Starter Molecule Research Articles

Investigation of the mechanism and steric course of the reaction catalyzed by 6-methylsalicylic acid synthase from Penicillium patulum using (R)-[1-13C;2-2H]- and (S)-[1-13C;2-2H]malonates.

Chiral malonyl-CoA derivatives, enzymically synthesized from (R)- and (S)-[1-13C;2-2H]malonates using succinyl-CoA transferase, were incorporated into 6-methylsalicylic acid with homogeneous 6-methylsalicylic acid synthase isolated from Penicillium patulum. Analysis of the 6-methylsalicylic acid formed established that the hydrogen atoms at the 3- and 5-positions are derived from opposite absolute configurations in malonyl-CoA. When acetoacetyl-CoA was used as the starter molecule, a single hydrogen atom is incorporated from the chiral malonates into the 3-position of the 6-methylsalicylic acid. Mass spectrometric analysis of the 6-methylsalicylic acid indicates that this hydrogen atom originates from HRe of malonyl-CoA or HSi in the polyketide intermediate. It is thus concluded that the hydrogen atom at the 5-position of 6-methylsalicylic acid originates from HSi of malonyl-CoA or HRe in the polyketide intermediate. During the reaction the enzyme also catalyzes the stereospecific exchange of hydrogen atoms in the polyketide intermediates. The implications of the stereochemical information from these experiments are discussed in relation to the mechanism of the 6-methylsalicylic acid synthase reaction.

Use of chiral malonates to determine the absolute configuration of the hydrogen atoms eliminated during the formation of 6-methylsalicylic acid by 6-methylsalicylic acid synthase from Penicillium patulum

When acetoacetyl-CoA was used as the starter molecule together with chiral malonyl-CoA derivatives, prepared from (R)- and (S)-[1-13C; 2-2H]malonates, for the biosynthesis of 6-methylsalicylic acid using 6-methylsalicylic acid synthase from Penicillium patulum, mass spectrometric analysis of the products established that the hydrogen atom incorporated at the 3-position of 6-methylsalicylic acid originates from HRe of malonyl-CoA allowing the deduction that the hydrogen atom at the 5-position must originate from HSi.

Rubber transferase activity in rubber particles of guayule

Rubber transferase (RuT) activity, measured as incorporation of radiolabelled isoprene from [14C]isopentenyl pyrophosphate (IPP) into rubber, was assayed in suspensions of washed rubber particles (WRPs) purified from stembark tissue of Parthenium argentatum. Isolated WRPs had high RuT activity which was not diminished even after repeated washing, demonstrating the firm association of the enzyme with the particles. The activity of RuT was characterized with respect to substrate and WRP concentration. The rate of IPP-incorporation was dependent upon the concentration of two substrates, IPP and the allylic pyrophosphate starter molecule E,E-farnesyl pyrophosphate (FPP). The enzyme present in 6 × 1010 WRPs per ml was saturated by 1 mM IPP and 20 μM FPP. Under saturating cosubstrate concentrations the apparent Km of RuT was ca 300 μM IPP and 3 μM FPP. Analysis of WRPs by SDS-PAGE revealed a simple protein profile characteristic of guayule rubber particles. A successful and facile assay for IPP-polymerization by isolated rubber particles is described.

Studies of precursor-directed biosynthesis with streptomyces. Part 2. New and unusual manumycin analogues produced by Streptomyces parvulus

The application of a new type of precursor-directed biosynthesis, in which artificial aromatic starter molecules were fed to the manumycin producer Streptomyces parvulus, resulted in new and unusual manumycin analogues for which structures are reported. The new compounds can be divided into three classes; the artificial starter molecule elongated (i) with the triene chain including the C5N moiety, (ii) with the chiral manumycin C13-side chain only, and (iii) with both substituents. Based on these results it is plausible to differentiate between an amidase linking the chiral C13-side chain to the artificial precursor, and a CoA-transferase activating the aromatic carboxy group for further elongation via the polyketide pathway. The specificity of the enzymes involved with regard to the structure of the aromatic precursor is discussed.

