Ertosterol biosynthesis in Saccharomyces cerevisiae: mutants deficient in the early steps of the pathway.

Summary Thermosensitive mutants, auxotrophic for ergosterol synthesis, have been isolated, analyzed genetically and their enzymatic deficiencies investigated. These mutants were classified into seven unlinked complementation groups. These groupes lack the following enzymatic activities: squalene epoxidase (erg 1), 2,3-oxidosqualene-lanosterol cyclase (erg 7), phosphomevalonic kinase (erg 8), mevalonic kinase (erg 12) and squalene synthetase (erg 9, erg 10, erg 11).     

Conserved metabolic steps for sporopollenin precursor formation in tobacco and rice

The development of pollen wall with proper sporopollenin deposition is essential for pollen viability and male fertility in flowering plants. Sporopollenin is a complex biopolymer synthesized from fatty acid and phenolic derivatives. Recent investigations in Arabidopsis have identified a number of anther‐specific genes involved in the production of fatty‐acyl monomers potentially required for exine formation. The existence of ancient biochemical pathways for sporopollenin biosynthesis has been widely proposed but experimental evidence from plant species other than Arabidopsis is not extensively available. Here, we investigated the metabolic steps catalyzed by the anther‐specific acyl‐CoA synthetase (ACOS), polyketide synthase (PKS) and tetraketide α‐pyrone reductase (TKPR). Using fatty acids as starting substrates, sequential activities of heterologously expressed tobacco enzymes NtACOS1, NtPKS1 and NtTKPR1 resulted in the production of reduced tetraketide α‐pyrones. Transgenic RNA interference lines were then generated for the different tobacco genes which were demonstrated to be indispensable for normal pollen development and male fertility. Similarly, recombinant rice OsPKS1 and OsTKPR1 were shown to function as downstream enzymes of NtACOS1. In addition, insertion mutant lines for these rice genes displayed different levels of impaired pollen and seed formation. Taken together, reduced tetraketide α‐pyrones appear to represent common sporopollenin fatty‐acyl precursors essential for male fertility in taxonomically distinct plant species.     

Compartmentation of the early steps of cholesterol biosynthesis in mammalian liver

Summary Cholesterol synthesis was studied in the isolated perfused rat liver and with cell-free preparations by incorporation measurements of³H from³HOH and of carbon label from [1-¹⁴C]-acetate. Using specific inhibitors such as (-)-hydroxycitrate, kynurenate, and avidin the following conclusions were reached:Fatty acid and cholesterol biosynthesis share a common substrate pool of cytoplasmic acetyl-CoA. The substrate of mevalonate synthesis is furnished by an extramitochondrial pathway of-hydroxy--methylgluraryl-CoA synthesis, which does not include malonyl-CoA. This favors the assumption of a sequence including cytoplasmic thiolase and-hydroxy--methylglutaryl-CoA synthase.Besides its inhibitory action on ATP citrate lyase (-)-hydroxycitrate was found to stimulate acetyl-CoA carboxylase.Acetyl-CoA synthetase activity of liver is localized predominantly in the cytoplasm. The regulatory behavior of the cytoplasmic enzyme points to a lipogenetic function.The control of cholesterol biosynthesis and the role of cytoplasmic acetyl-CoA synthetase in the maintenance of the extramitochondrial acetyl-CoA pool are considered in light of the reported findings.     

Genetic evidence for a multi-subunit complex in the O-methyltransferase steps of coenzyme Q biosynthesis

Coq3 O-methyltransferase carries out both O-methylation steps in coenzyme Q (ubiquinone) biosynthesis. The degree to which Coq3 O-methyltransferase activity and expression are dependent on the other seven COQ gene products has been investigated. A panel of yeast mutant strains harboring null mutations in each of the genes required for coenzyme Q biosynthesis (COQ1-COQ8) have been prepared. Mitochondria have been isolated from each member of the yeast coq mutant collection, from the wild-type parental strains and from respiratory deficient mutants harboring deletions in ATP2 or COR1 genes. These latter strains constitute Q-replete, respiratory deficient controls. Each of these mitochondrial preparations has been analyzed for COQ3-encoded O-methyltransferase activity and steady state levels of Coq3 polypeptide. The findings indicate that the presence of the other COQ gene products is required to observe normal levels of O-methyltransferase activity and the Coq3 polypeptide. However, COQ3 steady state RNA levels are not decreased in any of the coq mutants, relative to either wild-type or respiratory deficient control strains, suggesting either a decreased rate of translation or a decreased stability of the Coq3 polypeptide. These data are consistent with the involvement of the Coq polypeptides (or the Q-intermediates formed by the Coq polypeptides) in a multi-subunit complex. It is our hypothesis that a deficiency in any one of the COQ gene products results in a defective complex in which the Coq3 polypeptide is rendered unstable.     

Ecdysone biosynthesis: Pathways,enzymes, and the early steps problem

Summary

In this review, the different aspects of the ecdysone biosynthesis have been described. The “early step problem” and the enzymes which are involved in the final sequence of hydroxylations are discussed in detail. The recent results obtained on ecdysone biosynthesis in embryos are also summarized.     

