Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin

The peptide synthetase gene operon, which consists of mcyA, mcyB, and mcyC, for the activation and incorporation of the five amino acid constituents of microcystin has been identified [T. Nishizawa et al. (1999) J. Biochem. 126, 520-529]. By sequencing an additional 34 kb of DNA from microcystin-producing Microcystis aeruginosa K-139, we identified the residual microcystin synthetase gene operon, which consists of mcyD, mcyE, mcyF, and mcyG, in the opposite orientation to the mcyABC operon. McyD consisted of two polyketide synthase modules, and McyE contained a polyketide synthase module at the N-terminus and a peptide synthetase module at the C-terminus. McyF was found to exhibit similarity to amino acid racemase. McyG consisted of a peptide synthetase module at the N-terminus and a polyketide synthase at the C-terminus. The microcystin synthetase gene cluster was conserved in another microcystin-producing strain, Microcystis sp. S-70, which produces Microcystin-LR, -RR, and -YR. Insertional mutagenesis of mcyA, mcyD, or mcyE in Microcystis sp. S-70 abolished microcystin production. In conclusion, the mcyDEFG operon is presumed to be responsible for 3-amino-9-methoxy-2,6, 8-trimethyl-10-phenyldeca-4,6-dienoic acid (Adda) biosynthesis, and the incorporation of Adda and glutamic acid into the microcystin molecule.     

黑曲霉中生物合成赭曲霉毒素A的非核糖体肽合成酶基因的鉴定

赭曲霉毒素A(ochratoxin A,OTA)是国际癌症研究机构认定的"2B"类致癌物。黑曲霉Aspergillus niger是美国食品药品监督局认可的食品安全菌。然而近年来陆续发现某些黑曲霉菌株能够产生OTA,这会对人类健康构成潜在威胁。阐明黑曲霉生物合成OTA的关键基因有助于理解OTA生物合成机制,这对OTA污染的防控具有重要意义。本研究克隆了产OTA黑曲霉中非核糖体肽合成酶(NRPS)编码基因(An15g07910),并对其进行了生物信息学分析,在此基础上采用同源重组的方法敲除了该基因,获得了一株性能稳定的敲除突变株Δnrps。与野生株相比,Δnrps突变株的表型在CYA培养基中并无明显改变,但在7d培养期间完全失去了合成赭曲霉毒素α(ochratoxinα,OTα)和OTA的能力,而赭曲霉毒素β(ochratoxinβ,OTβ)的合成不受影响。在野生株培养过程中,该nrps基因前4d表达量逐渐增大,并在第4天达到最高,随后基因表达量逐渐下降并趋于稳定,这与OTA的含量变化基本一致。结果表明该nrps基因(An15g07910)参与OTA的生物合成,其编码的NRPS可能负责催化苯丙氨酸部分和二氢异香豆素部分的交联。     

恶臭假单胞菌丙氨酸消旋酶基因的克隆、序列分析及表达

从恶臭假单胞菌(Pseudomonas putida)200的基因组出发,用PCR方法克隆到两个独立作用的丙氨酸消旋酶基因,称之为dadX和alr。DadX编码357个氨基酸长的多肽,计算分子量为38.82kDa,alr编码409个氨基酸长的多肽,计算分子量为44.182kDa。序列分析显示,DadX的氨基酸序列与Pseudomonas putidaKT2440,铜绿假单胞菌(Pseudomonas aeruginosa),鼠伤寒沙门氏菌(Salmonella typhimurium)和大肠杆菌(Escherichia coli)的DadX比较,相似性分别为96.64%、71.99%、44.88%和47.37%。Alr的氨基酸序列与Pseudomonas putidaKT2440比较,同源性为94.38%,而与铜绿假单胞菌(P.aeruginosa)、鼠伤寒沙门氏菌(S.typhimurium)和大肠杆菌(E.coli)的Alr比较,同源性均较低,分别为22.89%、25.72%和26.44%。在P.putida200的DadX和Alr氨基酸序列中部发现有对于酶活性至关重要的保守区域,如磷酸吡哆醛(PLP)结合位点。DadX和alr在大肠杆菌中得到表达,DadX丙氨酸消旋酶只对丙氨酸有消旋作用,而Alr丙氨酸消旋酶可以作用于丙氨酸和丝氨酸两种底物,且对丝氨酸特异性更高。Alr的表达不依赖于外源启动子,说明在其结构基因上游存在启动子结构。     