The Separation of Two Different Enzymes Catalyzing the Formation of Hydroxycinnamic A dd Glucosides and Esters

Protein extracts from anthers of Tulipa cv. Apcldoorn catalyze the formation of glucosidcs and glucose esters of hydroxycinnamic acid with UDP-glucose as the glucosyl donor. By chromatofocusing and by HPLC (anion exchange chromatography) it could be demonstrated for the first time that two different enzymes are involved in these reactions. By using the molecular sieving (HPLC) a molecular weight of about 45000 D was determined for the GT (E) (= catalyz­ing the formation of esters) and of about 25000 D for the GT (G) (= catalyzing the formation of glucosidcs). Both enzymes exhibit a high spezificity for free hydroxycinnamic acids. It is assumed that these transferases arc involved in the formation of feruloylglucose and ferulic acid glucoside, the latter of which can be isolated from anthers at early developmental stages. Furthermore, it is hypothesized that feruloylglucose functions as a starter molecule for the transacylation reaction by which di- and triferulovlsucrose are formed.

Substrate specificity of chalcone synthase from Petroselinum hortense. Formation of phloroglucinol derivatives from aliphatic substrates.

The substrate specificity of chalcone synthase, the key enzyme of flavonoid biosynthesis, was investigated. A purified enzyme preparation from cell suspension cultures of parsley (Petroselinum hortense) catalyzed chain elongations with acetate units from malonyl-CoA, using various aromatic and aliphatic CoA esters as starter molecules. Malonyl-CoA could not be replaced by malonyl acyl carrier protein in the standard chalcone synthase assay. Butyryl-CoA, hexanoyl-CoA, and benzoyl-CoA served as substrates for the condensation reaction with similar efficiency as 4-coumaroyl-CoA, the natural substrate of the enzyme. Acetyl-CoA and octanoyl-CoA were relatively poor substrates. Among the products formed with the two most efficient aliphatic substrates tested, butyryl-CoA and hexanoyl-CoA, were the respective chalcone analogues, phlorobutyrophenone and phlorocaprophenone. The possibility is discussed that chalcone synthase and the corresponding enzyme of fatty acid synthesis in higher plants, beta-ketoacyl-acyl carrier protein synthase, have a common evolutionary origin.

Biosynthesis of cyclopentenyl fatty acids. Cyclopentenylglycine, a non-proteinogenic amino acid as precursor of cyclic fatty acids in Flacourtiaceae.

In seeds of Hydnocarpus anthelminthica of Flacourtiaceae, cyclopentenylglycine and cyclopentenyl fatty acids are found naturally. The non-proteinogenic amino acid may serve as precursor of cyclopentenyl fatty acids via aleprolic acid, the starter molecule for these long-chain compounds. After administration of cyclopentenyl[2-14C]glycine to maturing seeds of H. anthelminthica, labelled cyclopentenyl fatty acids were synthesized. Comparative activities were observed, when [1-14C]aleprolic acid was supplied to the seeds. Incorporation studies with [1-14C]acetate revealed that the chain-lengthening systems for straight-chain and cyclic fatty acids were still functioning in mature seeds. Endosperm and embryo of H. Anthelminthica seeds synthesized cyclopentenyl fatty acids from cyclopentenyl[2-14C]glycine, [1-14C]aleprolic acid and [1-14C]acetate. In embryonic tissue, a dilution experiment proved the following path for cyclopentenyl fatty acid biosynthesis: cyclopentenylglycine leads to aleprolic acid leads to cyclopentenyl fatty acids. The conversion of cyclopentenylglycine to aleprolic acid may occur via transamination and oxidative decarboxylation; activated aleprolic acid is then lengthened by C2-units to cyclopentenyl fatty acids.