Order of steps in the cytochrome C folding pathway: evidence for a sequential stabilization mechanism

Previous work used hydrogen exchange (HX) experiments in kinetic and equilibrium modes to study the reversible unfolding and refolding of cytochrome c (Cyt c) under native conditions. Accumulated results now show that Cyt c is composed of five individually cooperative folding units, called foldons, which unfold and refold as concerted units in a stepwise pathway sequence. The first three steps of the folding pathway are linear and sequential. The ordering of the last two steps has been unclear because the fast HX of the amino acid residues in these foldons has made measurement difficult. New HX experiments done under slower exchange conditions show that the final two foldons do not unfold and refold in an obligatory sequence. They unfold separately and neither unfolding obligately contains the other, as indicated by their similar unfolding surface exposure and the specific effects of destabilizing and stabilizing mutations, pH change, and oxidation state. These results taken together support a sequential stabilization mechanism in which folding occurs in the native context with prior native-like structure serving to template the stepwise formation of subsequent native-like foldon units. Where the native structure of Cyt c requires sequential folding, in the first three steps, this is found. Where structural determination is ambiguous, in the final two steps, alternative parallel folding is found.     

A continuous fluorescent enzyme assay for early steps of lipid A biosynthesis

UDP-N-acetylglucosamine acyltransferase (LpxA) and UDP-3-O-(R-3-hydroxyacyl)-glucosamine acyltransferase (LpxD) catalyze the first and third steps of lipid A biosynthesis, respectively. Both enzymes have been found to be essential for survival among gram-negative bacteria that synthesize lipopolysaccharide and are viable targets for antimicrobial development. Catalytically, both acyltransferases catalyze an acyl-acyl carrier protein (ACP)-dependent transfer of a fatty acyl moiety to a UDP-glucosamine core ring. Here, we exploited the single free thiol unveiled on holo-ACP after transfer of the fatty acyl group to the glucosamine ring using the thiol-specific labeling reagent, ThioGlo. The assay was continuously monitored as a change in fluorescence at λ(ex)=379 nm and λ(em)=513 nm using a microtiter plate reader. This assay marks the first continuous and nonradioactive assay for either acyltransferase.     

Studies on the late steps of (+) pisatin biosynthesis: evidence for (-) enantiomeric intermediates

Pisatin, a 6a-hydroxyl-pterocarpan phytoalexin from pea (Pisum sativum L.), is relatively unique among naturally occurring pterocarpans by virtue of the (+) stereochemistry of its 6a-11a C-C bond. However, pisatin synthesizing pea tissue has an isoflavone reductase, first identified in alfalfa, which acts on the (-) antipode. In order to establish the natural biosynthetic pathway to (+) pisatin, and to evaluate the possible involvement of intermediates with a (-) chirality in its biosynthesis, we administered chiral, tritium-labeled, isoflavanones and pterocarpans to pisatin-synthesizing pea cotyledons and compared the efficiency of their incorporation. Pea incorporated the isoflavanone, (-) sophorol, more efficiently than either its (+) antipode, or the pterocarpans (+) or (-) maackiain. (-) Sophorol was also metabolized by protein extracts from pisatin-synthesizing pea seedlings in a NADPH-dependent manner. Three products were produced. One was the isoflavene (7,2'-dihydroxy-4',5'-methylenedioxyisoflav-3-ene), and another had properties consistent with the isoflavanol (7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol), the expected product for an isoflavanone reductase. A cDNA encoding sophorol reductase was also isolated from a cDNA library made from pisatin-synthesizing pea. The cloned recombinant sophorol reductase preferred (-) sophorol over (+) sophorol as a substrate and produced 7,2'-dihydroxy-4',5'-methylenedioxyisoflavanol. Although no other intermediates in (+) pisatin biosynthesis were identified, the results lend additional support to the involvement of intermediates of (-) chirality in (+) pisatin synthesis.     

Direct evidence for a xylose metabolic pathway in Saccharomyces cerevisiae

Xylose transport, xylose reductase, and xylitol dehydrogenase activities are demonstrated in Saccharomyces cerevisiae. The enzymes in the xylose catabolic pathway necessary for the conversion of xylose to xylulose are present, although S. cerevisiae cannot grow on xylose as a sole carbon source. Xylose transport is less efficient than glucose transport, and its rate is dependent upon aeration. Xylose reductase appears to be a xylose inducible enzyme and xylitol dehydrogenase activity is constitutive, although both are repressed by glucose. Both xylose reductase and xylitol dehydrogenase activities are five- to tenfold lower in S. cerevisiae as compared to Candida utilis. In vivo conversion of (14)C-xylose in S. cerevisiae is demonstrated and xylitol is detected, although no significant levels of any other (14)C-labeled metabolites (e. g., ethanol) are observed.     

