Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

Meneely, Kathleen M.; Luo, Qianyi; Lamb, Audrey L.

doi:10.1016/j.abb.2013.09.007

Cited by 5 publications

(20 citation statements)

References 47 publications

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“…The wild-type pchA ( 9 ) and irp9 ( 27 ) genes were cloned as previously described. The wild-type entc gene was amplified from E. coli strain JM109 genomic DNA by polymerase chain reaction using Vent DNA Polymerase (New England Biolabs).…”

Section: Methodsmentioning

confidence: 99%

“…Wild-type and variant Irp9, 27 wild-type and variant PchA, 9 and PchB 30 were purified as previously described. BL21 (DE3) E. coli containing the EntC wild-type or variant expression plasmid was grown in LB medium containing 200 μg/mL ampicillin at 37 °C with shaking (250 rpm) until an optical density at 600 nm (OD 600 ) of ∼0.8 was reached.…”

Section: Methodsmentioning

confidence: 99%

“…Partial occupancies were refined, with 0.54 for chorismate and 0.46 for isochorismate in molecule A and 0.56 for chorismate and 0.44 for isochorismate in molecule B, in close agreement with the solution equilibrium values for this enzyme. 2 , 27 The model includes 269 water molecules.…”

Section: Methodsmentioning

confidence: 99%

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An Open and Shut Case: The Interaction of Magnesium with MST Enzymes

et al. 2016

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The shikimate pathway of bacteria, fungi, and plants generates chorismate, which is drawn into biosynthetic pathways that form aromatic amino acids and other important metabolites, including folates, menaquinone, and siderophores. Many of the pathways initiated at this branch point transform chorismate using an MST enzyme. The MST enzymes (menaquinone, siderophore, and tryptophan biosynthetic enzymes) are structurally homologous and magnesium-dependent, and all perform similar chemical permutations to chorismate by nucleophilic addition (hydroxyl or amine) at the 2-position of the ring, inducing displacement of the 4-hydroxyl. The isomerase enzymes release isochorismate or aminodeoxychorismate as the product, while the synthase enzymes also have lyase activity that displaces pyruvate to form either salicylate or anthranilate. This has led to the hypothesis that the isomerase and lyase activities performed by the MST enzymes are functionally conserved. Here we have developed tailored pre-steady-state approaches to establish the kinetic mechanisms of the isochorismate and salicylate synthase enzymes of siderophore biosynthesis. Our data are centered on the role of magnesium ions, which inhibit the isochorismate synthase enzymes but not the salicylate synthase enzymes. Prior structural data have suggested that binding of the metal ion occludes access or egress of substrates. Our kinetic data indicate that for the production of isochorismate, a high magnesium ion concentration suppresses the rate of release of product, accounting for the observed inhibition and establishing the basis of the ordered-addition kinetic mechanism. Moreover, we show that isochorismate is channeled through the synthase reaction as an intermediate that is retained in the active site by the magnesium ion. Indeed, the lyase-active enzyme has 3 orders of magnitude higher affinity for the isochorismate complex relative to the chorismate complex. Apparent negative-feedback inhibition by ferrous ions is documented at nanomolar concentrations, which is a potentially physiologically relevant mode of regulation for siderophore biosynthesis in vivo.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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An Open and Shut Case: The Interaction of Magnesium with MST Enzymes

et al. 2016

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“…Despite very high sequence homology in the active site, three non-conserved amino acids were identified that differ systematically between salicylate synthases and isochorismate synthases[26]. However, mutation of one or more of these isochorismate synthase residues to the cognate salicylate synthase residues failed to make the isochorismate synthases competent for lyase function[2, 9, 27]. It is clear that the functional difference between the two classes of MST enzymes is not solely due to variation in active site sidechain composition.…”

Section: Salicylate Synthasesmentioning

confidence: 99%

Unraveling the Structure and Mechanism of the MST(ery) Enzymes

Shelton

Lamb

2018

Trends in Biochemical Sciences

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The menaquinone, siderophore, and tryptophan (MST) enzymes transform chorismate to generate precursor molecules for the biosynthetic pathways defined in their name. Kinetic data, both steady-state and transient-state, and X-ray crystal structures indicate that these enzymes are highly conserved both in mechanism and in structure. Because these enzymes are found in pathogens but not in humans, there is considerable interest in these enzymes as drug design targets. While great progress has been made in defining enzyme structure and mechanism, inhibitor design has lagged behind. This review provides a detailed description of the evidence that begins to unravel the mystery of how the MST enzymes work, and how that information has been used in inhibitor design.

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“…148, 149 Additional members of this family include PchA of pyochelin biosynthesis, BasJ of acinetobactin biosynthesis, and Irp9 of yersiniabactin biosynthesis. Interestingly while the initial step in the reaction—the isomerization to isochorismate—is shared, different members of the family form different products.…”

Section: Klebsiella Pneumoniaementioning

confidence: 99%

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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Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities

Cited by 5 publications

References 47 publications

An Open and Shut Case: The Interaction of Magnesium with MST Enzymes

An Open and Shut Case: The Interaction of Magnesium with MST Enzymes

Unraveling the Structure and Mechanism of the MST(ery) Enzymes

Nonribosomal peptide synthetase biosynthetic clusters of ESKAPE pathogens

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