Pipecolic acid biosynthesis in Rhizoctonia leguminicola. I. The lysine saccharopine, delta 1-piperideine-6-carboxylic acid pathway

The biosynthesis of pipecolic acid from L-lysine in the fungal parasite, Rhizoctonia leguminicola has been reinvestigated. Pipecolate is then utilized to form the toxic octahydroindolizine alkaloids, slaframine and swainsonine. Incorporation studies of L-versus D-[U-14C]lysine into R. leguminicola metabolites confirmed earlier findings that L-lysine is the predominant substrate for pipecolate formation and D-lysine for alpha-N-acetyllysine (concerned in lysine catabolism). However [alpha-15N]lysine, not [epsilon-15N]lysine as previously reported, labeled pipecolate. Such findings implied that delta 1-piperideine-6-carboxylate, not delta 1-piperideine-2-carboxylate, was formed from lysine and was the immediate precursor of pipecolate. Evidence from cell-free enzyme systems established the following biosynthetic events: L-lysine A----saccharopine B----delta 1-piperideine-6-carboxylate C----pipecolate. Products of reactions A and C were identified from biological and chemical considerations. Reaction B was carried out by a previously undescribed flavin enzyme termed saccharopine oxidase. The product of reaction B, which reacted with p-dimethylaminobenzaldehyde, was reduced with Na-CNB2H3. Its NMR spectrum was identical with that of deuteriated pipecolate prepared from authentic delta 1-piperideine-6-carboxylate, but not from authentic delta 1-piperideine-2-carboxylate. Reaction B represents a branching of primary lysine metabolism from saccharopine to a secondary pathway leading to pipecolate and to octahydroindolizine alkaloids in R. leguminicola.     

D-lysergic acid activation and cell-free synthesis of D-lysergyl peptides in enzyme fractions from the ergot fungus Claviceps purpurea

The D-lysergic acid activating enzyme from the ergot fungus Claviceps purpurea was purified to near homogeneity. It has a native Mr of about 245,000 and in its denatured form is a single polypeptide chain of Mr 62,000. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on D-lysergic acid and, though much less, that dependent on dihydrolysergic acid. Western blot analysis of SDS electropherograms of crude protein extracts from C. purpurea using monospecific antibodies directed against the D-lysergic acid activating enzyme revealed the immunostaining of one particular band which was identical with that of the D-lysergic acid activating enzyme. No significant immunoreactive band with higher molecular weight was seen, which precludes the possibility that the enzyme had arisen from the proteolysis of a high molecular weight ergot peptide synthetase. An ammonium sulfate fractionated enzyme fraction was prepared from C. purpurea strain C1 that catalyzed the incorporation of D-lysergic acid into two peptides which besides D-lysergic acid contained alanine, phenylalanine, and proline. Dihydrolysergic acid was efficiently incorporated into the corresponding dihydrolysergic acid containing analogues of the two compounds. Radiochemical analysis and degradation studies suggest that the two D-lysergic acid containing peptides most probably are N-[N-(D-lysergyl)-L-alanlyl]-L-phenylalanyl-L-proline lactam and N-[N-(D-lysergyl)-L-analyl]-L-phenylalanyl-D-proline lactam, respectively. N-[N-(D-Lysergyl)-L-alanyl]-L-phenylalanyl-L-proline lactam is considered to be the immediate precursor of ergotamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enantioselective synthesis of various D-amino acids by a multi-enzyme system

We have developed an effective method for the synthesis of various D-amino acids from the corresponding α-keto acids and ammonia by coupling four enzyme reactions catalyzed by D-amino acid aminotransferase, glutamate racemase, glutamate dehydrogenase, and formate dehydrogenase. In this system, D-glutamate is continuously regenerated from α-ketoglutarate, ammonia and NADH by the coupled reaction of glutamate dehydrogenase and glutamate racemase, and used as an amino donor for the enantioselective D-amino acid synthesis by the D-amino acid aminotransferase reaction. The unidirectional formate dehydrogenase reaction is also coupled to regenerate NADH consumed. Under the optimum conditions, D-enantiomers of valine, alanine, α-keto analogues with a molar yield higher than 80%.     

