Studies on new phosphate ester antifungal antibiotics phoslactomycins. I. Taxonomy, fermentation, purification and biological activities.

New antibiotics phoslactomycins A, B, C, D, E and F, which contain alpha, beta-unsaturated delta-lactone, phosphate ester, conjugated diene and cyclohexane ring moieties, were isolated from the culture broth of a soil isolate actinomycete. Morphological, cultural and physiological studies revealed that the isolate is a strain of Streptomyces nigrescens. Phoslactomycins were obtained by butanol extraction, gel filtration and reverse phase chromatography. The antibiotics show strong activity against various fungi, particularly phytopathogenic fungi (Botrytis cinerea and Alternaria kikuchiana).

Studies on new phosphate ester antifungal antibiotics phoslactomycins. II. Structure elucidation of phoslactomycins A to F.

Phoslactomycins A to F are new antifungal antibiotics produced by Streptomyces nigrescens SC-273. On the basis of physico-chemical properties and NMR studies, their structures have been determined as shown in Fig. 6. They are characterized by possessing alpha, beta-unsaturated delta-lactone, phosphate ester, conjugated diene and cyclohexane ring moieties. The structural difference between them is ascribed to a substituent bound to the cyclohexane ring.

Formation and utilization of formyl phosphate by N10-formyltetrahydrofolate synthetase: evidence for formyl phosphate as an intermediate in the reaction.

N10-Formyltetrahydrofolate synthetase from bacteria and yeast catalyzes a slow formate-dependent ADP formation in the absence of H4folate. The synthesis of formyl phosphate by the enzyme was detected by trapping the intermediate as formyl hydroxamate. That the "formate kinase" activity was part of the catalytic center of N10-formyltetrahydrofolate synthetase was shown by demonstrating coordinate inactivation of the "kinase" and synthetase activities by heat and a sulfhydryl reagent, similar effects of monovalent cations, similar Km values for substrates, and similar Ki values for the inhibitor phosphonoacetaldehyde for both activities. The relative rates of the kinase activities for the bacterial and yeast enzymes are about 10(-4) and 4 x 10(-6) of their respective synthetase activities. These slow rates for the kinase reaction can be explained by the slow dissociation of ADP and formyl phosphate from the enzyme. This conclusion is supported by rapid-quench studies where a "burst" of ADP formation (6.4 s-1) was observed that is considerably faster than the steady-state rate (0.024 s-1). The demonstration of enzyme-bound products by a micropartition assay and the lack of a significant formate-stimulated exchange between ADP and ATP provide further evidence for the slow release of the products from the enzyme. The synthesis of N10-CHO-H4folate when H4folate was added to the E-formyl phosphate-ADP complex is also characterized by a "burst" of product formation. The rate of this burst phase at 5 degrees C occurs with a rate constant of 18 s-1 compared to 14 s-1 for the overall reaction at the same temperature. These results provide further evidence for formyl phosphate as an intermediate in the reaction and are consistent with the sequential mechanism of the normal catalytic pathway. Positional isotope exchange experiments using [beta,gamma-18O]ATP showed no evidence for exchange during turnover experiments in the presence of either H4folate or the competitive inhibitor pteroyltriglutamate. The absence of scrambling of the 18O label as observed by 31P NMR suggests that the central complex may impose restraints to limit free rotation of the P beta oxygens of the product ADP.

The bialaphos resistance gene (bar) plays a role in both self-defense and bialaphos biosynthesis in Streptomyces hygroscopicus.

We inactivated the bialaphos (BA) resistance gene (bar) of a BA producer, Streptomyces hygroscopicus, by the gene replacement technique. The resulting BA-sensitive mutant (Bar-) was able to produce little BA but considerable amount of an intermediate demethylphosphinothricin (DMPT). The Bar- mutant was still able to convert the N-acetyl derivative (AcDMPT) of DMPT to BA. Introduction of normal bar containing plasmid restored both BA resistance and BA biosynthesis to levels as high as the parental BA producer. By contrast, introducing a multi copy glutamine synthetase gene (glnA) into the Bar- mutant restored BA resistance but not BA production. Thus, the bar gene plays a crucial role in both self-defense and a step of BA biosynthesis in the BA-producing S. hygroscopicus.

