Kirromycin is a potent protein biosynthesis inhibitor. But unlike many known antibiotics like erytrhomycin or tetracyclin, kirromycin does not target the bacterial ribosomes directly but instead interacts with an essential helper protein, the elongation factor EF-Tu. EF-Tu is an essential protein of the bacterial protein biosynthesis machinery which transfers the amino acyl tRNAs to the acceptor (A) site of the ribsome. In its GTP bound conformation it binds to the aminoacyl-tRNA substrate and delivers it to the ribosome. After interaction of the aa-tRNA with the ribosome-bound mRNA GTP is hydrolyzed which confers a conformational change to EF-Tu and the subsequent release of the factor. EF-Tu-GDP is then recycled via interaction with EF-Ts and reloaded with aa-tRNA.

Binding of kirromycin now leads to an uncoupling of GTP hydrolysis and the change of conformation. Thus kirromycin-bound EF-Tu does no longer dissociate from the target and therby blocks translation.

Kirromycin was firstly isolated from the actinomycete Streptomyces collinus Tü 365 by H. Wolf in H. Zähner's group in Tübingen (Wolf and Zähner, 1972, Arch. Microbiol. 83:147-154).

The linear kirromycin-molecule contains a pyridone moiety, a central tetrahydrofurane group and a goldinonic acid lactone that is linked via an amide bond to the rest of the molecule (Figure 1).

In our recent work, we identified and isolated the kirromycin biosynthetic gene cluster from a cosmid library of the producing strain Streptomyces collinus Tü 365 (Weber et al., 2003, J. Biotechnol. 106:221-232; Weber et al., 2008, Chem. Biol. 15:175-188). Based on genetic, biochemical and in silico data, 26 genes encoded on a 82 kb genomic fragment, were assigned to the kirromycin biosynthetic gene cluster (Figure 2).

The carbon skeleton of kirromycin is synthesized by a unique combination of trans-AT polyketide synthases (PKS), cis-AT PKS and non-ribosomal peptide synthetases, which is subsequently tailored to yield bioactive kirromycin (Figure 3).

During our work, we were able to firstly identify a novel route to pyridone formation which involves the condensation of the non-proteinogenic amino acid β-alanine to an polyketide precursor. We showed, that β-alanine is not only provided by primary metabolism but also through an specific enzyme of the kirromycin biosynthetic gene cluster (Laiple et al., 2009, J. Antibiotics 62:465-468)

In contrast to many other PKS or NRPS gene clusters, the enzyme which is required to activate the carrier protein domains of the PKS/NRPS is encoded within the kirromycin biosynthetic gene cluster. We could demonstrate that the phosphopantetheinyltransferase KirP is able to convert both, the ACP and PCP domains of KirAI-AVI and KirB from their inactive apo-form into the active holo form (Pavlidou et al., 2011, FEMS Microbiol. Lett. 319, 26-33).

The kirromycin PKS/NRPS is the first enzyme, where non-malonate incorporation by a trans-AT PKS was biochemically investigated: At module 5 of the PKS KirAIII an ethyl branch is introduced into the kirromycin precursor molecule. On molecular level, this is achieved by a second acyltransferase enzyme, KirCII, which is highly specific to load the extender unit ethylmalonyl-CoA onto ACP5 of KirAII (Musiol et al., 2011, Chem. Biol. 18, 438-444).

Current research interests

• Kirromycin resistance
• Biochemistry of kirromycin tailoring
• Trans-AT polyketide synthase mechanism
• Regulation of kirromycin production
• Comparative genomics of kirromycin/elfamycin producers
  
  
  

• Melanie Knobloch
• Ewa-Maria Musiol
• Thomas Härtner
• Thomas Schafhauser
• Tilmann Weber

• Prof. Dr. Stephanie Grond, Institute of Organic Chemistry, University of Tübingen
• Prof. Dr. Jörn Piel, Kekulé Institute of Organic Chemistry and Biochemisty, University of Bonn
• Prof. Dr. Gavin Williams, Dept. of Chemistry, North Carolina State University, Raleigh, NC, USA