Control of the actinomycin biosynthetic pathway in and actinomycin resistance of Streptomyces spp.

Using actinomycin-producing and nonproducing strains of Streptomyces antibioticus, I studied several steps in the biosynthetic pathway of this antibiotic. Actinomycin-nonproducing strains derived after acriflavine or novobiocin treatment showed activity of kynurenine formamidase and phenoxazinone synthase as high as that of the parental strain, but these nonproducing strains failed to convert 4-methyl-3-hydroxy-anthranilic acid to actinomycin. In addition, accumulation of 4-methyl-3-hydroxyanthranilic acid (in the presence of D-valine) was not detected in the nonproducing isolates. Actinomycin-nonproducing strains derived after acriflavine treatment of Streptomyces parvulus showed a drastic decrease of resistance to the antibiotic. However these strains regained resistance after preincubation with a small amount of actinomycin D.     

Evidence for a constitutive and inducible form of kynurenine formamidase in an actinomycin-producing strain of Streptomyces parvulus

Two forms of kynurenine formamidase have been found in an actinomycin-producing strain of Streptomyces parvulus. Formamidase I has a molecular weight of 42,000 and is synthesized constitutively. Formamidase II is smaller (24,000) and is present just prior to and during synthesis of actinomycin.     

The characterization of multiple forms of kynurenine formidase in Drosophila melanogaster.

Two enzymic forms of kynurenine formamidase (EC 3.5.1.9) from Drosophila melanogaster were separated and partially purified by pH fractionation, (NH4) 2SO4 fractionation and Sephadex G-75 gel filtration. The enzymes were also separated by DEAE-cellulose ion-exchange chromatography and distinguished by their different rates of thermal inactivation. The multiple forms are termed formamidase I and formamidase II. The molecular weight of formamidase I as measured by Sephadex G-75 chromatography is 60 000 and that of formamidase II is 31 000. The pH optima are broad, ranging between 6.7 and 7.8 for formamidase I and 6.5 and 8.0 for formamidase II. The apparent Km values are 5-10(-3) and 0.83-10(-3) M, resepctively. The possibility that formamidase II is an active subunit of formamidase I is discussed, although neither enzyme will convert to the other when separated and rechromatographed. Eight organisms were tested for the presence or absence of multiple forms of formamidase. Drosophila melanogaster and Drosophila virilis have both enzymes; cow, chicken, yeast and housefly have formamidase I only, and mouse and frog have formamidase II only.     

Actinomycin synthetases. Multifunctional enzymes responsible for the synthesis of the peptide chains of actinomycin

Two enzymes were purified from actinomycin-synthesizing Streptomyces chrysomallus which could be identified as peptide synthetases involved in the biosynthesis of actinomycin. Actinomycin synthetase II activates the first two amino acids of the peptide chains of the peptide lactone antibiotic, threonine and valine (or isoleucine), as thioesters via their corresponding adenylates. It is a single polypeptide chain of Mr 225,000. Similarly, actinomycin synthetase III activates proline, glycine, and valine (the remaining three amino acids in the antibiotic) as thioesters and is a single polypeptide chain of about Mr 280,000. It also carries the methyltransferase function(s) for N-methylation of thioesterified glycine and valine. In addition, it catalyzes the formation of cyclo(sarcosyl-N-methyl-L-valine) from glycine, L-valine, and S-adenosyl-L-methionine at the expense of ATP. Although the cell-free synthesis of the peptide lactone was not as yet accomplished, the data provide evidence that together with the 4-methyl-3-hydroxyanthranilic acid-activating enzyme (now designated as actinomycin synthetase I) all amino acid-activating protein components of the actinomycin-synthesizing enzyme complex are identified.     

Reduction of Methionine Sulfoxide by Plants

A relaxed (rel) mutant was found among thirty spontaneous thiopeptin-resistant isolates of Streptomyces antibioticus strain 3720, an actinomycin-producing strain, which showed severely reduced ability to accumulate ppGpp during a nutritional shift-down. The pool size of GTP decreased markedly in the parental strain, but to a lesser extent in the rel mutant. The rel mutant did not show the induction of an enzyme, phenoxazinone synthase, which is involved in the biosynthesis of actinomycin. No negative effect of the rel mutation was observed on a constitutive enzyme, kynurenine formamidase, which also plays a role in actinomycin synthesis. The mutant also failed to produce melanin, but still retained the ability to form aerial mycelium and spores, although the onset of the formation of aerial mycelium was markedly delayed. Neither the phenoxazinone synthase activity nor the kynurenine formamidase activity was affected by ppGpp in vitro. It is suggested tha the stringent response (ppGpp) may be generally essential for the induction of enzymes involved in secondary metabolism.     

