Abstract：
:Nonribosomal peptide synthetases (NRPSs) are megasynthetases that require complex and specific interactions between multiple domains and proteins to functionally produce a metabolite. MbtH-like proteins (MLPs) are integral components of many NRPSs and interact directly with the adenylation domain of the megasynthetases to stimulate functional enzymology. All of the MLP residues that are essential for functional interactions between the MLP and NRPS have yet to be defined. Here we probe the interactions between YbdZ, an MLP, and EntF, an NRPS, from Escherichia coli by performing a complete alanine scan of YbdZ. A phenotypic screen identified 11 YbdZ variants that are unable to replace the wild-type MLP, and these YbdZ variants were characterized using a series of in vivo and in vitro assays in an effort to explain why functional interactions with EntF were disrupted. All of the YbdZ variants enhanced the solubility of overproduced EntF, suggesting they were still capable of direct interactions with the megasynthase. Conversely, we show that EntF also influences the solubility of YbdZ and its variants. In vitro biochemical analyses of EntF function with each of the YbdZ variants found the impact that an amino acid substitution will have on NRPS function is difficult to predict, highlighting the complex interaction between these proteins.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Schomer RA,Park H,Barkei JJ,Thomas MGdoi
10.1021/acs.biochem.8b00552subject
Has Abstractpub_date
2018-07-17 00:00:00pages
4125-4134issue
28eissn
0006-2960issn
1520-4995journal_volume
57pub_type
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