A gene encoding alanine racemase is involved in spore germination in <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis>

Alanine racemase is a major component of the exosporium of Bacillus cereus spores. A gene homologous to that of alanine racemase (alrA) was cloned from Bacillus thuringiensis subsp. kurstaki, and RT-PCR showed that alrA was transcribed only in the sporulating cells. Disruption of alrA did not affect the growth and sporulation of B. thuringiensis, but promoted l-alanine-induced spore germination. When the spore germination rate was measured by monitoring DPA release, complementation of the alrA disruptant reduced the rate of l-alanine-induced spore germination below that of even wild-type spores. As previously reported for spores of other Bacillus species, d-alanine was an effective and competitive inhibitor of l-alanine-induced germination of B. thuringiensis spores. d-cycloserine alone stimulated inosine-induced germination of B. thuringiensis spores in addition to increasing l-alanine-induced germination by inhibiting alanine racemase. d-Alanine also increased the rate of inosine-induced germination of wild-type spores. However, d-alanine inhibited inosine-induced germination of the alrA disruptant spores. It is possible that AlrA converted d-alanine to l-alanine, and this in turn, stimulated spore germination in B. thuringiensis. These results suggest that alrA plays a crucial role in moderating the germination rate of B. thuringiensis spores.     

The alanine racemase gene is essential for growth of Lactobacillus plantarum.

The Lactobacillus plantarum alr gene encoding alanine racemase was cloned by complementation of an Escherichia coli Alr- DadX- double mutant strain. Knockout of the alr gene abolished all measurable alanine racemase activity, and the mutant was shown to be strictly dependent on D-alanine for growth.     

A side reaction of alanine racemase: transamination of cycloserine

Alanine racemase (EC 5.1.1.1) catalyzes the interconversion of alanine enantiomers, and thus represents the first committed step involved in bacterial cell wall biosynthesis. Cycloserine acts as a suicide inhibitor of alanine racemase and as such, serves as an antimicrobial agent. The chemical means by which cycloserine inhibits alanine racemase is unknown. Through spectroscopic assays, we show here evidence of a pyridoxal derivative (arising from either isomer of cycloserine) saturated at the C4' carbon position. We additionally report the L- and D-cycloserine inactivated crystal structures of Bacillus stearothermophilus alanine racemase, which corroborates the spectroscopy via evidence of a 3-hydroxyisoxazole pyridoxamine derivative. Upon the basis of the kinetic and structural properties of both the L- and D-isomers of the inhibitor, we propose a mechanism of alanine racemase inactivation by cycloserine. This pathway involves an initial transamination step followed by tautomerization to form a stable aromatic adduct, a scheme similar to that seen in cycloserine inactivation of aminotransferases.     

鹅膏环肽毒素生源合成的研究进展

在蘑菇目Agaricales中,已知鹅膏属Amanita、环柄菇属Lepiota和盔孢伞属Galerina 3个属的部分物种能产生剧毒的鹅膏环肽毒素。全球90%以上的致死性蘑菇中毒事件是由含鹅膏环肽蘑菇导致。上述3个属的剧毒蘑菇虽然亲缘关系较远,但却可以合成同一种鹅膏环肽毒素,且使用了大致相同的生源合成代谢途径,涉及多个毒素合成基因,采用了特殊的组合式合成机制。本文总结了鹅膏环肽毒素合成途径研究的最新进展,指出了当前工作中遇到的一些难题,对未来研究的趋势进行了展望。     

Markerless mutagenesis in Methanococcus maripaludis demonstrates roles for alanine dehydrogenase, alanine racemase, and alanine permease