Biosynthesis of quinoxaline antibiotics: purification and characterization of the quinoxaline-2-carboxylic acid activating enzyme from Streptomyces triostinicus

A quinoxaline-2-carboxylic acid activating enzyme was purified to homogeneity from triostin-producing Streptomyces triostinicus. It could also be purified from quinomycin-producing Streptomyces echinatus. Triostins and quinomycins are peptide lactones that contain quinoxaline-2-carboxylic acid as chromophoric moiety. The enzyme catalyzes the ATP-pyrophosphate exchange reaction dependent on quinoxaline-2-carboxylic acid and the formation of the corresponding adenylate. Besides quinoxaline-2-carboxylic acid, the enzyme also catalyzes the formation of adenylates from quinoline-2-carboxylic acid and thieno[3,2-b]pyridine-5-carboxylic acid. No adenylates were seen from quinoline-3-carboxylic acid, quinoline-4-carboxylic acid, pyridine-2-carboxylic acid, and 2-pyrazinecarboxylic acid. Previous work [Gauvreau, D., & Waring, M. J. (1984) Can. J. Microbiol. 30, 439-450] revealed that quinoline-2-carboxylic acid and thieno[3,2-b]pyridine-5-carboxylic acid became efficiently incorporated into the corresponding quinoxaline antibiotic analogues in vivo. Together with the data described here, this suggests that the enzyme is part of the quinoxaline antibiotics synthesizing enzyme system. The enzyme displays a native molecular weight of 42,000, whereas in its denatured form it is a polypeptide of Mr 52,000-53,000. It resembles in its behavior actinomycin synthetase I, the chromophore activating enzyme involved in actinomycin biosynthesis [Keller, U., Kleinkauf, H., & Zocher, R. (1984) Biochemistry 23, 1479-1484].     

4-Methyl-3-hydroxyanthranilic acid activating enzyme from actinomycin-producing Streptomyces chrysomallus

A 4-methyl-3-hydroxyanthranilic acid (4-MHA) activating enzyme was purified 24-fold from a crude protein extract of Streptomyces chrysomallus . The enzyme catalyzes both 4-MHA-dependent ATP/PPi exchange and the formation of the corresponding adenylate. No AMP was formed during the reaction, indicating that no covalent binding of 4-MHA takes place. Besides 4-MHA, the enzyme also catalyzes the formation of adenylates from 3-hydroxyanthranilic acid (3-HA), anthranilic acid (AA), benzoic acid (BA), 3-hydroxybenzoic acid (3-HB), 4-methyl-3-hydroxybenzoic acid (4-MHB), 4-methyl-3-methoxybenzoic acid (4- MMB ), and 4-aminobenzoic acid (4-AB). No such adenylates were formed from 2-aminophenol (2-AP), 2-hydroxybenzoic acid (2-HB), 3-hydroxykynurenine (3-HK), and tryptophan (Trp). 3-HA, 4-MHB, and 4-AB were among the structural analogues of 4-MHA that were the most effective for adenylate synthesis. In the case of 3-HA, considerable AMP release was observed, most probably due to nonenzymatic hydrolysis of the corresponding adenylate. A molecular weight between 53 000 and 57 000 was estimated. The specific activity of the enzyme was correlated with the titer of antibiotic in the cultures, and feeding experiments with whole mycelium of S. chrysomallus showed that 4-MHB was a strong inhibitor of actinomycin synthesis in vivo. The data strongly suggest that the enzyme is involved in the biosynthesis of actinomycin.     

Reciprocal Control of Thyroid Binding and the Pipecolate Pathway in the Brain

Thyroid hormones have long been known to play an essential role in brain growth and development, with cytoplasmic thyroid hormone binding proteins (THBPs) playing a critical role in thyroid hormone bioavailability. A major mammalian THBP is μ-crystallin (CRYM), which was originally characterized by its ability to strongly bind thyroid hormones in an NADPH-dependent fashion. However, in 2011 it was discovered that CRYM is also an enzyme, namely ketimine reductase (KR), which catalyzes the NAD(P)H-dependent reduction of –C=N– (imine) double bonds of a number of cyclic ketimine substrates including sulfur-containing cyclic ketimines. The enzyme activity was also shown to be potently inhibited by thyroid hormones, thus suggesting a novel reciprocal relationship between enzyme catalysis and thyroid hormone bioavailability. KR is involved in a number of amino acid metabolic pathways. However, the best documented biological function of KR is its role as a ?¹-piperideine-2-carboxylate (P2C) reductase in the pipecolate pathway of lysine metabolism. The pipecolate pathway is the main l-lysine degradation pathway in the adult brain, whereas the saccharopine pathway predominates in extracerebral tissues and in infant brain, suggesting that KR has evolved to perform specific and important roles in neural development and function. The potent regulation of KR activity by thyroid hormones adds further weight to this suggestion. KR is also involved in l-ornithine/l-glutamate/l-proline metabolism as well as sulfur-containing amino acid metabolism. This review describes the pipecolate pathway and recent discoveries related to mammalian KR function, which have important implications in normal and pathological brain functions.     

