The role of MbtH-like proteins in nonribosomal peptide synthetase (NRPS) assembly-lines # Tools for biosynthesis/bioengineering of novel antibiotics.

Many of the naturally derived antibiotics in current use (along with many other pharmaceuticals such as anti-tumor agents and immuno-suppressants) are complex secondary metabolites biosynthesized on nonribosomal peptide synthetase (NRPS) assembly-lines by bacteria. These assembly-lines contain many proteins usually encoded in gene clusters on the chromosome and comprise multiple modules each containing three core domains for condensation (C), adenylation (A), and thiolation (T). The most studied systems are from Streptomyces coelicolor, a soil-dwelling filamentous bacteria that contains 18 complex secondary metabolite gene clusters1. Common to most NRPS gene clusters is a small protein comprised of ~70 amino acids, which plays an important role in stimulating the adenylating activity of A domains in NRPS modules2,3. Garneau-Tsodikova and co-workers have shown that three such proteins (SAMR0548, KtzJ, and TioT) were essential for the activation of congocidine, kutzneride, and thiocoraline NRPS systems, respectively, of S. coelicolor-4-7. Furthermore, they recently found that co-expression of TioT homologues from other NRPS systems with TioK resulted in a drastic change in the biochemical function of the complex (manuscript in preparation). While the kinetic parameters for the main substrate remained excellent, even in the presence of a non-cognate partners for TioK, the substrate profile of the A domain of the A-T TioK di-domain changed and was broadened. Thus, by combining a variety of MbtH-like proteins with different NRPS partners, it may be possible to create a toolbox for the bioengineering of novel pharmaceuticals. In Mycobacterium tuberculosis, the NRPS assembly-line is responsible for producing two types of siderophores, called mycobactins, responsible for the sequestration of iron and assisting M. tuberculosis survival under the low-iron conditions of human infection. SSGCID has recently determined the solution structure of three members of this gene family (from M. tuberculosis (MtbH, MytuD.01649, 2KHR), M. murinum (MymaA.01649.c, 2MYY), and M. avium, (Myav.01649.c, not yet submitted to PDB); as well as Burkholderia pseudomallei, BupsA.13472.b, 2LPD). All contain a three-strand anti-parallel â-sheet (â3:â1:â2) nestled against one C-terminal á-helix8. The aim of our functional study is to use these MtbH-like proteins to expand the NRPS toolbox and determine the structural basis driving the functional variability. A structure for M. tuberculosis MbtB has not been determined because, like TioN and TioK, it is cannot be expressed in soluble and active form by itself.