Effect of levulinic acid on pigment biosynthesis in Agmenellum quadruplicatum.

When levulinic acid was added to a growing culture of the cyanobacterium (blue-green alga) Agmenellum quadruplicatum PR-6, delta-aminoelevulinic acid accumulated in the medium and chlorophyll a synthesis and cell growth were inhibited, but there was a small amount of c-phycocyanin synthesis. The amount of delta-aminolevulinic acid produced in the treated culture did not fully account for the amount of pigment synthesized in the untreated control. Levulinic acid and either sodium nitrate or ammonium chloride were added to nitrogen-starved cultures of PR-6, and delta-aminolevulinic acid production and chlorophyll a and c-phycocyanin content were monitored. When ammonium chloride was added as a nitrogen source after nitrogen starvation, the cells recovered more rapidly than when sodium nitrate was added as a nitrogen source. In cultures recovering from nitrogen starvation, synthesis of c-phycocyanin occurred before synthesis of chlorophyll a.     

Modulation of cytochrome oxidase kinetics by indirect antibody action

Polyclonal antibodies raised against isolated subunit V from beef heart cytochrome oxidase or against the intact enzyme increase its apparent affinity for the substrate cytochrome c at the high-affinity site while diminishing the turnover at that site. At the low-affinity site the major action of both types of antibody is to reduce the apparent affinity for cytochrome c. At high ionic strengths the kinetic effect of anti-subunit V is very small although it still binds to the enzyme. The results are interpreted in terms of a model for the enzyme in which antibodies can modulate cytochrome oxidase kinetics by affecting the binding of cytochrome c, even if the antibody-binding site is on a subunit not directly involved in substrate binding.     

Fatty acid biosynthesis in yeast

Summary Fatty acid synthetase and acetyl CoA carboxylase mutants have been used to study several aspects of fatty acid biosynthesis in yeast: the contribution of the various enzymes of fatty acid biosynthesis and modification to the overall cellular fatty acid composition, the mechanism of fatty acyl chain elongation in yeast, the molecular structure and the reaction mechanism of the fatty acid synthetase complex and the genetic control of the biosynthesis of this multi-enzyme system. Genetic and biochemical evidence suggest an ₆₆ molecular structure of this complex, where and are multifunctional proteins comprising, respectively, 3 and 5 of the various fatty acid synthetase component functions. The two subunits and are synthesized on two different, unliked genes, fas 2 and fas 1. The biosynthesis of both is coordinated. The various component enzyme activities reside in distinct domains on the multifunctional chains. While most domains appear to be functionally independent, the three acyl transferases exhibit extensive mutual interactions. It is suggested that the biosynthesis of a multifunctional protein is favoured on the grounds of kinetics and regulation as compared with the formation of a complex of the corresponding individual enzymes.     

Genetic manipulation of a latent defect in yeast cytochrome biosynthesis utilizing cytoduction.

The effects of a mit^? mutation, oxi2, and the ρ° mutation on expression of a defective nuclear structural gene for δ-aminolevulinic acid synthase (cyd1) were compared. The technique of cytoduction was used to introduce oxi2 mitochondria into a cyd1ρ° recipient cell, thereby permitting comparison of isonuclear strains. Like ρ°, the oxi2 mutation caused an apparent unmasking of the cytochrome deficiency associated with the cyd1 mutation, provided cells were grown on glucose. When cyd1 strains with ρ⁺, ρ° or oxi2 mitochondrial genotypes were grown on galactose medium, substantial cytochrome formation occurred in each case. It is concluded that the exacerbation of the cyd1-dependent cytochrome deficiency by ρ° or oxi2 mutations depends upon glucose repression. However, derepression resulting from growth on galactose medium does not fully overcome the cyd1 defect, since both cyd1ρ°oxi2 strains require added δ-aminolevulinic acid for maximum cytochrome biosynthesis.     

Modulation of fatty acid biosynthesis by antisense beta-keto reductase expression

Plants with an antisense construct to beta-ketoacyl-acyl carrier protein reductase have reduced seed and leaf fatty acid contents and show distinct phenotypes, including leaf curl, stunted growth and wrinkled seeds. The carbon resource allocation is altered in both of these tissues. In leaves there is an inability to synthesize transient starch and in seeds embryogenesis is foreshortened and storage proteins accumulate prematurely.     