The ectopic overexpression of a citrus gibberellin 20-oxidase enhances the non-13-hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated phenotype in tobacco

Transgenic plants of Nicotiana tabacum overexpressing a gibberellin (GA) 20-oxidase cDNA ( CcGA20ox1 ) from citrus, under the control of the 35S promoter, were taller (up to twice) and had larger inflorescences and longer flower peduncles than those of control plants. Hypocotyls of transgenic seedlings were also longer (up to 4 times), and neither the seedlings nor the growing plants elongated further after application of GA₃. Hypocotyl and stem lengths were reduced by application of paclobutrazol, and this inhibition was reversed by exogenous GA₃. The ectopic overexpression of CcGA20ox1 enhanced the non-13-hydroxylation pathway of GA biosynthesis leading to GA₄, apparently at the expense of the early-13-hydroxylation pathway. The level of GA₄ (the active GA from the non-13-hydroxylation pathway) in the shoot of transgenic plants was 3–4 times higher than in control plants, whereas that of GA₁, formed via the early-13-hydroxylation pathway (the main GA biosynthesis pathway in tobacco), decreased or was not affected. GA₄ applied to the culture medium or to the expanding leaves was found to be at least equally active as GA₁ on stimulating hypocotyl and stem elongation of tobacco plants. The results suggest that the tall phenotype of tobacco transgenic plants was due to their higher content of GA₄, and that the GA response was saturated by the presence of the transgene.     

Palmitate modulates the early steps of insulin signalling pathway in pancreatic islets

Insulin stimulates its own secretion and synthesis by pancreatic beta-cells. Although the exact molecular mechanism involved is unknown, changes in beta-cell insulin signalling have been recognized as a potential link between insulin resistance and its impaired release, as observed in non-insulin-dependent diabetes. However, insulin resistance is also associated with elevated plasma levels of free fatty acids (FFA) that are well known modulators of insulin secretion by pancreatic islets. This information led us to investigate the effect of FFA on insulin receptor signalling in pancreatic islets. Exposure of pancreatic islets to palmitate caused up-regulation of several insulin-induced activities including tyrosine phosphorylation of insulin receptor and pp185. This is the first evidence that short exposure of these cells to 100 microM palmitate activates the early steps of insulin receptor signalling. 2-Bromopalmitate, a carnitine palmitoyl-CoA transferase-1 inhibitor, did not affect the effect of the fatty acid. Cerulenin, an acylation inhibitor, abolished the palmitate effect on protein levels and phosphorylation of insulin receptor. This result supports the proposition that protein acylation may be an important mechanism by which palmitate exerts its modulating effect on the intracellular insulin signalling pathway in rat pancreatic islets.     

The methymycin/pikromycin pathway: A model for metabolic diversity in natural product biosynthesis

The methymycin/pikromycin (Pik) macrolide pathway represents a robust metabolic system for analysis of modular polyketide biosynthesis. The enzymes that comprise this biosynthetic pathway display unprecedented substrate flexibility, combining to produce six structurally diverse macrolide antibiotics in Streptomyces venezuelae. Thus, it is appealing to consider that the pikromycin biosynthetic enzymes could be leveraged for high-throughput production of novel macrolide antibiotics. Accordingly, efforts over the past decade have focused on the detailed investigation of the six-module polyketide synthase, desosamine sugar assembly and glycosyl transfer, and the cytochrome P450 monooxygenase that is responsible for hydroxylation. This review summarizes the advances in understanding of pikromycin biosynthesis that have been gained during the course of these investigations.     

Gibberellin biosynthesis and the regulation of plant development

Gibberellins (GAs) form a large family of plant growth substances with distinct functions during the whole life cycle of higher plants. The rate of GA biosynthesis and catabolism determines how the GA hormone pool occurs in plants in a tissue and developmentally regulated manner. With the availability of genes coding for GA biosynthetic enzymes, our understanding has improved dramatically of how GA plant hormones regulate and integrate a wide range of growth and developmental processes. This review focuses on two plant systems, pumpkin and Arabidopsis, which have added significantly to our understanding of GA biosynthesis and its regulation. In addition, we present models for regulation of GA biosynthesis in transgenic plants, and discuss their suitability for altering plant growth and development.     

Plant development: three steps for stomata

Three basic helix-loop-helix proteins regulate sequential steps in the formation of stomata: SPEECHLESS initiates entry into the stomatal lineage; MUTE controls asymmetric divisions of stomatal precursor cells; and FAMA promotes guard cell differentiation.     

Functional analyses of cytochrome P450 genes responsible for the early steps of brassicicene C biosynthesis

We previously revealed that Orf8 and Orf6, which were identified in the brassicicene C biosynthetic gene cluster in Alternaria brassicicola strain ATCC96836, were fusicoccadiene (FD) synthase and 16-O-methyltransferase, respectively. In the present Letter, the early biosynthetic steps after the formation of FD were investigated. Plasmids carrying the FD synthase gene, one (or two) of five cytochrome P450 genes (orf1, orf2, orf5, orf7, and orf11) identified in the cluster and a cytochrome P450 reductase gene cloned from strain ATCC96836 were constructed and introduced into Saccharomyces cerevisiae. Based on the structures of the compounds produced by the transformants, Orf1 is suggested to be an 8β-hydroxylation enzyme that yields FD 8β-ol (4), followed by 16-hydroxylation by Orf7 to produce FD 8β16-diol (5).