Modification of Brassica napus seed oil by expression of the Escherichia coli fabH gene,encoding 3-ketoacyl-acyl carrier protein synthase III

The Escherichia coli fabH gene encoding 3-ketoacyl-acyl carrier protein synthase III (KAS III) was isolated and the effect of overproduction of bacterial KAS III was compared in both E. coli and Brassica napus. The change in fatty acid profile of E. coli was essentially the same as that reported by Tsay et al. (J Biol Chem 267 (1992) 6807–6814), namely higher C14:0 and lower C18:1 levels. In our study, however, an arrest of cell growth was also observed. This and other evidence suggests that in E. coli the accumulation of C14:0 may not be a direct effect of the KAS III overexpression, but a general metabolic consequence of the arrest of cell division. Bacterial KAS III was expressed in a seed- and developmentally specific manner in B. napus in either cytoplasm or plastid. Significant increases in KAS III activities were observed in both these transformation groups, up to 3.7 times the endogenous KAS III activity in mature seeds. Only the expression of the plastid-targeted KAS III gene, however, affected the fatty acid profile of the storage lipids, such that decreased amounts of C18:1 and increased amounts of C18:2 and C18:3 were observed as compared to control plants. Such changes in fatty acid composition reflect changes in the regulation and control of fatty acid biosynthesis. We propose that fatty acid biosynthesis is not controlled by one rate-limiting enzyme, such as acetyl-CoA carboxylase, but rather is shared by a number of component enzymes of the fatty acid biosynthetic machinery.     

Chitin synthetase activity is bound to the plasma membrane and to a cytoplasmic particulate fraction in Candida albicans germ tube cells

Abstract Subcellular distribution of chitin synthetase has been studied in germ tubes of Candida albicans . Two fractions with synthetase activity were separated from cell homogenates: (i) a mixed membrane fraction where the enzyme, partly in an active form, is associated with the plasma membrane (isopycnic centrifugation of mixed membrane fraction on linear sucrose gradients resolved a unique peak of activity matching with [³H]ConA-labelled membranes at a buoyant density of 1.195 g/ml); and (ii) a cytoplasmic fraction containing fully zymogenic enzyme associated with particles whose buoyant density (determined by isopycnic centrifugation on linear sucrose gradients) depended on the cell breakage conditions. The actual cytoplasmic fraction-enzyme may correspond to particles with buoyant density 1.135 g/ml (chitosomes), whereas the enzyme particles with other densities (1.085 and 1.165 g/ml) probably originated during cell disruption, as has been reported previously to occur during the preparation of yeast cell homogenates.     

Cloning and Functional Analysis of an Allene Oxide Synthase in Physcomitrella patens

Jasmonic acid (JA) is a plant hormone that plays important roles in a large number of processes in stress adaptation and development in flowering plants. A search of genome database indicated the existence of allene oxide synthase (AOS), an enzyme of JA biosynthesis, in Physcomitrella patens, a model plant among mosses. In this study, the presence of JA was detected in P. patens. The recombinant AOS of P. patens, which was overexpressed in Escherichia coli, showed AOS activity. These data suggest that the octadecanoid pathway also exists in P. patens.     

Partial Assembly of the Yeast Mitochondrial ATP Synthase 1

The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F₁ catalytic domain, the F₀ proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F₁ to F₀, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F₀ into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F₀. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.     

Purification and Characterization of a Cold-adapted Isocitrate Lyase and a Malate Synthase from Colwellia maris,a Psychrophilic Bacterium

Isocitrate lyase (ICL) and malate synthase (MS) of a psychrophilic marine bacterium, Colwellia maris, were purified to electrophoretically homogeneous state. The molecular mass of the ICL was found to be 240 kDa, composed of four identical subunits of 64.7 kDa. MS was a dimeric enzyme composed of 76.3 kDa subunits. N-Terminal amino acid sequences of the ICL and MS were analyzed. Purified ICL had its maximum activity at 20°C and was rapidly inactivated at the temperatures above 30°C, but the optimum temperature for the activity of MS was 45°C. NaCl was found to protect ICL from heat inactivation above 30°C, but the salt did not stabilize MS. Effects of temperatures on the kinetic parameters of both the enzymes were examined. The K_m for the substrate (isocitrate) of ICL was decreased with decreasing temperature. On the other hand, the K_m for the substrate (glyoxylate) of MS was increased with decreasing temperature. The calculated value of free energy of activation of ICL was on the same level as that of MS.     

Distribution of oxoacyl synthase homology sequences within Streptomyces DNA

Abstract A genomic DNA sequence of Streptomyces strain ISP 5485 was cloned, sequenced and compared with corresponding information from nucleic acid data banks. The DNA sequence was unique, but showed homology to DNA coding for the condensing enzyme, 2-oxoacyl synthase, of the deoxyerythronolide B synthase complex (DEBS) from Saccharopolyspora erythraea NRRL 2338. A subfragment of the sequenced DNA was used to construct a gene-specific probe that formed part of the putative 2-oxoacyl synthase gene. The PCR-amplified and labelled probe was used in hybridization experiments involving 33 streptomycete strains that produced different classes of antibiotics. The probe showed widespread homology with DNA considered to be part of analogous genes within genomes of different polyketide producers. The implications of the probe homology to bacterial chromosomal DNA are discussed.     

Histidine-272 of isopenicillin N synthase of Cephalosporium acremonium, which is possibly involved in iron binding, is essential for its catalytic activity

Abstract Amino acid sequence alignment of the Cephalosporium acremonium isopenicillin N synthase (cIPNS) to similar non-heme Fe²⁺-containing enzymes from 28 different sources (bacterial, fungal, plant and animals) revealed a homologous region of high sequence conservation containing an invariant histidine residue at position 272 in cIPNS. The importance of this histidine residue in cIPNS was investigated through site-directed mutagenesis by replacing the histidine residue with leucine. The mutated gene was verified by DNA sequence analysis and expressed in Escherichia coli . When analyzed by denaturing gel electrophoresis and immunoblotting, the mutant cIPNS had identical mobility as that of the wild-type enzyme. Enzyme studies on the mutant enzyme showed loss of enzymatic activity indicating that His272 is essential for the catalytic function of cIPNS, possibly as a ligand for iron binding.     

Substrate Locking Promotes Dimer-Dimer Docking of an Enzyme Antibiotic Target

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Structural and Functional Analysis of E. coli Cyclopropane Fatty Acid Synthase

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Concise synthesis of artemisinin from a farnesyl diphosphate analogue

Artemisinin is one of the most potent anti-malaria drugs and many often-lengthy routes have been developed for its synthesis. Amorphadiene synthase, a key enzyme in the biosynthetic pathway of artemisinin, is able to convert an oxygenated farnesyl diphosphate analogue directly to dihydroartemisinic aldehyde, which can be converted to artemisinin in only four chemical steps, resulting in an efficient synthetic route to the anti-malaria drug.