Biosynthesis of cyclopentenyl fatty acids: (2-Cyclopentenyl)carboxylic acid (aleprolic acid) as a special primer for fatty acid biosynthesis in Flacourtiaceae

The biosynthesis of cyclopentenyl fatty acids from (2-cyclopentenyl)carboxylic acid (aleprolic acid) via chain-lengthening by C 2-units was tested in seeds and leaves of Caloncoba echinata and Hydnocarpus anthelminthica of Flacourtiaceae and in various preparations of higher plants other than Flacourtiaceae. Only tissues of Flacourtiaceae, where cyclopentenyl fatty acids are found naturally, were able to accept aleprolic acid as a starter molecule for the synthesis of cyclic fatty acids. Labelling patterns of straight chain and cyclic fatty acids, synthesized after incubation of Flacourtiaceae seeds with [1- 14C]-acetate, indicated de novo synthesis of C 16 fatty acids in either case, followed by elongation to higher homologs.

Substrate specificity of flavanone synthase from cell suspension cultures of parsley and structure of release products in vitro

The substrate specificity of an extensively purified flavanone synthase from light-induced cell suspension cultures of Petroselinum hortense was investigated. p-Coumaroyl-CoA was found to be the only efficient substrate for flavanone synthesis, producing naringenin (5,7,4′-trihydroxyflavanone). Besides 4-hydroxy-6[4-hydroxystyryl]2-pyrone (F. Kreuzaler and K. Hahlbrock (1975) Arch. Biochem. Biophys. 169, 84–90) two further release products of the synthase reaction in vitro were identified as 4-hydroxy-5,6-dihydro-6(4-hydroxyphenyl)2-pyrone and p-hydroxybenzalacetone. The apparent K m values for malonyl-CoA and p-coumaroyl-CoA in the reaction leading to naringenin, and for p-coumaroyl-CoA in the reaction leading to the styrylpyrone derivative were 35, 1.6, and 2.6 μ m, respectively. With caffeoyl-CoA as substrate only a very small amount of eriodictyol (5,7,3′,4′-tetrahydroxyflavanone) was formed besides relatively large amounts of the corresponding styrylpyrone, dihydropyrone, and benzalacetone derivatives. No flavanone formation was observed with feruloyl-CoA as substrate, but again appreciable amounts of the three types of short-chain release products were formed. No reaction at all took place with cinnamoyl-CoA, p-methoxycinnamoyl-CoA, isoferuloyl-CoA, or p-hydroxybenzoyl-CoA. None of the styrylpyrone, dihydropyrone, and benzalacetone derivatives has been detected in the cell cultures in vivo. The present results suggest that naringenin is the only natural product of the synthase reaction and that further substitution in the B-ring of the flavonoids occurs in parsley at or after the flavanone stage. The nature of the smaller release products is consistent with the assumption of a stepwise addition of acetate units from malonyl-CoA to the acyl moiety of the starter molecule, p-coumaroyl-CoA.

Biosynthesis of Cyclopentenyl Fatty Acids. Chain Elongation and Desaturation

In seeds of Hydnocarpus anthelminthica of Flacourtiaceae, cyclopentenylglycine and cyclopentenyl fatty acids are found naturally. The non-proteinogenic amino acid may serve as precursor of cyclo-pentenyl fatty acids via aleprolic acid, the starter molecule for these long-chain compounds. After administration of cyclopentenyl[2-14C]glycine to maturing seeds of H. anthelminthica, labelled cyclopentenyl fatty acids were synthesized. Comparative activities were observed, when [1-14C]aleprolic acid was supplied to the seeds. Incorporation studies with [1-14C]acetate revealed that the chain-lengthening systems for straight-chain and cyclic fatty acids were still functioning in mature seeds. Endosperm and embryo of H. anthelminthica seeds synthesized cyclopentenyl fatty acids from cyclopentenyl[2-14C]glycine, [1-14C]aleprolic acid and [1-14C]acetate. In embryonic tissue, a dilution experiment proved the following path for cyclopentenyl fatty acid biosynthesis: cyclopentenylglycine aleprolic acid cyclopentenyl fatty acids. The conversion of cyclopentenylglycine to aleprolic acid may occur via transamination and oxidative decarboxylation; activated aleprolic acid is then lengthened by C2-units to cyclopentenyl fatty acids.