Among the archaea, Methanococcus maripaludis has the unusual ability to use L- or D-alanine as a nitrogen source. To understand how this occurs, we tested the roles of three adjacent genes encoding homologs of alanine dehydrogenase, alanine racemase, and alanine permease. To produce mutations in these genes, we devised a method for markerless mutagenesis that builds on previously established genetic tools for M. maripaludis. The technique uses a negative selection strategy that takes advantage of the ability of the M. maripaludis hpt gene encoding hypoxanthine phosphoribosyltransferase to confer sensitivity to the base analog 8-azahypoxanthine. In addition, we developed a negative selection method to stably incorporate constructs into the genome at the site of the upt gene encoding uracil phosphoribosyltransferase. Mutants with in-frame deletion mutations in the genes for alanine dehydrogenase and alanine permease lost the ability to grow on either isomer of alanine, while a mutant with an in-frame deletion mutation in the gene for alanine racemase lost only the ability to grow on D-alanine. The wild-type gene for alanine dehydrogenase, incorporated into the upt site, complemented the alanine dehydrogenase mutation. Hence, the permease is required for the transport of either isomer, the dehydrogenase is specific for the L isomer, and the racemase converts the D isomer to the L isomer. Phylogenetic analysis indicated that all three genes had been acquired by lateral gene transfer from the low-moles-percent G+C gram-positive bacteria.     

Purification of peptide synthetases involved in pristinamycin I biosynthesis.

Several assays of pristinamycin I synthetases based on adenylate or thioester formation were developed. Purification to near homogeneity of these enzymatic activities from cell extracts of Streptomyces pristinaespiralis showed that three enzymes could activate all pristinamycin I precursors. SnbA, a 3-hydroxypicolinic acid: AMP ligase activating the first pristinamycin I residue, was purified 200-fold, using an ATP-pyrophosphate exchange assay. This enzyme was shown to be a monomer with an Mr of 67,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Then a multifunctional enzyme, consisting of two identical subunits (SnbC) with Mrs of 240,000 and able to bind covalently L-threonine as a thioester, was purified 100-fold. This protein also activated L-aminobutyric acid, which is further epimerized to generate the third residue of the pristinamycin I macrocycle. A third protein, consisting of two identical subunits (SnbD) with Mrs estimated to be between 250,000 and 350,000, was purified 200-fold. This large enzyme catalyzed thioesterification and subsequent N-methylation of 4-dimethylamino-L-phenylalanine, the fifth pristinamycin I residue. SnbD could also activate L-proline, the fourth pristinamycin I residue, and some preparations retained a low but significant activity for the last two pristinamycin I precursors. Finally, a single polypeptide chain (SnbE) with an Mr of 170,000, catalyzing L-phenylglycine-dependent ATP-pyrophosphate exchange, was purified 3,000-fold and characterized. Stepwise Edman degradation of the entire polypeptides or some of their internal fragments provided amino acid sequences for the four isolated proteins. The purified SnbE protein was further shown to be a proteolytic fragment of SnbD.     

A nonribosomal peptide synthetase involved in the biosynthesis of ampullosporins in Sepedonium ampullosporum.

Recently, the saprophytic ascomycete Sepedonium ampullosporum strain HKI-0053 was isolated from a basidiomycete on account of its premature induction of pigment formation in Phoma destructiva, a process often related to the neuroleptic activity of the inducing compound. The active substance was identified as the 15-membered peptaibol type peptide Ampullosporin. Although to date more than 300 peptaibols have been discovered, their biosynthetic machinery has not been characterized yet. By improving the culture conditions it was possible to grow S. ampullosporum in a submerged culture and to increase Ampullosporin production by more than three times to 33 mg/l at reduced fermentation times. The appearance of two high molecular weight proteins, HMWP1 (1.5 MDa) and HMWP2 (350 kDa) was closely related to the production of Ampullosporin during the course of fermentation. Both proteins showed a cross-reaction with antibodies against a core fragment of nonribosomal peptide synthetases (NRPSs). Biochemical characterization of the partially purified enzymes exhibited selectivity for the substrate amino acid alpha-aminoisobutyric acid (Aib). substantiating their involvement in Ampullosporin biosynthesis. Our data suggest that Ampullosporin synthetase has been isolated, and provides the basis for the characterization of the entire biosynthetic gene cluster. Furthermore, this knowledge will enable the manipulation of its NRPS template, in order to engineer mutant strains of Sepedonium ampullosporum which could produce more potent analogues of Ampullosporin.     