Microbial transformation of immunosuppressive compounds III. Glucosylation of immunomycin (FR 900520) and FK 506 byBacillus subtilis ATCC 55060

Summary The regiospecific glucosylation of FK 506 and immunomycin (FR 900520) at the 24-hydroxy position was performed using resting cells ofBacillus subtilis ATCC 55060. 24-Glucopyranosyl FK 506 and 24-glucopyranosyl immunomycin were isolated by methylene chloride extraction and purification using reverse phase HPLC. The metabolite structures were established using spectroscopic techniques including MS and NMR. The glucose conjugate was further confirmed by chemical degradation. Enzymatic glucosylation was demonstrated using cell-free extracts derived fromBacillus subtilis ATCC 55060. The 24-glucosyltransferase, which appears UDP-glucose dependent, was solubilized from cell membranes by treatment with 0.1% Nonidet P-40 detergent. The optimal conditions for assay of the enzyme have been determined.     

Enzymatic tRNA acylation by acid and alpha-hydroxy acid analogues of amino acids

Incorporation of unnatural amino acids with unique chemical functionalities has proven to be a valuable tool for expansion of the functional repertoire and properties of proteins as well as for structure-function analysis. Incorporation of alpha-hydroxy acids (primary amino group is substituted with hydroxyl) leads to the synthesis of proteins with peptide bonds being substituted by ester bonds. Practical application of this modification is limited by the necessity to prepare corresponding acylated tRNA by chemical synthesis. We investigated the possibility of enzymatic incorporation of alpha-hydroxy acid and acid analogues (lacking amino group) of amino acids into tRNA using aminoacyl-tRNA synthetases (aaRSs). We studied direct acylation of tRNAs by alpha-hydroxy acid and acid analogues of amino acids and corresponding chemically synthesized analogues of aminoacyl-adenylates. Using adenylate analogues we were able to enzymatically acylate tRNA with amino acid analogues which were otherwise completely inactive in direct aminoacylation reaction, thus bypassing the natural mechanisms ensuring the selectivity of tRNA aminoacylation. Our results are the first demonstration that the use of synthetic aminoacyl-adenylates as substrates in tRNA aminoacylation reaction may provide a way for incorporation of unnatural amino acids into tRNA, and consequently into proteins.     

Significance of Altered Carbon Flow in Aromatic Amino Acid Synthesis: an Approach to the Isolation of Regulatory Mutants in Pseudomonas aeruginosa

Pseudomonas aeruginosa displays a native resistance to a variety of inhibitory compounds, including many analogues of amino acids, purines, and pyrimidines. Therefore, it has been difficult to isolate analogue-resistant regulatory mutants which have been so valuable in other microbial species for the study of enzyme control mechanisms and for the study of amino acid transport and its regulation. However, we have found that increased sensitivity to growth inhibition by analogues can be demonstrated by manipulation of the nutritional environment. When P. aeruginosa is grown with fructose as the nutritional source of carbon and energy, the cells become sensitive to growth inhibition by beta-2-thienylalanine and p-amino-phenylalanine, analogues of phenylalanine and tyrosine, respectively. Thus, mutants were isolated which are resistant to growth inhibition by beta-2-thienylalanine and p-amino-phenylalanine when fructose is the carbon source, and many of the beta-2-thienylalanine-resistant mutants overproduce phenylalanine. Several lines of evidence suggest that the increased sensitivity to growth inhibition by analogues of phenylalanine and tyrosine reflects a decreased rate of synthesis of aromatic amino acids or their precursors when fructose is the carbon source. This general approach promises to be valuable in the study of regulatory phenomena in microorganisms which, like P. aeruginosa, are naturally resistant to many metabolite analogues.     

Oxodiene formation during the Vicia sativa lipoxygenase-catalyzed reaction: occurrence of dioxygenase and fatty acid lyase activities associated in a single protein

An enzyme with at least dual activities, lipoxygenase and fatty acid lyase, has been isolated from Vicia sativa seeds. The enzyme utilizes directly linoleic acid as substrate. The enzyme had a pH optimum at 5.8 for the two activities and converted linoleic acid into two products: 9-hydroperoxylinoleic acid and trans-2, cis-4 decadienal. The enzyme does not act on 13- or 9- fatty acid hydroperoxide isomers. An enzymatic reaction for the biogenesis of trans-2, cis-4- decadienal is proposed. This involves the synthesis of an intermediate peroxyl radical due to oxygen insertion in carbon 9 of linoleic acid. This intermediate peroxyl radical may be converted into 9-HPOD and 2,4-decadienal.     