Purification and characterization of tryptophan dioxygenase from Streptomyces parvulus

Tryptophan dioxygenase, derived from Streptomyces parvulus, was purified to near homogeneity and shown to have a native Mr of 88,000. Kinetic parameters of the enzyme were determined and evidence suggesting that it is a hemoprotein was obtained. Tryptophan dioxygenase has a high specificity toward L-tryptophan with an apparent Km of 0.3 mM. L-3-Hydroxykynurenine was a competitive inhibitor with respect to L-tryptophan with a Ki of 0.16 mM. In vitro, the enzyme displayed little activity in the absence of a reducing agent; ascorbate, at 50 mM, was the preferred reductant providing almost a 50-fold increase in enzyme activity. The regulation of tryptophan dioxygenase synthesis and activity is described. The expression of the enzyme is correlated with the biosynthesis of actinomycin D in S. parvulus. These results support the hypothesis that tryptophan dioxygenase functions as the first enzyme in the sequence converting L-tryptophan to the chromophore of this antibiotic.     

Metabolic regulation in Streptomyces parvulus during actinomycin D synthesis, studied with 13C- and 15N-labeled precursors by 13C and 15N nuclear magnetic resonance spectroscopy and by gas chromatography-mass spectrometry.

Recent studies have suggested that the onset of synthesis of actinomycin D in Streptomyces parvulus is due to a release from L-glutamate catabolic repression. In the present investigation we showed that S. parvulus has the capacity to maintain high levels of intracellular glutamate during the synthesis of actinomycin D. The results seem contradictory, since actinomycin D synthesis cannot start before a release from L-glutamate catabolic repression, but a relatively high intracellular pool of glutamate is needed for the synthesis of actinomycin D. Utilizing different labeled precursors, D-[U-13C]fructose and 13C- and 15N-labeled L-glutamate, and nuclear magnetic resonance techniques, we showed that carbon atoms of an intracellular glutamate pool of S. parvulus were not derived biosynthetically from the culture medium glutamate source but rather from D-fructose catabolism. A new intracellular pyrimidine derivative whose nitrogen and carbon skeletons were derived from exogenous L-glutamate was obtained as the main glutamate metabolite. Another new pyrimidine derivative that had a significantly reduced intracellular mobility and that was derived from D-fructose catabolism was identified in the cell extracts of S. parvulus during actinomycin D synthesis. These pyrimidine derivatives may serve as a nitrogen store for actinomycin D synthesis. In the present study, the N-trimethyl group of a choline derivative was observed by 13C nuclear magnetic resonance spectroscopy in growing S. parvulus cells. The choline group, as well as the N-methyl groups of sarcosine, N-methyl-valine, and the methyl groups of an actinomycin D chromophore, arose from D-fructose catabolism. The 13C enrichments found in the peptide moieties of actinomycin D were in accordance with a mechanism of actinomycin D synthesis from L-glutamate and D-fructose.     

Biochemical identification and crystal structure of kynurenine formamidase from Drosophila melanogaster

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the α/β hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.     

Phenoxazinone biosynthesis: accumulation of a precursor, 4-methyl-3-hydroxyanthranilic acid, by mutants of Streptomyces parvulus.

Mutants of Streptomyces parvulus that are blocked in the synthesis of the phenoxazinone-containing antibiotic, actinomycin, were isolated by the 'agar piece' method (after ultraviolet irradiation or treatment with 8-methoxypsoralen plus near-ultraviolet light). Radiolabelling experiments in conjunction with paper, thin-layer and column chromatography revealed that 4-methyl-3-hydroxyanthranilic acid (MHA) is a major metabolite accumulated by these mutants. Studies in vitro and in vivo provided evidence that MHA is a precursor of the phenoxazinone chromophore, actinocin. Normally MHA does not accumulate during growth or antibiotic synthesis by the parental strains. Protoplasts derived from the mutant strain AM5 synthesized MHA in significant amounts. A scheme is proposed for the biosynthesis of actinomycin D that accounts for the accumulation of MHA by the mutants.     

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.     