Studies on the biosynthesis of bialaphos (SF-1293). 8. Purification and characterization of 2-phosphinomethylmalic acid synthase from Streptomyces hygroscopicus SF-1293.

2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM. The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism, to an electrophoretically homogeneous state. The purified PMM synthase has a subunit molecular weight of 48,000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90,000 approximately 98,000 by gel filtration. PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30 degrees C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA. Enzyme activity suppressed by EDTA was completely restored by adding Co++ or Mn++ and partially restored by addition of Ca++, Fe++ or Mg++. The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA. The enzyme does not catalyze the liberation of CoA from acetyl-CoA in the presence of alpha-keto acids, such as pyruvate, alpha-ketoglutarate, deamino-alpha-ketodemethylphosphinothricin or phosphonopyruvate. The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA. The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate. PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike (R)-citrate synthase PMM synthase was not inhibited by oxygen.

Elucidation of the 2-aminoethylphosphonate biosynthetic pathway in Tetrahymena pyriformis.

The biosynthetic reaction pathway leading to the natural product, 2-aminoethylphosphonate in Tetrahymena pyriformis has been elucidated. Incubation of [32P]PEP and [14C]PEP with T.pyriformis cellular homogenate fortified with Mg2+ and alanine/pyridoxal phosphate, yielded 2-aminoethylphosphonate as the minor reaction product (2-5% yield) and phosphoglycerate and pyruvate plus orthophosphate as the major products. Inclusion of thiamine pyrophosphate in the reaction mixture increased the yield of 2-aminoethylphosphonate by a factor of 10. Incubation of phosphonoacetaldehyde or phosphonopyruvate in the cellular homogenate also provided 2-aminoethylphosphonate. The cellular homogenate catalyzed the transformation of phosphonoacetaldehyde to 2-aminoethylphosphonate in an ca. 80% yield. However, the maximum yield of 2-aminoethylphosphonic acid obtained by use of phosphonopyruvate was only 15%. The major reaction pathways induced by treatment of phosphonopyruvate with the cellular extract involved its competitive conversion to PEP and pyruvate plus orthophosphate.

The bialaphos biosynthetic genes of Streptomyces hygroscopicus: cloning and analysis of the genes involved in the alanylation step.

We have isolated and studied the genes involved in the alanylation step in the biosynthesis of a herbicide, bialaphos which is produced by Streptomyces hygroscopicus. Three bialaphos-nonproducing mutants, NP60, NP61 and NP62, isolated from S. hygroscopicus by treatment with N-methyl-N'-nitro-N-nitrosoguanidine were defective for the alanylation step and were not restored to productivity by any locus of the gene cluster previously cloned. Three plasmids were isolated using NP60, NP61 and NP62 as recipients. The genes which restored productivity to NP61 and NP62 hybridized to the contiguous region of the bialaphos biosynthetic gene cluster. The gene cluster involved in the bialaphos production was about 35 kb long. The gene which restored productivity to NP60 did not hybridize to the bialaphos biosynthetic gene cluster. VM3 and VM4, putative alanylation blocked mutants, were derived from a bialaphos producer by gene replacement of an unidentified region of the biosynthetic gene cluster with an in vitro altered DNA sequence. The genes which restored productivity to VM3 and VM4 were located between the genes which code for phosphinomethylmalic acid synthase and demethylphosphinothricin acetyltransferase in the cluster. These results suggest that multiple genes are involved in the alanylation step.

Replacement of Streptomyces hygroscopicus genomic segments with in vitro altered DNA sequences.

We have developed a method for gene replacement in Streptomyces hygroscopicus which permits introduction of an in vitro derived mutation carried on a plasmid into the chromosome. We constructed the plasmid pMSB212 which can replicate in S. hygroscopicus and contains the step5 gene of the bialaphos biosynthetic pathway which was inactivated by a frame-shift mutation caused by filling in the cohesive ends of the EcoR I site in the structural gene. pMSB212 was introduced into a bialaphos producer strain and by protoplast regeneration of the primary thiostrepton-resistant transformants, non-producing mutants, were obtained. Biochemical and genetical analyses indicated that these mutants were specifically blocked by introduction of the frame-shift mutation in the step5 gene on the chromosome. This method will enable us to obtain isogenic mutants of known genes and to identify new genes encoded on a cloned fragment.