Sheep kidney pyruvate carboxylase. Studies on the coupling of adenosine triphosphate hydrolysis and CO2 fixation.

Initial velocity and isotope exchange studies confirmed that the over-all reaction, like that catalyzed by pyruvate carboxylase purified from rat liver and chicken liver, was a nonclassical Ping Pong Bi Bi Uni Uni sequence with ATP and HCO3-binding randomly in the Bi Bi partial reaction. Three possible mechanisms for the coupling of ATP hydrolysis and CO2 fixation are considered: (i) Mechanism i, a concerted mechanism without the formation of a kinetically significant or detectable intermediate; (ii) Mechanism ii, activation of the enzyme by ATP to form an activated phosphoenzyme complex which can react with HCO3- by formation of a phosphorylated intermediate. On the basis of other evidence, an activated intermediate containing the ADP moiety was considered improbable. Evidence is presented which indicates that an isotopic exchange between ATP and ADP in the absence of added orthophosphate is not a property of the sheep kidney enzyme. This observation removed the need to postulate either that this exchange is an abortive reaction, or that there is a compulsory formation of a phosphoenzyme intermediate. Two analogues of ADP, alpha,beta-methylene adenosine diphosphate, and adenosine 5'-phosphosulfate, have been used to provide further evidence against Mechanism ii. Both compounds were competitive inhibitors with respect to MgATP2- (Ki values respectively, 0.58 mM and 3.0 mM, compared with 0.17 mM for ADP), but neither could be phosphorylated by the enzyme. Neither analogue could replace ADP in the HCO3-: oxalacetate isotopic exchange reaction, indicating that phosphorylation of ADP is necessary for this exchange to occur, and that Mechanism ii is not applicable. Since Mechanism iii involves formation of a carbonly phosphate intermediate, analogues of this compound, namely, carbamyl phosphate and phosphonacetic acid were used to examine this pathway. The fact that the enzyme catalyzed the synthesis of ATP from ADP and carbamyl phosphate, and that phosphonacetic acid was a noncompetitive inhibitor with respect to MgATP2- (Ki = 0.5 mM) provides strong evidence that a carbonyl phosphate derivative is involved in the reaction mechanism. However, we have not found from initial velocity studies evidence for the formation of this intermediate, and it may therefore have only a transient existence in an essentially concerted reaction.     

Direct solid-phase synthesis of octreotide conjugates: precursors for use as tumor-targeted radiopharmaceuticals.

Somatostatin analogues, such as octreotide, are useful for the visualization and treatment of tumors. Unfortunately, these compounds were produced synthetically using complex and inefficient procedures. Here, we describe a novel approach for the synthesis of octreotide and its analogues using p-carboxybenzaldehyde to anchor Fmoc-threoninol to solid phase resins. The reaction of the two hydroxyl groups of Fmoc-threoninol with p-carboxybenzaldehyde was catalyzed with p-toluenesulphonic acid in chloroform using a Dean-Stark apparatus to form Fmoc-threoninol p-carboxybenzacetal in 91% yield. The Fmoc-threoninol p-carboxybenzacetal acted as an Fmoc-amino acid derivative and the carboxyl group of Fmoc-threoninol p-carboxybenzacetal was coupled to an amine-resin via a DCC coupling reaction. The synthesis of protected octreotide and its conjugates were carried out in their entirety using a conventional Fmoc protocol and an autosynthesizer. The acetal was stable during the stepwise elongation of each Fmoc-amino acid as shown by the averaged coupling yield (> 95%). Octreotide (74 to 78% yield) and five conjugated derivatives were synthesized with high yields using this procedure, including three radiotherapy octreotides (62 to 75% yield) and two cellular markers (72 to 76% yield). This novel approach provides a strategy for the rapid and efficient large-scale synthesis of octreotide and its analogues for radiopharmaceutical and tagged conjugates.     

