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Applied and Environmental Microbiology, August 2001, p. 3603-3609, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3603-3609.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

E. Heinzelmann, G. Kienzlen, S. Kaspar, J. Recktenwald, W. Wohlleben, and D. Schwartz*

Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, D-72076 Tübingen, Germany

Received 31 January 2001/Accepted 7 May 2001

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.

* Corresponding author. Mailing address: Mikrobiologie/Biotechnologie, Eberhard-Karls-Universität Tübingen, Auf der Morgenstelle 28, D-72076 Tübingen, Germany. Phone: 49 7071 29-74638. Fax: 49 7071 29-5979. E-mail: schwartz{at}molbio.biol.biologie.uni-tuebingen.de.

Applied and Environmental Microbiology, August 2001, p. 3603-3609, Vol. 67, No. 8
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.8.3603-3609.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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