3-Phosphonopropionic acids inhibit carboxypeptidase A as multisubstrate analogues or transition-state analogues.

A series of phosphonic acid analogues of 2-benzylsuccinate were tested as inhibitors of carboxypeptidase A. The most potent of these, (2RS)-2-benzyl-3-phosphonopropionic acid, had a Ki of 0.22 +/- 0.05 microM, equipotent to (2RS)-2-benzylsuccinate and thus one of the most potent reversible inhibitors known for this enzyme. Lengthening by one methylene group to (2RS)-2-benzyl-4-phosphonobutyric acid increased the Ki to 370 +/- 60 microM. The monoethyl ester (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid was nearly as potent as (2RS)-2-benzyl-3-phosphonopropionic acid, with a Ki of 0.72 +/- 0.3 microM. The sulphur analogue of the monoethyl ester, 2-ambo-P-ambo-2-benzyl-3-(O-ethylthiophosphono)propionic acid, had a Ki of 2.1 +/- 0.6 microM, nearly as active as (2RS)-2-benzyl-3-(O-ethylphosphono)propionic acid. These phosphonic acids and esters could be considered to be multisubstrate inhibitors of carboxypeptidase A by virtue of their structural analogy with 2-benzylsuccinate. Alternatively, the tetrahedral hybridization at the phosphorus atom suggests that they could be mimicking a tetrahedral transition state for the enzyme-catalysed hydrolysis of substrate.

2,3-Cyclopyrophosphoglycerate in methanogens: evidence by 13C NMR spectroscopy for a role in carbohydrate metabolism.

The novel compound 2,3-cyclopyrophosphoglycerate (CPP) is the major small molecule carbon pool in Methanobacterium thermoautotrophicum. High-field 13C NMR 13CO2 pulse/unenriched CO2 chase experiments have shown that the labeled CPP rapidly loses its 13C to an insoluble pool, while the CPP steady-state concentration is maintained (as monitored by 31P NMR spectroscopy). The biosynthesis of CPP from CO2, acetyl coenzyme A, and pyruvate as precursors has been established by a 13C NMR study of ethanol extracts of Mb. thermoautotrophicum fed with 13CO2, [1-13C]- and [2-13C]acetate, and [1-13C]pyruvate. That CPP is a post-phosphoenolpyruvate metabolite has been confirmed by in vitro experiments with cell extracts. A role for CPP in carbohydrate metabolism was established when [1-13C]glucose fed to cells resulted in the formation of [3-13C]CPP exclusively. Possible functions of CPP within the cell are discussed.

Studies on phosphagen synthesis by mitochondrial preparations.

Mitochondrial preparations from muscles of a crab (Cancer pagurus), two fish (Trachurus trachurus and Scyliorhinus canicula) and a bird (Columba livia) are able to synthesise, through ATP, the phosphagen related to that species. This indicates the presence of a bound phosphagen kinase. Addition of creatine kinase and creatine to crab mitochondria results in the synthesis of phosphocreatine. Similarly, the addition of arginine kinase and arginine to mitochondrial preparations from the fish and bird results in the synthesis of phosphoarginine. In the crab, the mitochondrial form of arginine kinase released by sonication had the same kinetic affinity constants and electrophoretic mobility and could not be distinguished immunologically from the cytosolic form. The close similarity of bound and cytosolic forms of arginine kinase in this crustacean suggests that the two forms have not evolved separately as has creatine kinase in the mammal.

Synthesis of prenyl lipids in cells of spinach leaf. Compartmentation of enzymes for formation of isopentenyl diphosphate.