Factors affecting the level of alanine racemase in Escherichia coli

Alanine racemase occupies a key position in the alanine branch of peptidoglycan biosynthesis. The level of this enzyme in Escherichia coli W is a function of the carbon source. For example, growth on l-alanine causes a 25-fold higher level of alanine racemase when compared with growth on glucose. When potential inducers of this enzyme are added to either a glucose or succinate medium, a low specificity is observed with those compounds that cause higher levels of enzyme. Growth of E. coli W on either pyruvate, d-alanine, or l-alanine resulted in lower levels of l- and d-alanine in the internal pool. With each of these carbon sources, the level of alanine racemase was markedly elevated when compared to glucose-grown cells; thus, with single carbon sources, the concentration of alanine in the pool is inversely related to the specific activity of alanine racemase. These observations support derepression as a possible mechanism that gives rise to higher levels of alanine racemase. Since multiple forms of the alanine racemase were not detected in extracts from E. coli W grown on various carbon sources, it would appear that this type of heterogeneity is not a consideration in interpreting the above results.     

The relation of d-alanine and alanine racemase activity in molluscs

• 1.1. Eighteen molluscan species were examined for the presence of d-alanine and alanine racemase activity to probe the probable relation between them.
• 2.2. Two bivalve species had high concentration of d-alanine and l-alanine (1:1) and showed high activities of alanine racemase. In these species, the occurrence of d-alanine could be explained by the action of alanine racemase.
• 3.3. In other species, the levels of d-alanine and enzyme activity were low, and the occurrence of d-alanine did not correspond with the presence of alanine racemase activity.
• 4.4. The mechanism of the occurrence of d-alanine in molluscan tissues seems to vary from species to species and seems not to be associated with the phylogenic situation or habitats of the respective species.

     

Knockout of the alanine racemase gene in Lactobacillus plantarum results in septation defects and cell wall perforation

A stable mutant of Lactobacillus plantarum deficient in alanine racemase (Alr) was constructed by two successive homologous recombination steps. When the mutant was supplemented with D-alanine, growth and viability were unaffected. Surprisingly, deprivation of d-alanine during exponential growth did not result in a rapid and extensive lysis as observed in Alr-deficient strains of Escherichia coli or Bacillus subtilis. Rather, the starved mutant cells underwent a growth arrest and were gradually affected in viability with a decrease in colony forming units over 99% in less than 24 h. Additionally, fluorescent techniques demonstrated a loss of cell envelope integrity in the starved cells. Prolonged d-alanine starvation resulted in cells with an aberrant morphology. Scanning and transmission electron microscopy analyses revealed an increase in cell length, deficiencies in septum formation, thinning of the cell envelope and perforation of the cell wall in the septum region. We discuss the involvement of peptidoglycan hydrolases in these phenotypic defects in the context of the crucial role played by D-alanine in peptidoglycan biosynthesis and teichoic acids substitution.     

Streptomyces genes involved in biosynthesis of the peptide antibiotic valinomycin.

We have identified genes from Streptomyces levoris A-9 involved in the biosynthesis of the peptide antibiotic valinomycin. Two segments of chromosomal DNA were recovered from genomic libraries, constructed by using the low-copy-number plasmid pIJ922, by complementation of valinomycin-deficient (vlm) mutants of S. levoris A-9. One set of plasmids restored valinomycin production to only one mutant, that carrying vlm-1, whereas a second set of plasmids restored productivity to seven vlm mutants, those carrying vlm-2 through vlm-8. Additional complementation studies using subcloned restriction enzyme fragments showed that the vlm-1+ gene was contained within a 2.5-kilobase (kb) DNA region, whereas alleles vlm-2+ through vlm-8+ were contained in a 12-kb region, representing at least three genes. Physical mapping experiments based on the isolation of cosmid clones showed that the two vlm loci were 50 to 70 kb apart. Southern hybridization experiments demonstrated that the vlm-2+ gene cluster was highly conserved among other valinomycin-producing Streptomyces strains, whereas the vlm-1+ gene was ubiquitous among Streptomyces species tested. Increasing the copy number of the vlm-2+ gene cluster in S. levoris A-9 by the introduction of low-copy-number recombinant plasmids resulted in a concomitant increase in the level of valinomycin production.     

