Structural and functional analysis of enzymes associated with iron uptake.
With the exception of a few microbial organisms, iron is required for life on earth. Iron serves as an important cofactor for a variety of enzymes that perform crucial reactions, including roles in cellular respiration, nucleic acid synthesis, and resistance to reactive oxygen intermediates. Fe(III) is very insoluble and frequently biologically inaccessible such that the concentration of available iron in the human host is ~10-9 mM. Since a typical pathogenic bacterium requires ~1 mM iron for optimal growth, these organisms have developed elaborate systems to scavenge iron from the host. The pathogens that we study use low molecular weight iron chelators called siderophores. The bacteria synthesize, secrete, and then selectively take up the iron-loaded siderophore to colonize human tissues. Non-ribosomal peptide synthetases (NRPSs) and their accessory proteins are a fascinating collection of enzymes required for the production of these bioactive peptides. Siderophore biosynthetic enzymes are found in plants, fungi and bacteria, frequently having no human homologues, making them attractive targets for the development of new antimicrobial compounds. The goal of the lab is to understand the structure-function relationships that drive the biosynthesis of siderophores, compounds linked to virulence and pathogenesis in a variety of deadly bacteria. The ultimate outcome of the work will be the structural biology and mechanistic enzymology required for the development of new antibiotics to fight many bacterial infections, including P. aeruginosa, an opportunistic pathogen that is problematic for cystic fibrosis and other susceptible patients, as well as for bacteria that generate chemically-related siderophores such as Yersinia pestis (plague), Vibrio cholera (cholera) and Mycobacterium tuberculosis (tuberculosis). The enzymes we study are also of interest to the protein engineering community, as many bioactive peptides contain similar chemical moieties and homologous enzymes are found in biosynthetic pathways of a variety of natural products. Two examples are epothilone and cyclosporin. Therefore, our work may also impact the design of new anti-cancer and immunosuppressive therapeutics.
- Meneely KM, Ronnebaum TA, Riley AP, Prisinzano TE, Lamb AL. (2016) Holo structure and steady state kinetics of the thiazolinyl imine reductases for siderophore biosynthesis. Biochemistry 55, 5423-33..
- Meneely KM, Sundlov JA, Gulick AM, Moran GR, Lamb AL. (2016) An open and shut case: the interaction of magnesium with MST enzymes. Journal of the American Chemical Society 138, 9277-93.
- Lamb AL. (2015) Breaking a pathogen's iron will: inhibiting siderophore production as an antimicrobial strategy. Biochimica Biophysica Acta: Proteins and Proteomics 1854, 1054-1070.
- Lamb AL, Kappock TJ, Silvaggi NR (2015) You are lost without a map: navigating the sea of protein structures. Biochimica Biophysica Acta: Proteins and Proteomics 1854, 258-268.
- Meneely KM, Luo Q, Riley AP, Taylor B, Roy A, Stein RL, Prisinzano TE, Lamb AL. (2014) Expanding the results of a high throughput screen against an isochorismate-pyruvate lyase to enzymes of a similar scaffold or mechanism. Bioorg Med Chem. 22, 5961-69.
- Chilton AS, Ellis AL, Lamb AL. (2014) Structure of an Aspergillus fumigatus old yellow enzymes (EasA) involved in ergot alkaloid biosynthesis. Acta Crystallogr F Struct Biol Commun. 70, 1328-32.
- Frederick RE, Ojha S, Lamb AL, Dubois JL. (2014) How pH modulates the reactivity and selectivity of a siderophore-associated flavin monooxygenase. Biochemistry 53, 2007-16.
- Lothrop AP, Snider GW, Flemer S Jr, Ruggles EL, Davidson RS, Lamb AL, Hondal RJ. (2014) Compensating for the absence of selenocysteine in high-molecule weight thioredoxin reductases: the electrophilic activation hypothesis. Biochemistry 53, 664-74.
- Meneely KM, Luo Q, Lamb AL. (2013) Redesign of MST enzymes to target lyase activity instead promotes mutase and dehydratase activities. Archives of Biochemistry and Biophysics. 539, 70-80.
- Meneely KM, Luo Q, Dhar P, Lamb AL. (2013) Lysine221 is the general base residue of the isochorismate synthase from Pseudomonas aeruginosa (PchA) in a reaction that is diffusion limited. Archives of Biochemistry and Biophysics. 538, 49-56.
- Meneely KM, Lamb AL. (2012) Two structures of a thiazolinyl imine reductase from Yersinia enterocolitica provide insight into catalysis and binding to the nonribosomal peptide synthetase module of HMWP1. Biochemistry. 52, 9002-13.
- Olucha J, Meneely KM, Lamb AL. (2012) Modification of residue 42 of the active site loop with a lysine-mimetic sidechain rescues isochorismate-pyruvate lyase activity in Pseudomonas aeruginosa PchB. Biochemistry. 51, 7525-38.
- Lamb AL. (2011) Pericyclic reactions catalyzed by chorismate-utilizing enzymes. Biochemistry. 50, 7476-7483.
- Olucha J, Lamb AL. (2011) Mechanistic and structural studies of the N-hydroxylating flavoprotein monooxygenases. Bioorganic Chemistry. 39, 171-177.
- Olucha J, Ouellette AN, Luo Q, Lamb AL. (2011). pH dependence of catalysis by Pseudomonas aeruginosa isochorismate-pyruvate lyase: implications for transition state stabilization and the role of lysine 42. Biochemistry. 50, 7198-207.
- Olucha J, Meneely KM, Chilton AS, Lamb AL. (2011). Two structures of an N-hydroxylating flavoprotein monooxygenase: the ornithine hydroxylase from Pseudomonas aeruginosa. J. Biol. Chem. 286, 31789-31798,
- Luo Q, Meneely KM, Lamb, AL. (2011). Entropic and enthalpic components of catalysis in the mutase and lyase activities of Pseudomonas aeruginosa PchB. J. Am. Chem. Soc. 133, 7229-7233.
- Luo Q, Olucha J, Lamb AL. (2009). Stucture-function analyses of isochorismate-pyruvate lyase from Pseudomonas aeruginosa suggest differing catalytic mechanisms for the two pericyclic reactions of this bifunctional enzyme. Biochemistry 48,5239-5245.
- Meneely KM, Barr E, Bollinger JM, Lamb AL. (2009). Kinetic mechanism of ornithine hydroxylase (PvdA) from Pseudomonas aeruginosa: substrate triggering of O2 addition but not flavin reduction. Biochemistry 48, 4371-4376.
- Meneely KM Lamb AL. (2007). Biochemical characterization of a flavin adenine dinculeotide-dependent monooxygenase, ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism. Biochemistry 46, 11930–7.
- Zaitseva J, Lu J, Olechoski KL, Lamb AL (2006). Two crystal structures of the isochorismate-pyruvate lyase from Pseudomonas aeruginosa. J. Biol. Chem. 281, 33441–33449.
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