Citrate and the conversion of carbohydrate into fat. The regulation of fatty acid synthesis by rat liver extracts

1. The rate of fatty acid synthesis by particle-free extracts prepared from rat liver is increased greatly if the enzyme system is first activated with citrate. 2. The extent of the activation depends on the citrate concentration and on the time of activation in an interdependent manner. 3. Citrate activation is strongly dependent on temperature. 4. Tricarballylate can replace citrate as an activator, but its presence in the assay inhibits fatty acid synthesis. 5. Mg(2+) ions can replace citrate in the activation but not in the complete reaction system. 6. ATP prevents the activating effect of citrate and Mg(2+) ions. 7. The rate of fatty acid synthesis is increased by palmitoyl-dl-carnitine. This type of activation, additional to that caused by citrate, is rapid and does not depend on prior incubation. 8. Inhibition of fatty acid synthesis by palmitoyl-CoA can be prevented by palmitoyl-dl-carnitine or by increasing the concentration of protein.     

Purification of PCNA as a nucleotide excision repair protein.

Human cell free extracts carry out nucleotide excision repair in vitro. The extract is readily separated into two fractions by chromatography on a DEAE column. Neither the low salt (0.1 M KCl) nor the high salt (0.8 M KCl) fractions are capable of repair synthesis but the combination of the two restore the repair synthesis activity. Using the repair synthesis assay we purified a protein of 37 kDa from the high salt fraction which upon addition to the low salt fraction restores repair synthesis activity. Amino acid sequence analysis, amino acid composition and immunoblotting with PCNA antibodies revealed that the 37 kDa protein is the proliferating cell nuclear antigen (PCNA) known to stimulate DNA Polymerases delta and epsilon. By using an assay which specifically measures the excision of thymine dimers we found that PCNA is not required for the actual excision reaction per se but increases the extent of excision by enabling the excision repair enzyme to turn over catalytically.     

Purification and characterization of actinomycin synthetase I, a 4-methyl-3-hydroxyanthranilic acid-AMP ligase from Streptomyces chrysomallus.

Actinomycin synthetase I was purified to homogeneiety from actinomycin-producing Streptomyces chrysomallus. The purified enzyme is a single polypeptide chain of M(r) 45,000. It catalyzes the formation of the adenylate of 4-methyl-3-hydroxyanthranilic acid (4-MHA) from the free acid and ATP in an equilibrium reaction. 4-MHA is the precursor of the chromophoric part of actinomycin. By using the 4-MHA analogue, 4-methyl-3-hydroxybenzoic acid, as a model substrate it could be established that the equilibrium constant Keq is independent on enzyme concentration, which suggests that no stoichiometric acyladenylate-enzyme complex is formed in contrast to observations made with aminoacyl adenylates formed by aminoacyl-tRNA synthetases or multifunctional peptide synthetases. Actinomycin synthetase I does not charge itself with substrate carboxylic acid via a covalent thioester bond as is usual for amino acid activation in non-ribosomal peptide synthesis. In addition, the enzyme does not act as an acyl-coenzyme A ligase as revealed by its inability to release AMP in the presence of 4-MHA or other structurally related aromatic carboxylic acids, coenzyme A and ATP. Additional analysis of the activation reaction showed that it is exothermic, whereas the free enthalpy change delta G0 is positive due to a negative entropy change indicating a strong influence of restriction of random motion on the course of the reaction. Determinations of Km and kcat of various substrate carboxylic acids revealed the highest kcat/Km ratio for the natural substrate 4-MHA. From these properties, actinomycin synthetase I represents the prototype of novel chromophore activating enzymes involved in non-ribosomal synthesis of chromopeptide lactones in streptomycetes.     

Histidine in the nucleotide-binding site of NADP-linked isocitrate dehydrogenase from pig heart.

Incubation of pig heart NADP-dependent isocitrate dehydrogenase with ethoxyformic anhydride (diethylpyrocarbonate) at pH 6.2 results in a 9-fold greater rate of loss of dehydrogenase than of oxalosuccinate decarboxylase activity. The rate constants for loss of dehydrogenase and decarboxylase activities depend on the basic form of ionizable groups with pK values of 5.67 and 7.05, respectively, suggesting that inactivation of the two catalytic functions results from reaction with different amino acid residues. The rate of loss of dehydrogenase activity is decreased only slightly in the presence of manganous isocitrate, but is reduced up to 10-fold by addition of the coenzymes or coenzyme analogues, such as 2'-phosphoadenosine 5'-diphosphoribose (Rib-P2-Ado-P). Enzyme modified at pH 5.8 fails to bind NADPH, but exhibits manganese-enhanced isocitrate binding typical of native enzyme, indicating that reaction takes place in the region of the nucleotide binding site. Dissociation constants for enzyme . coenzyme-analogue complexes have been calculated from the decrease in the rate of inactivation as a function of analogue concentration. In the presence of isocitrate, activating metals (Mn2+, Mg2+, Zn2+) decrease the Kd value for enzyme . Rib-P2-Ado-P, while the inhibitor Ca2+ increases Kd. The strengthened binding of nucleotide produced by activating metal-isocitrate complexes may be essential for the catalytic reaction, reflecting an optimal orientation of NADP+ to facilitate hydride transfer. Measurements of ethoxyformyl-histidine formation at 240 nm and of incorporation of [14C]ethoxy groups in the presence and absence of Rib-P2-Ado-P indicate that loss of activity may be related to modification of approximately one histidine. The critical histidine appears to be located in the nucleotide binding site in a region distal from the substrate binding site.     