Aerobic tryptophan degradation pathway in bacteria: novel kynurenine formamidase

While a variety of chemical transformations related to the aerobic degradation of L-tryptophan (kynurenine pathway), and most of the genes and corresponding enzymes involved therein have been predominantly characterized in eukaryotes, relatively little was known about this pathway in bacteria. Using genome comparative analysis techniques we have predicted the existence of the three-step pathway of aerobic L-tryptophan degradation to anthranilate (anthranilate pathway) in several bacteria. Based on the chromosomal gene clustering analysis, we have identified a previously unknown gene encoding for kynurenine formamidase (EC 3.5.1.19) involved with the second step of the anthranilate pathway. This functional prediction was experimentally verified by cloning, expression and enzymatic characterization of recombinant kynurenine formamidase orthologs from Bacillus cereus, Pseudomonas aeruginosa and Ralstonia metallidurans. Experimental verification of the inferred anthranilate pathway was achieved by functional expression in Escherichia coli of the R. metallidurans putative kynBAU operon encoding three required enzymes: tryptophan 2,3-dioxygenase (gene kynA), kynurenine formamidase (gene kynB), and kynureninase (gene kynU). Our data provide the first experimental evidence of the connection between these genes (only one of which, kynU, was previously characterized) and L-tryptophan aerobic degradation pathway in bacteria.     

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].     

Tryptophan catabolism via kynurenine production in <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis>: identification of three genes coding for the enzymes of tryptophan to anthranilate pathway

Most enzymes involved in tryptophan catabolism via kynurenine formation are highly conserved in Prokaryotes and Eukaryotes. In humans, alterations of this pathway have been related to different pathologies mainly involving the central nervous system. In Bacteria, tryptophan and some of its derivates are important antibiotic precursors. Tryptophan degradation via kynurenine formation involves two different pathways: the eukaryotic kynurenine pathway, also recently found in some bacteria, and the tryptophan-to-anthranilate pathway, which is widespread in microorganisms. The latter produces anthranilate using three enzymes also involved in the kynurenine pathway: tryptophan 2,3-dioxygenase (TDO), kynureninase (KYN), and kynurenine formamidase (KFA). In Streptomyces coelicolor, where it had not been demonstrated which genes code for these enzymes, tryptophan seems to be important for the calcium- dependent antibiotic (CDA) production. In this study, we describe three adjacent genes of S. coelicolor (SCO3644, SCO3645, and SCO3646), demonstrating their involvement in the tryptophan-to-anthranilate pathway: SCO3644 codes for a KFA, SCO3645 for a KYN and SCO3646 for a TDO. Therefore, these genes can be considered as homologous respectively to kynB, kynU, and kynA of other microorganisms and belong to a constitutive catabolic pathway in S. coelicolor, which expression increases during the stationary phase of a culture grown in the presence of tryptophan. Moreover, the S. coelicolor ΔkynU strain, in which SCO3645 gene is deleted, produces higher amounts of CDA compared to the wild-type strain. Overall, these results describe a pathway, which is used by S. coelicolor to catabolize tryptophan and that could be inactivated to increase antibiotic production.     

Identification and molecular characterization of the beta-ketoacyl-[acyl carrier protein] synthase component of the Arabidopsis mitochondrial fatty acid synthase

Substrate specificity of condensing enzymes is a predominant factor determining the nature of fatty acyl chains synthesized by type II fatty acid synthase (FAS) enzyme complexes composed of discrete enzymes. The gene (mtKAS) encoding the condensing enzyme, beta-ketoacyl-[acyl carrier protein] (ACP) synthase (KAS), constituent of the mitochondrial FAS was cloned from Arabidopsis thaliana, and its product was purified and characterized. The mtKAS cDNA complemented the KAS II defect in the E. coli CY244 strain mutated in both fabB and fabF encoding KAS I and KAS II, respectively, demonstrating its ability to catalyze the condensation reaction in fatty acid synthesis. In vitro assays using extracts of CY244 containing all E. coli FAS components, except that KAS I and II were replaced by mtKAS, gave C(4)-C(18) fatty acids exhibiting a bimodal distribution with peaks at C(8) and C(14)-C(16). Previously observed bimodal distributions obtained using mitochondrial extracts appear attributable to the mtKAS enzyme in the extracts. Although the mtKAS sequence is most similar to that of bacterial KAS IIs, sensitivity of mtKAS to the antibiotic cerulenin resembles that of E. coli KAS I. In the first or priming condensation reaction of de novo fatty acid synthesis, purified His-tagged mtKAS efficiently utilized malonyl-ACP, but not acetyl-CoA as primer substrate. Intracellular targeting using green fluorescent protein, Western blot, and deletion analyses identified an N-terminal signal conveying mtKAS into mitochondria. Thus, mtKAS with its broad chain length specificity accomplishes all condensation steps in mitochondrial fatty acid synthesis, whereas in plastids three KAS enzymes are required.     