Inactivation of alanine racemase by beta-chloro-L-alanine released enzymatically from amino acid and peptide C10-esters of deacetylcephalothin

The reactions of a set of amino acid and peptidyl C10-esters of deacetylcephalothin (1-5) have been examined with purified enzymes in vitro. Each of the compounds examined is a substrate for the Escherichia coli TEM-2 beta-lactamase, and enzyme-catalyzed hydrolysis of the lactam bond gives release of an amino acid or a peptidyl fragment from a cephem nucleus. 7 beta-(2-Thienylacetamido)-3-[[(beta-chloro-L-alanyl)oxy]methyl]-3- cephem-4-carboxylate (4) gives time-dependent inactivation of E. coli JSR-O alanine racemase in a process that requires beta-lactamase for the initial liberation of beta-chloro-L-alanine from the cephalosporin. Alanine racemase is similarly inactivated by 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-beta-chloro- L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (1), but this inhibition requires the sequential action of both beta-lactamase and alanine aminopeptidase. Analysis of the enzymatic transformations of 7 beta-(2-thienylacetamido)-3-[[[(beta-chloro-L-alanyl)-L- alanyl]oxy]methyl]-3-cephem-4-carboxylate (3), monitored by high-field 1H NMR, reveals that (1) beta-lactamase releases the dipeptide beta-chloro-L-alanyl-L-alanine from 3 and (2) leucine aminopeptidase effects stoichiometric hydrolysis of the dipeptide to beta-chloro-L-alanine and L-alanine. These biochemical findings are discussed with reference to the mechanism of antibacterial action of 1 against beta-lactamase-producing, penicillin-resistant microorganisms [Mobashery, S., Lerner, S. A., & Johnston, M. (1986) J. Am. Chem. Soc. 108, 1685].     

Silica-immobilized His6-tagged enzyme: alanine racemase in hydrophobic solvent

A new immobilization method for enzymes is presented to facilitate synthetic applications in aqueous as well as organic media. The enzyme Alanine racemase (AlaR) from Geobacillus stearothermophilus was cloned, overexpressed and then immobilized on a silica-coated thin-layer chromatography plate to create an enzyme surface. The enzyme, fused to a His(6)-tag at its N-terminal, was tethered to the chemically modified silica-coated TLC plate through cobalt ions. The immobilized enzyme showed unaltered kinetic parameters in small-scale stirred reactions and retained its activity after rinsing, drying, freezing or immersion in n-hexane. This practical method is a first step towards a general immobilization method for synthesis applications with any enzyme suitable for His6-tagging.     

sanC--a novel gene involved in nikkomycin biosynthesis in Streptomyces ansochromogenes

AIMS: To investigate the genes involved in the nikkomycin biosynthesis and their molecular mechanism. METHODS AND RESULTS: A 0.9 kbp SmaI fragment was cloned and sequenced which contains a complete open reading frame designated sanC (GenBank accession no. AF228522). In search of database, the deduced product of sanC was not homologous with any known proteins. The disruption and complementation of sanC showed that sanC is essential for nikkomycin biosynthesis in Streptomyces ansochromogenes. CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: sanC is a novel and essential gene involved in nikkomycin biosynthesis in S. ansochromogenes.     

A B-cell mitogen from a pathogenic trypanosome is a eukaryotic proline racemase

Lymphocyte polyclonal activation is a generalized mechanism of immune evasion among pathogens. In a mouse model of Trypanosoma cruzi infection (American trypanosomiasis), reduced levels of polyclonal lymphocyte responses correlate with resistance to infection and cardiopathy. We report here the characterization of a parasite protein with B-cell mitogenic properties in culture supernatants of infective forms, the cloning of the corresponding gene and the analysis of the biological properties of its product. We characterized the protein as a co-factor-independent proline racemase, and show that its expression as a cytoplasmic and/or membrane-associated protein is life-stage specific. Inhibition studies indicate that availability of the racemase active site is necessary for mitogenic activity. This is the first report to our knowledge of a eukaryotic amino acid racemase gene. Our findings have potential consequences for the development of new immune therapies and drug design against pathogens.