Aminoacyl-tRNA synthetases from yeast: generality of chemical proofreading in the prevention of misaminoacylation of tRNA.

The specificity of valyl-, phenylalanyl-, and tyrosyl-tRNA synthetases from yeast has been examined by a series of stringent tests designed to eliminate the possibility of artefactual interference. Valyl-tRNA synthetase, as well as activating a number of amino acid analogues, will accept alanine, cysteine, isoleucine, and serine in addition to threonine as substrates for both ATP-PPi exchange and transfer to some tRNAVal species. The transfer is not observed if atempts are made to isolate the appropriate aminoacyl-tRNAVal-C-C-A but its role in the overall aminoacylation can be suspected from both the formation of a stable aminoacyl-tRNAVal-C-C-A(3'NH2) compound and from the stoichiometry of ATP hydrolysis during the aminoacylation of the native tRNA. Similar tests with phenylalanyl-tRNA synthetase indicate that this enzyme will also activate and transfer other naturally occurring amino acids, namely, leucine, methionine, and tyrosine. The tyrosine enzyme, which lacks the hydrolytic capacity of the other two enzymes (von der Haar, F., & Cramer, F (1976) Biochemistry 15, 4131--4138) is probably absolutely specific for tyrosine. It is concluded that chemical proofreading, in terms of an enzymatic hydrolysis of a misacylated tRNA, plays an important part in maintaining the specificity in the overall reaction and that this activity may be more widespread than has so far been suspected.     

Chromophore activating enzyme involved in the biosynthesis of the mikamycin B antibiotic etamycin from Streptomyces griseoviridus

A 3-hydroxypicolinic acid activating enzyme from etamycin producing Streptomyces griseoviridus has been purified to apparent homogeneity. Etamycin is a member of mikamycin B antibiotics, chromopeptide lactones, which contain 3-hydroxypicolinic acid (3-HPA) as the chromophoric group. The enzyme catalyzes both the 3-HPA-dependent ATP-pyrophosphate exchange and the formation of 3-HPA adenylate from 3-HPA and ATP. SDS-polyacrylamide gel electrophoresis indicates that the enzyme is a single polypeptide chain with a Mr between 56,000 and 58,000. The molecular mass of the native enzyme was in the same range. In addition to 3-HPA, the enzyme catalyzes the formation of adenylates from picolinic acid, nicotinic acid, and 2-pyrazinecarboxylic acid. Nicotinic acid and picolinic acid when added externally to etamycin producing S. griseoviridus cultures gave rise to the formation of etamycin analogues each containing nicotinic acid or picolinic acid instead of the genuine 3-HPA. The data strongly suggest that the enzyme is involved in the biosynthesis of the chromopeptide lactone etamycin and possibly in that of other mikamycin B antibiotics.     

Factors affecting the initial rate of lipoxygenase catalysis

The rate of peroxidation of linoleic acid by soybean type-1 lipoxygenase was studied under conditions which assured that the substrate was present as a monomolecular solution and that the first 5% of the reaction was observed. In order to achieve this, the kinetics were carried out at pH 10.0 in borate buffer using linoleic acid and enzyme concentrations of less than 75 μM and 0.2 nM respectively. The initial rate was increased by the presence of added product (13-hydroperoxy-9(Z),11(E)-octadecadienoic acid) in the substrate solutions in a concentration dependent and saturatable fashion. Product analogues lacking the hydroperoxide group (13-hydroxy-9(Z),11(E)-octadecadienoic acid and 13-methoxy-9(Z),11(E)-octadecadienoic acid) did not evoke this rate enhancing effect. These compounds reduced the initial rate when preincubated with enzyme prior to mixing with substrate. The results indicated that the chemical reactivity of the product was a necessary requirement for its activating effect on the enzyme.