The Phosphinomethylmalate Isomerase Gene pmi, Encoding an Aconitase-Like Enzyme, Is Involved in the Synthesis of Phosphinothricin Tripeptide in Streptomyces viridochromogenes

Abstract

Streptomyces viridochromogenes Tü494 produces the antibiotic phosphinothricin tripeptide (PTT). In the postulated biosynthetic pathway, one reaction, the isomerization of phosphinomethylmalate, resembles the aconitase reaction of the tricarboxylic acid (TCA) cycle. It was speculated that this reaction is carried out by the corresponding enzyme of the primary metabolism (C. J. Thompson and H. Seto, p. 197-222, in L. C. Vining and C. Stuttard, ed., Genetics and Biochemistry of Antibiotic Production, 1995). However, in addition to the TCA cycle aconitase gene, a gene encoding an aconitase-like protein (the phosphinomethylmalate isomerase gene, pmi) was identified in the PTT biosynthetic gene cluster by Southern hybridization experiments, using oligonucleotides which were derived from conserved amino acid sequences of aconitases. The deduced protein revealed high similarity to aconitases from plants, bacteria, and fungi and to iron regulatory proteins from eucaryotes. Pmi and the S. viridochromogenes TCA cycle aconitase, AcnA, have 52% identity. By gene insertion mutagenesis, a pmi mutant (Mapra1) was generated. The mutant failed to produce PTT, indicating the inability of AcnA to carry out the secondary-metabolism reaction. A His-tagged protein (Hispmi*) was heterologously produced in Streptomyces lividans. The purified protein showed no standard aconitase activity with citrate as a substrate, and the corresponding gene was not able to complement an acnA mutant. This indicates that Pmi and AcnA are highly specific for their respective enzymatic reactions.The structurally identical antibiotics phosphinothricin tripeptide (PTT) and bialaphos are produced by Streptomyces viridochromogenes and by Streptomyces hygroscopicus (4, 18), respectively. They consist of two molecules, L-alanine and one molecule of the unusual amino acid phosphinothricin (PT). A biosynthetic pathway for bialaphos, consisting of at least 13 steps, was postulated following analysis of nonproducing S. hygroscopicus mutants (summarized in reference 35). Several enzymes were purified, and various genes of the PTT biosynthetic gene cluster were mapped in S. hygroscopicus (35), as well as in S. viridochromogenes (1,12,28,32). It was shown that the respective genes and enzymes were highly similar (up to 80%) on the DNA and amino acid levels (29,39,40). As the genetic organizations of the two clusters are basically identical, it has been concluded that the biosynthesis in both producing strains proceeds in the same way.The biosynthetic steps 6, 7, and 8 were found to be similar to the citrate synthase, aconitase, and isocitrate dehydrogenase reactions of the tricarboxylic acid (TCA) cycle, respectively (Fig. 1). In contrast to the step 6 reaction, for which a specific PTT biosynthetic gene and protein were identified (15), the subsequent steps, especially the isomerization of phosphinomethylmalate in step 7, were speculated to be catalyzed by the enzymes of the primary metabolism (35). Three facts supported this. First, inhibition of aconitase resulted i...

Biosynthetic Gene Cluster of the Herbicide Phosphinothricin Tripeptide from Streptomyces viridochromogenes Tü494

“…In this plasmid, trs is transcribed from the constitutive erythromycin resistance gene promoter (ermEp). Since the vector pEM4 has already been successfully used as an expression plasmid (11,35) and since there is no termination structure between the AatII site and the trs gene, expression of the trs gene should take place. No difference in PTT sensitivity was detected between S. lividans TK23(pDS400) and the control strain S. lividans TK23(pEM4).…”

“…Plasmid pDS204, which carries an 8.9-kb EcoRI/KpnI fragment of the PTT biosynthetic gene cluster (orf1-phsBorfM-phsC gene region) was digested with BlpI. An internal 0.7-kb BlpI DNA fragment of the orfM gene was replaced by the aprP resistance gene cassette (1.8-kb EcoRV/StuI fragment of pEH13), which carries the apramycin resistance gene and the constitutive promoter ermEp to drive the expression of downstream genes (11), resulting in plasmid pDS301. To avoid polar effects by disruption of an operon structure, we cloned the cassette in the transcriptional direction of the phsC gene.…”

“…The aconitase-like gene pmi encodes a protein that specifically catalyzes the isomerization of the intermediate phosphinomethylmalate (step 7) (Fig. 2) (11).…”

Identification of a topoisomerase IV in actinobacteria: purification and characterization of ParYR and GyrBR from the coumermycin A1 producer Streptomyces rishiriensis DSM 40489

“…In order to allow replication of these vectors and expression of GyrB R and ParY R in S. lividans, they were fused with the vector pGM9, which contains an origin of replication for Streptomyces (Muth et al, 1989). The resulting vectors were introduced into S. lividans T7, which contains a thiostrepton-inducible gene for T7 RNA polymerase (Heinzelmann et al, 2001). After induction, crude protein extracts were prepared and subjected to nickel affinity chromatography.…”

“…For preparation of genomic DNA from S. coelicolor, YMG medium was used. Protoplast preparation and transformation of S. lividans T7 (Heinzelmann et al, 2001) for protein expression was carried out according to standard methods (Kieser et al, 2000). E. coli transformants with pET-24a(+) or pGM9 fusion plasmids were selected with kanamycin (50 mg ml 21 ); transformants with pRSET B were selected with carbenicillin (50 mg ml 21 ).…”