The identification and characterization of two separate carboxylesterases in guinea-pig serum.

The esterase activity of guinea-pig serum was investigated. A 3-fold purification was achieved by removing the serum albumin by Blue Sepharose CL-6B affinity chromatography. The partially purified enzyme preparation had carboxylesterase and cholinesterase activities of 1.0 and 0.22 mumol of substrate/min per mg of protein respectively. The esterases were labelled with [3H]di-isopropyl phosphorofluoridate (DiPF) and separated electrophoretically on sodium dodecyl sulphate/polyacrylamide gels. Two main labelled bands were detected: band I had Mr 80 000 and bound 18-19 pmol of [3H]DiPF/mg of protein, and band II had Mr 58 000 and bound 7 pmol of [3H]DiPF/mg of protein. Bis-p-nitrophenyl phosphate (a selective inhibitor of carboxylesterase) inhibited most of the labelling of bands I and II. The residual labelling (8%) of band I but not band II (4%) was removed by preincubation of partially purified enzyme preparation with neostigmine (a selective inhibitor of cholinesterase). Paraoxon totally prevented the [3H]DiPF labelling of the partially purified enzyme preparation. Isoelectrofocusing of [3H]DiPF-labelled and uninhibited partially purified enzyme preparation revealed that there were at least two separate carboxylesterases, which had pI3.9 and pI6.2, a cholinesterase enzyme (pI4.3) and an unidentified protein that reacts with [3H]DiPF and has a pI5.0. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of these enzymes showed that the carboxylesterase enzymes at pI3.9 and pI6.2 corresponded to the 80 000-Mr subunit (band I) and 58 000-Mr subunit (band II). The cholinesterase enzyme was also composed of 80 000-Mr subunits (i.e. the residual labelling in band I after bis-p-nitrophenyl phosphate treatment). The unidentified protein at pI5.0 corresponded to the residual labelling in band II (Mr 58 000), which was insensitive to neostigmine and bis-p-nitrophenyl phosphate. These studies show that the carboxylesterase activity of guinea-pig serum is the result of at least two separate and distinct enzymes.     

Opposite facial specificity for two hydroquinone epoxidases: (3-si,4-re)-2,5-dihydroxyacetanilide epoxidase from Streptomyces LL-C10037 and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase from Streptomyces MPP 3051

(3-si,4-re)-2,5-Dihydroxyacetanilide epoxidase (DHAE I), a key enzyme in the biosynthesis of the epoxysemiquinone antibiotic LL-C10037 alpha by Streptomyces LL-C10037 [Gould, S.J., & Shen, B. (1991) J. Am. Chem. Soc. 113, 684-686], and (3-re,4-si)-2,5-dihydroxyacetanilide epoxidase (DHAE II) isolated from Streptomyces MPP 3051--which yields the (3R,4S)-epoxyquinone mirror image product of DHAE I--are described. DHAE I was purified 640-fold. Gel permeation chromatography indicated an Mr of 117,000 +/- 10,000; SDS-PAGE gave a major band of 22,300 daltons, indicating that DHAE I is either a pentamer or hexamer in solution. The enzyme had a pH optimum of 6.5, a Km of 8.4 +/- 0.5 microM, and a Vmax of 3.7 +/- 0.2 mumol min-1 mg-1. DHAE II was purified 1489-fold. The enzyme was shown to be a dimer of Mr 33,000 +/- 2000, with 16,000-dalton subunits, with a pH optimum of 5.5 and a Km of 7.2 +/- 0.4 microM. Both enzymes required only O2 and substrate; flavin and nicotinamide coenzymes had little or no effect. Neither catalase nor EDTA affected the activity of either enzyme, but complete inhibition of both was obtained with 1,10-phenanthroline. The activity of the purified DHAE I could be enhanced, but only by Mn2+ (relative V = 246 at 0.04 mM), Ni2+ (relative V = 266 at 0.2 mM), or Co2+ (relative = 498 at 0.2 mM). Reconstitution from a DHAE I apoenzyme, generated by treatment with 1,10-phenanthroline followed by Sephadex G-25 chromatography, occurred only by addition of one of these three metals.(ABSTRACT TRUNCATED AT 250 WORDS)     