Analysis of heme biosynthesis in catalase and cytochrome deficient yeast mutants

Summary Mutants of Saccharomyces cerevisiae, described as catalase and cytochromes deficient (Pachecka et al., 1974), have been analyzed for heme biosynthesis ability. Some enzymatic activities involved in protoheme synthesis were measured in acellular extracts, whereas whole cells were analyzed for cytochrome spectra and for possible accumulation of porphyrin synthesis intermediates. A good correlation was found between these in vitro and in vivo studies. Results show that two mutants were impaired in 5-aminolevulinate synthesis, two mutants were devoid of uroporphyrinogen I synthetase activity and one mutant presented defects in coproporphyrinogen III oxidase activity.     

Modulation of abscisic acid signal transduction and biosynthesis by an Sm-like protein in Arabidopsis.

The phytohormone abscisic acid (ABA) regulates plant growth and development as well as stress tolerance. The Arabidopsis sad1 (supersensitive to ABA and drought) mutation increases plant sensitivity to drought stress and ABA in seed germination, root growth, and the expression of some stress-responsive genes. sad1 plants are also defective in the positive feedback regulation of ABA biosynthesis genes by ABA and are impaired in drought stress induction of ABA biosynthesis. SAD1 encodes a polypeptide similar to multifunctional Sm-like snRNP proteins that are required for mRNA splicing, export, and degradation. These results suggest a critical role for mRNA metabolism in the control of ABA signaling as well as in the regulation of ABA homeostasis.     

The role of oxygen in the biosynthesis of cytochrome c oxidase of yeast mitochondria.

The formation of cytochrome c oxidase in yeast is dependent on oxygen. In order to examine the oxygen-dependent formation of the active enzyme, the effect of oxygen on the synthesis and the assembly of cytochrome c oxidase subunits was studied. Pulse-labeling experiments revealed that oxygen has no significant immediate effect on the synthesis of the three mitochondrially made subunits I to III; however, its presence causes subunits I and II to form a complex with the cytoplasmically made subunits VI and VII. This "assembly-inducing" effect can be demonstrated with intact yeast cells as well as with isolated mitochondria. It is independent of cytoplasmic or mitochondrial protein synthesis. After anaerobic growth for 10 or more generations, the intracellular concentrations of individual cytochrome c oxidase subunits drop 10- to 100-fold. Most of these residual subunits are not assembled within a functional cytochrome c oxidase molecule.     

Regulation of the biosynthesis of cytochrome P-450 in brewer's yeast. Role of cyclic AMP.

The drug metabolising enzyme cytochrome P-450 has been studied in great detail in mammalian systems and its presence in microorganisms is also well established. However, neither its function nor its means of control in brewer's yeast, Saccharomyces cerevisiae, has been investigated. We demonstrate here using yeast protoplasts that it is the intracellular concentration of cyclic AMP which controls, by repression, the de novo synthesis of the enzyme, and also that cyclic AMP concentrations are in turn inversely related to the concentration of glucose in the yeast growth medium.     

Two species of cytochrome P-450 involved in ergosterol biosynthesis of yeast

Discrimination of cytochrome P-450 involved in delta 22-desaturation of ergosta-5,7-dien-3 beta-o1 (P-450(22)-DS) from that involved in lanosterol 14 alpha-demethylation (P-450(14)-DM) in ergosterol biosynthesis was investigated with microsomes of several strains of Saccharomyces cerevisiae. In mutant N22 which is partially defective in the delta 22-desaturation, the 14 alpha-demethylation was not blocked. In contrast, mutant SG1 which is known to lack the 14 alpha-demethylation showed a significant activity of the delta 22-desaturation. The delta 22-desaturation activity was markedly increased upon aerobic adaptation of yeast cells but the 14 alpha-demethylation was not affected. Buthiobate, a specific inhibitor of P-450(14)-DM, and rabbit antibodies against P-450(14)-DM did not inhibit the delta 22-desaturation activity at all. It is evident from the obtained observations that these phenomena are not explainable in terms of NADPH-cytochrome P-450 reductase. These results indicate that P-450(22)-DS is different from P-450(14)-DM in molecular species.     