Purified spinach chloroplasts incorporate [1-14C]isopentenyl diphosphate into prenyl lipids in high yields. The immediate biosynthetic precursors of isopentenyl diphosphate (hydroxymethylglutaryl-CoA, mevalonate, mevalonate-5-phosphate, mevalonate-5-diphosphate), on the other hand, are not accepted as substrates and the corresponding enzymes hydroxymethylglutaryl-CoA reductase, mevalonate kinase, phosphomevalonate kinase, and diphosphomevalonate decarboxylase are not present in the organelles. These enzymes can only be detected in a membrane-bound form at the endoplasmic reticulum (hydroxymethylglutaryl-CoA reductase) and as soluble activities in the cytoplasm. The concept is developed that isopentenyl diphosphate is formed in the cytoplasm as a 'central intermediate' and is distributed then to other cellular compartments (endoplasmic reticulum, plastids, mitochondria) for further biosynthetic utilization.

Alteration of acylphosphate formation of cardiac sarcoplasmic reticulum ATPase by calmodulin-dependent phosphorylation.

The calcium-dependent acylphosphate formed by the calcium transport ATPase of cardiac sarcoplasmic reticulum and the calcium-, calmodulin-dependent phosphoester(s) of sarcoplasmic reticulum fractions formed by a calcium-, calmodulin-dependent membrane-bound protein kinase can be distinguished by removal of calcium and/or magnesium by EDTA or hydroxylamine treatment of the acid denaturated membranes. Both procedures decompose the acylphosphate with little effect on the phosphoester(s). Calmodulin-dependent phosphorylation (2.44 nmol/mg SR protein) reduces the apparent K(Ca) of the acylphosphate steady state level of the calcium transport ATPase from 0.56 to 0.34 microM free calcium, without affecting the maximum phosphoenzyme level (0.93 versus 0.89 nmol/mg protein), and has little, if any, effect on the Hill-coefficient (1.32 versus 1.54).

Cosynthesis and protoplast fusion by mutants of bialaphos (AMPBA) producing Streptomyces hygroscopicus.

Cosynthesis in mixed culture and protoplast fusion of non-producing mutants of Streptomyces hygroscopicus which may produce biosynthetic intermediates of bialaphos (AMPBA) were studied. Non-producing mutants were obtained by mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (NTG) treatment, and six stable non-producing mutants were used for cosynthesis and protoplast fusion studies. The biosynthetic block sites of the non-producers were consistent with studies of the biosynthetic pathway of AMPBA.

Studies on the biosynthesis of bialaphos (SF-1293) Part 3. Production of phosphinic acid derivatives, MP-103, MP-104 and MP-105, by a blocked mutant of Streptomyces hygroscopicus SF-1293 and their roles in the biosynthesis of bialaphos.

During biosynthetic studies on bialaphos to reveal the formation mechanisms of carbon-phosphorous bonds in detail, three new metabolites containing a H-P-C bond structure were isolated from the fermentation broth of a mutant of Streptomyces hygroscopicus SF-1293. Based on the spectroscopic analysis, the structures of these compounds have been determined as shown in Fig. 1. Transformation experiments of these metabolites to bialaphos suggested that the reduction of the phosphorous atom in phosphate will take place at an early biosynthetic stage.

Synthesis of phosphocreatine and phosphoarginine by mitochondria from various sources.

Mitochondria from heart, skeletal muscle and the liver of the rat have been shown to synthesise phosphoarginine through ATP if supplied with arginine and lobster arginine kinase. Liver mitochondria have been shown to synthesise phosphocreatine through ATP with the aid of the cytosolic isomer of creatine kinase. Mitochondria prepared from muscles of a crustacean, a fish and a bird have been shown oxidatively to synthesise phosphocreatine (crustacean) and phosphoarginine (fish and bird) provided they are supplied with the appropriate kinase and catalytic amounts of ATP. Within one second of the addition of either cytosolic kinases, mitochondria from skeletal muscle and liver begin a steady state synthesis of phosphoarginine or phosphocreatine. The results suggest that, with respect to phosphagen synthesis, the addition of the cytosolic enzymes can substitute for the mitochondrial enzyme. It is difficult therefore to accept a special vectorial function for the bound mitochondrial enzyme at the biological concentrations of ATP and the cytosolic enzymes normally associated with phosphagen synthesis.