACP, revealing its pathway of acyl chain binding. Although the acyl chain resides inside the pocket, Thr-39 and Glu-60 at the entrance stabilize the phosphopantetheine linker through hydrogen bonding. Comparisons of the different ACP forms indicate that the loop region between helices II and III and the prosthetic linker may aid in substrate recognition by enzymes of fatty acid synthase systems. The MD simulations consistently show that the hydrophobic binding pocket of ACP is best suited to accommodate an octanoyl group and is capable of adjusting in size to accommodate chain lengths as long as decanoic acid. The simulations also reveal a second, novel binding mode of the acyl chains inside the hydrophobic binding pocket directed toward helix I. This study provides a detailed dynamic picture of acyl-ACPs that is in excellent agreement with available experimental data and, thereby, provides a new understanding of enzyme-ACP interactions.
| S-EPMC2662278 | biostudies-literature
Project description:Escherichia coli flavohemoglobin is endowed with the notable property of binding specifically unsaturated and/or cyclopropanated fatty acids both as free acids or incorporated into a phospholipid molecule. Unsaturated or cyclopropanated fatty acid binding to the ferric heme results in a spectral change observed in the visible absorption, resonance Raman, extended x-ray absorption fine spectroscopy (EXAFS), and x-ray absorption near edge spectroscopy (XANES) spectra. Resonance Raman spectra, measured on the flavohemoglobin heme domain, demonstrate that the lipid (linoleic acid or total lipid extracts)-induced spectral signals correspond to a transition from a five-coordinated (typical of the ligand-free protein) to a hexacoordinated, high spin heme iron. EXAFS and XANES measurements have been carried out both on the lipid-free and on the lipid-bound protein to assign the nature of ligand in the sixth coordination position of the ferric heme iron. EXAFS data analysis is consistent with the presence of a couple of atoms in the sixth coordination position at 2.7 A in the lipid-bound derivative (bonding interaction), whereas a contribution at 3.54 A (nonbonding interaction) can be singled out in the lipid-free protein. This last contribution is assigned to the CD1 carbon atoms of the distal LeuE11, in full agreement with crystallographic data on the lipid-free protein at 1.6 A resolution obtained in the present work. Thus, the contributions at 2.7 A distance from the heme iron are assigned to a couple of carbon atoms of the lipid acyl chain, possibly corresponding to the unsaturated carbons of the linoleic acid.
| S-EPMC1304290 | biostudies-literature
Project description:A novel human homologue of Escherichia coli, yeast and plant 1-acylglycerol-3-phosphate acyltransferase has been isolated from U937 cell cDNA. Expression of the cloned sequence in 1-acylglycerol-3-phosphate acyltransferase-deficient E. coli resulted in increased incorporation of oleic acid into cellular phospholipids. Membranes made from COS7 cells transfected with the cDNA exhibited higher acyltransferase activity towards a range of donor fatty acyl-CoAs and lysophosphatidic acid. Northern-blot analysis of the cDNA sequence indicated high levels of expression in immune cells and epithelium. Rapid amplification of cDNA ends revealed differentially expressed splice variants, which suggests regulation of the enzyme by alternative splicing. This cDNA therefore represents the first described sequence of a mammalian gene homologous to non-mammalian lysophosphatidic acid acyltransferases.
| S-EPMC1218691 | biostudies-other
Project description:Recent results have suggested that plant lysophosphatidylcholine:acyl-coenzyme A acyltransferases (LPCATs) can operate in reverse in vivo and thereby catalyse an acyl exchange between the acyl-coenzyme A (CoA) pool and the phosphatidylcholine. We have investigated the abilities of Arabidopsis AtLPCAT2, Arabidopsis lysophosphatidylethanolamine acyltransferase (LPEAT2), S. cerevisiae lysophospholipid acyltransferase (Ale1) and S. cerevisiae lysophosphatidic acid acyltransferase (SLC1) to acylate lysoPtdCho, lysoPtdEtn and lysoPtdOH and act reversibly on the products of the acylation; the PtdCho, PtdEtn and PtdOH. The tested LPLATs were expressed in an S. cervisiae ale1 strain and enzyme activities were assessed in assays using microsomal preparations of the different transformants. The results show that, despite high activity towards lysoPtdCho, lysoPtdEtn and lysoPtdOH by the ALE1, its capacities to operate reversibly on the products of the acylation were very low. Slc1 readily acylated lysoPtdOH, lysoPtdCho and lysoPtdEtn but showed no reversibility towards PtdCho, very little reversibility towards PtdEtn and very high reversibility towards PtdOH. LPEAT2 showed the highest levels of reversibility towards PtdCho and PtdEtn of all LPLATs tested but low ability to operate reversibly on PtdOH. AtLPCAT2 showed good reversible activity towards PtdCho and PtdEtn and very low reversibility towards PtdOH. Thus, it appears that some of the LPLATs have developed properties that, to a much higher degree than other LPLATs, promote the reverse reaction during the same assay conditions and with the same phospholipid. The results also show that the capacity of reversibility can be specific for a particular phospholipid, albeit the lysophospholipid derivatives of other phospholipids serve as good acyl acceptors for the forward reaction of the enzyme.
| S-EPMC4700060 | biostudies-literature
Project description:Camelina sativa is an emerging biotechnology oil crop. However, more information is needed regarding its innate lipid enzyme specificities. We have therefore characterized several triacylglycerol (TAG) producing enzymes by measuring in vitro substrate specificities using different combinations of acyl-acceptors (diacylglycerol, DAG) and donors. Specifically, C. sativa acyl-CoA:diacylglycerol acyltransferase (DGAT) 1 and 2 (which both use acyl-CoA as acyl donor) and phospholipid:diacylglycerol acyltransferase (PDAT, with phosphatidylcoline as acyl donor) were studied. The results show that the DGAT1 and DGAT2 specificities are complementary, with DGAT2 exhibiting a high specificity for acyl acceptors containing only polyunsaturated fatty acids (FAs), whereas DGAT1 prefers acyl donors with saturated and monounsaturated FAs. Furthermore, the combination of substrates that resulted in the highest activity for DGAT2, but very low activity for DGAT1, corresponds to TAG species previously shown to increase in C. sativa seeds with downregulated DGAT1. Similarly, the combinations of substrates that gave the highest PDAT1 activity were also those that produce the two TAG species (54:7 and 54:8 TAG) with the highest increase in PDAT overexpressing C. sativa seeds. Thus, the in vitro data correlate well with the changes in the overall fatty acid profile and TAG species in C. sativa seeds with altered DGAT1 and PDAT activity. Additionally, in vitro studies of C. sativa phosphatidycholine:diacylglycerol cholinephosphotransferase (PDCT), another activity involved in TAG biosynthesis, revealed that PDCT accepts substrates with different desaturation levels. Furthermore, PDCT was unable to use DAG with ricineoleyl groups, and the presence of this substrate also inhibited PDCT from using other DAG-moieties. This gives insights relating to previous in vivo studies regarding this enzyme.
| S-EPMC7456936 | biostudies-literature