Phenylalanyl-tRNA synthetase of baker's yeast. Modulation of adenosine triphosphate-pyrophosphate exchange by transfer ribonucleic acid

Native and modified phenylalanine transfer ribonucleic acid (tRNAPhe) can modulate phenylalanine-dependent adenosine triphosphate--inorganic [32P]pyrophosphate (ATP--[32P]PPi) exchange activity via inhibition of adenylate synthesis. Inhibition is visualized if concentrations of L-phenylalanine, ATP, and pyrophosphate are subsaturating. In the proposed mechanism, tRNAPhe is a noncompetitive inhibitor at conditions where only one of the two active sites per molecule of enzyme is occupied by L-phenylalanine, ATP, and pyrophosphate. At saturating concentrations of these reactants, both active sites are occupied and, according to the model, inhibition is eliminated. Occupation by these reactants is assumed to follow homotropic negative cooperativity. The type of effects depends on modification of tRNAPhe. Native tRNAPhe, tRNA2'-dAPhe, and tRNAoxi-redPhe are inhibitors, tRNAPhepCpC has no effect, and tRNAoxPhe is an activator. Kinetics of activation by tRNAoxPhe are slow, following the time course of Schiff base formation and subsequent reduction by added cyanoborohydride. Besides showing that a putative enzyme amino group is nonessential for substrate binding and adenylate synthesis, this result may suggest that an enzyme amino group could interact with the 3'-terminal adenyl group of cognate tRNA. In the case of asymmetrical occupation of the enzyme active sites by all of the small reactants ATP, L-phenylalanine, and pyrophosphate, the interaction with the amino group might trigger the observed noncompetitive inhibition of the pyrophosphate exchange by tRNAPhe.     

Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis

To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.     

Induction of rosmarinic acid biosynthesis in Lithospermum erythrorhizon cell suspension cultures by yeast extract

Summary A transient increase in rosmarinic acid (RA) content in cultured cells of Lithospermum erythrorhizon was observed after addition of yeast extract (YE) to the suspension cultures, reaching a maximum at 24 hr. The highest increase of the RA content (2.5-fold) was obtained when 6-day-old cells in the exponential growth phase were treated with YE. Preceding the induced RA accumulation, phenylalanine ammonia-lyase (PAL) activity increased rapidly, whereas tyrosine aminotransferase (TAT) activity was largely unaffected by the treatment. The incorporation of both ¹⁴C-phenylalanine and ¹⁴C-tyrosine into RA was enhanced in the YE-treated cells, consistent with increased synthesis of the ester.Abbreviations 2,4-D 2,4 dichlorophenoxyacetic acid - PAL phenylalanine ammonia-lyase - TAT tyrosine aminotransferase - RA rosmarinic acid - YE yeast extract     

Primary structure of yeast cytochrome c peroxidase. II. The complete amino acid sequence

The complete amino acid sequence of 293 residues in the single polypeptide chain of yeast cytochrome c peroxidase has been determined. Sequence analyses were performed on fragments obtained by cleavage with cyanogen bromide and by tryptic digestion of citraconylated protein. These fragments were aligned with sequential and compositional data as well as those obtained with intact protein, tryptic, and chymotryptic peptides (Takio and Yonetani, 1980, Arch. Biochem. Biophys.203, 605–614). Residues critical for catalysis by the enzyme were identified as arginine 48, tryptophan 51, histidine 52, and histidine 174 by fitting the sequence to the electron density map derived by others. Sequence comparison with other proteins show limited homology with horseradish peroxidase and myoglobin.     

酵母微生物油脂的生物合成研究

摘要：目的 微生物油脂可作为制备绿色能源生物柴油的原料。对酵母微生物油脂的生物合成方法进行研究。方法 以斯达油脂酵母Lipomyces starkeyi AS 2.1560为菌种进行微生物油脂生物合成。首先获得大量细胞,将细胞收集后,转移至葡萄糖溶液中进行油脂合成。结果 斯达油脂酵母可在不含有其他营养成分的葡萄糖溶液中快速合成油脂,细胞油脂含量可达到细胞干重的60%以上。菌龄对油脂生成影响不明显,糖浓度过高抑制油脂生成,40 g/L葡萄糖溶液中60 h合成油脂最多,达到65.2%,并有进一步积累的可能,在（0.5～6）×108个/mL,接种细胞的密度越大,油脂合成能力越低。合成油脂成分主要为棕榈酸和油酸。结论 斯达油脂酵母细胞增殖与油脂生物合成可分开进行,其油脂成分与普通动植物油脂成分相似。