relA Is Required for Actinomycin Production in Streptomyces antibioticus

The relA gene from Streptomyces antibioticus has been cloned and sequenced. The gene encodes a protein with an Mr of 93,653, which is 91% identical to the corresponding protein from Streptomyces coelicolor. Disruption of S. antibioticus relA produces a strain which grows significantly more slowly on actinomycin production medium than the wild type or a disruptant to which the intact relA gene was restored. Moreover, the disruptant was unable to accumulate ppGpp to the levels observed during the normal course of growth and actinomycin production in the wild type. The strain containing the disrupted relA gene did not produce actinomycin and contained significantly lower levels of the enzyme phenoxazinone synthase than the wild-type strain. Actinomycin synthetase I, a key enzyme in the actinomycin biosynthetic pathway, was undetectable in the relA disruptant. Growth of the disruptant on low-phosphate medium did not restore actinomycin production.     

Glutamyl-tRNA synthetases from wheat. Isolation and characterization of three dimeric enzymes

Three dimeric glutamyl-tRNA synthetases (GluRS) were isolated from extracts of quiescent wheat germ and wheat chloroplasts. The chloroplast enzyme (Mr = 110 000), called GluRS C, exhibits a prokaryotic (Escherichia coli) tRNA specificity. Two enzymes were found in the quiescent germ and were separated on phosphocellulose P11: one called GluRS P, probably the mitochondrial enzyme, has the same tRNA specificity as GluRS C; the other, called GluRS E, has eukaryotic (wheat germ) tRNA specificity. Both enzymes exhibit a molecular weight close to 160 000. Each of these enzymes co-eluate on hydroxyapatite and phosphocellulose chromatographies with an unstable active monomer whose molecular weight is approximately half that of the corresponding dimer. Two assumptions are discussed about these monomers.     

Pleiotropic effects of a relC mutation in Streptomyces antibioticus.

Ochi (Agric. Biol. Chem. 51:829-835, 1987) has isolated a relaxed mutant of Streptomyces antibioticus, designated relC49, relC49 accumulates significantly lower levels of ppGpp than the parent stain, IMRU3720. At its maximum, the ppGpp level in relC49 was only one-fourth that observed in strain IMRU3720. Interestingly, a burst of ppGpp synthesis between 18 and 22 h of growth in IMRU3720 coincided with the onset of actinomycin production in that strain. As shown previously, the activity in protein synthesis of ribosomes from strain IMRU3720 decreases with the age of the culture. The decrease in activity was less pronounced in cultures of relC49. relC49 mycelium contains reduced levels of phenoxazinone synthase, a key enzyme involved in actinomycin biosynthesis. The rel mutation prevents the normal increase in the activity of one of the other enzymes required for production of the antibiotic, 3-hydroxyanthanilate-4-methyltransferase, and a third enzyme, actinomycin synthetase I, appears to be completely absent from relC49 mycelium. Levels of phenoxazinone synthease mRNA were examined by RNA dot blotting with the cloned phenoxazinone synthase gene as a probe. mRNA levels for phenoxazinone synthase were dramatically reduced in relC49 compared with strain IMRU3720. These results are discussed in terms of the possible regulation of the onset of actinomycin production by ppGpp.     

Biochemical and genetic characterization of kynurenine formamidase from Drosophila melanogaster

The molecular weight forms of kynurenine formamidase were studied both genetically and biochemically. Formamidase I (native molecular weight 60,000) was purified using (NH₄)₂SO₄ and pH fractionation, DEAE-cellulose chromatography at two different pH's, hydroxylapatite chromatography, and Sephadex G-100 gel filtration. Its subunit molecular weight, as determined by SDS gel electrophoresis, is 34,000, indicating that formamidase I is a dimer. Its K _m is 1.87×10^–3 m. Its isoelectric point is pH 5.3. Its amino acid composition is reported. Formamidase II (native molecular weight 31,000) was partially purified using techniques similar to those above. Its K _m is 2.31×10^–3 m. The response of formamidase activity to change in gene dosage was measured in segmental aneuploids generated in the second, third, and X chromosomes. Two separate chromosomal regions were identified which when present in extra dosage result in an elevation of the level of formamidase activity close to that predicted for the addition of a structural gene in a two-gene system. These tentative map positions were substantiated by demonstration that addition of one of the regions, 25A–27E, causes a 50% elevation in the relative amount of formamidase II. Addition of the other region, 91B–93F, causes a similar elevation in the relative amount of formamidase I. A model of the evolutionary origin of the two forms is presented, and the significance of these results to this model is discussed.This work was supported by USPHS Grant GM-21286.