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RESEARCH - H. D. Mootz - Protein chemistry / biotechnology

Coordination in multi-domain biosynthesis enzymes  
 

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The biosynthesis of complex natural products is one of the most fascinating aspects of protein function from a chemists' perspective. Multi-step reactions are orchestrated by multiple enzymes that catalyze the synthesis from simple precursors. Arguably, the producing microorganisms or plants can be regarded as excellent synthetic chemists. We are interested in the biosynthesis of a certain class of natural products, the nonribosomal peptides. This group of natural products include important compounds like the penicillins, vancomycin and cyclosporine (Figure 1). They can display antibiotic, antifungal, and immunosuppressive activites, for example. According to their name, these are not synthesized in an mRNA-templated fashion on the ribosome, but instead, on protein templates called nonribosomal peptide synthetases (NRPS). Nonribosomal peptides are not limited to the 20 proteinogenic amino acids but can contain a huge number of different building blocks as well as various modifications such as lipidation or glycosylation. Furthermore, most peptides are cyclic or branched cyclic to increase their bioactivity. NRPSs synthesize their products in a manner reminiscent of an assembly-line (Figure 2). NRPSs are composed of modules, each of which is typically responsible for the incorporation of one amino acid into the growing peptide chain (1). Modules can be further subdivided into single domains that harbour the catalytic activities, for example the adenylation domain selects an amino acid and activates it to the aminoacyl adenylate under consumption of ATP, or serve as carrier domains to covalently bind the intermediate as a 4'-phosphopantetheinyl thioester.

We are interested in how these mega-enzymes work. How is a directed coordination between the multiple domains achieved? For example, the NRPS responsible for the biosynthesis of the cyclic decapeptide antibiotic tyrocidine consists of 32 domains, which are located on three subunits TycA, TycB, and TycC (Figure 2)(2). Although crystal structures of isolated domains of each type, of a few bi-domain constructs, and even of one entire module are known, their interplay in space and time is not well understood. Each peptidyl-carrier protein (PCP) or thiolation domain has to interact with several catalytic centers, however, these are too far apart to be reached by a simple movement of the 4'phosphopantetheine prosthetic group. Consequently, significant conformational changes and domain movements are postulated (3). To address these questions, we use a combination of biochemical and biophysical techniques.

Selected references

  1. Mootz, H. D., Schwarzer, D., and Marahiel, M. A. (2002)
    Ways of assembling complex natural products on modular nonribosomal peptide synthetases, Chembiochem 3, 490-504.

  2. Mootz, H. D., and Marahiel, M. A. (1997)
    The tyrocidine biosynthesis operon of Bacillus brevis: complete nucleotide sequence and biochemical characterization of functional internal adenylation domains, J Bacteriol 179, 6843-6850.

  3. Zettler, J., and Mootz, H. D. (2010)
    Biochemical evidence for conformational changes in the cross-talk between adenylation and peptidyl-carrier protein domains of nonribosomal peptide synthetases, The FEBS journal 277, 1159-1171.

Last update: 2015/11


Technology development for
protein semisynthesis
Posttranslational modifications
of the Ubiquitin-type
Coordination in multi-domain
biosynthesis enzymes

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