Characterization of the Functional Variance in MbtH-like Protein Interactions with a Nonribosomal Peptide Synthetase

Abstract

Many nonribosomal peptide synthetases (NRPSs) require MbtH-like proteins (MLPs) for solubility or for activation of amino acid substrate by the adenylation domain. MLPs are capable of functional crosstalk with noncognate NRPSs at varying levels. Using enterobactin biosynthesis in Escherichia coli as a model MLP-dependent NRPS system, we use in vivo and in vitro techniques to characterize how seven noncognate MLPs influence the function of the enterobactin NRPS EntF when the cognate MLP, YbdZ, is absent. Using a series of in vitro assays to analyze EntF solubility, adenylation, aminoacylation, and in vitro enterobactin production, we show that interactions between MLPs and NRPSs are multifaceted and more complex than previously appreciated. We separate MLP influence on solubility and function in a manner that shows altered solubility is not indicative of a functional MLP/NRPS pair. Although much of the functional variation among these noncognates can be explained by differences in EntF affinity for an MLP or the extent an MLP alters EntF l-Ser affinity, we demonstrate that MLPs can have a broader impact beyond solubility and adenylation. First, we show that a noncognate MLP can affect formation of l-Ser-S-EntF. Second, under in vitro conditions saturating for substrate and MLP, enterobactin production remains compromised in the absence of an appropriate MLP partner. These data suggest that we expand our investigations into how the MLPs influence NRPS enzymology. A more detailed understanding of these influences will be essential for downstream engineering of hybrid NRPS systems.

TxtH is a key component of the thaxtomin biosynthetic machinery in the potato common scab pathogen Streptomyces scabies

“…It has been reported that MLPs from different pathways can functionally complement each other (Boll et al, 2011;Lautru et al, 2007;Mori et al, 2018;Schomer and Thomas, 2017;Wolpert et al, 2007;Zhang et al, 2010). In organisms where multiple MLPs are encoded in a single genome, the deletion of a single MLP often does not abolish the production of the cognate metabolite, but instead metabolite production is eliminated only when all copies of MLP-encoding genes are removed from the host genome (Lautru et al, 2007;Wolpert et al, 2007).…”

“…MLPs are small (~70 amino acids) proteins that are usually encoded within NRPS gene clusters (Baltz, 2011). Recent studies have shown that they play essential roles in promoting the proper folding and activity of the NRPS A-domains, though for reasons currently unknown, not all NRPS A-domains require an MLP for proper function (Boll et al, 2011;Felnagle et al, 2010;McMahon et al, 2012;Schomer and Thomas, 2017;Zhang et al, 2010). It has also been revealed that MLPs from different pathways can, in some instances, functionally complement each other with varying efficiencies (Boll et al, 2011;Lautru et al, 2007;Mori et al, 2018;Schomer and Thomas, 2017;Wolpert et al, 2007;Zhang et al, 2010).…”

Using MbtH‐Like Proteins to Alter the Substrate Profile of a Nonribosomal Peptide Adenylation Enzyme

“…The effect of MLPs on kinetic parameters is opposite to that of a previous study about the activity of the A domain EntF with different MLPs, which displayed much greater dependence on K m than k cat . [8, 10b] However, as stated before, the activity of the EntF enzyme is not fully dependent on MLPs. These substrate profiles and kinetic data, when compared to previous studies, suggest that the exact role of MLPs cannot be explained by a single function, although it is obvious that MLPs are necessary for some A domains for their optimal activity.…”

“…By amino acid sequence analyses, we concluded that the amino acid residues involved in MLP-A domain interactions are fairly conserved, but not identical in A domains with a shared cognate MLP, and that sequence homology of MLPs does not directly correlate with their ability to alter A domain function, which is consistent with a previous study. [8] All these results indicated that MLP-A domain interactions are not simple and a lot remains to be understood. Based on the encouraging broadening of the substrate versatility of TioK by noncognate MLPs, future studies focused on engineering MLPs to enhance the promiscuity of A domains while retaining their activity will be warranted.…”

“…[4b, 5a] It was also shown that various MLPs can flexibly interact with a heterologously expressed A domain to change the kinetics of activation of its natural substrate. [6a, 8] Structural studies of MLPs [9] and MLP-A domain complexes [5g, 10] have provided insights as to how A domains interact with and are affected by MLPs. The MLP structures revealed that they contain three anti-parallel β-sheets followed by one or two α-helices.…”