X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture

Tarry, M.J.; Haque, Asfarul S.; Bui, Khanh Huy; Schmeing, T.M.

doi:10.1016/j.str.2017.03.014

Cited by 90 publications

(137 citation statements)

References 71 publications

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“…13,15,17 Residues involved in NRPS–MLP interaction in these structures were cross-referenced with the sequence alignments (Figure 3a). Among these residues, there is significant commonality between functional and nonfunctional MLPs.…”

Section: Resultsmentioning

confidence: 99%

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

Schomer

Thomas

2017

Biochemistry

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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.

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Section: Resultsmentioning

confidence: 99%

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

Schomer

Thomas

2017

Biochemistry

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“…The NRPS fragment contains an adenylation-PCP-condensation domain, crossing the border between modules and extending to the downstream condensation domain. 63 The structure shows that no interactions exist between the adenylation domain and the condensation domain of the subsequent module. Instead the condensation domain cradles the “back face” of the carrier protein domain.…”

Section: The Biochemistry and Structural Biology Of The Nonribosommentioning

confidence: 99%

“…Indeed, this is an exciting time for the structural studies of NRPSs as many structures of multidomain proteins were determined in 2016. 56-63 This review will briefly describe the structures of individual domains and the implications of the recent structures on the necessary dynamics to transfer the substrates and intermediates between different catalytic domains (Fig. 1).…”

Section: The Biochemistry and Structural Biology Of The Nonribosommentioning

confidence: 99%

“…224 These small auxiliary proteins interact with NRPS adenylation domains to enhance adenylation activity. 225, 226 Crystal structures of MLPs bound to an adenylation domain 227 or to multidomain NRPS proteins 59, 63 illustrate how the MLP interacts with the N-terminal subdomain over 15 Å away from the active site. Structures of NRPS proteins in the presence and absence of the partner MLP have not yet identified the basis for activation.…”

Section: Pseudomonas Aeruginosamentioning

confidence: 99%

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Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

Gulick

2017

Nat. Prod. Rep.

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Overview Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

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“…[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.…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2018

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MbtH-like proteins (MLPs) are required for soluble expression and/or optimal activity of some adenylation (A) domains of nonribosomal peptide synthetases. Because A domains can interact with noncognate MLP partners, how the function of an A domain, TioK, involved in the biosynthesis of the bisintercalator thiocoraline, is altered by noncognate MLPs has been investigated. Measuring TioK activity with 12 different MLPs from a variety of bacterial species by using a radiometric assay suggested that the A domain substrate promiscuity could be altered by foreign MLPs. Kinetic studies and bioinformatics analysis expanded the complexity of MLP functions and interactions.

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X-Ray Crystallography and Electron Microscopy of Cross- and Multi-Module Nonribosomal Peptide Synthetase Proteins Reveal a Flexible Architecture

Cited by 90 publications

References 71 publications

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

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

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

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

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