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Title:Investigation of the biosynthesis of the phosphonate antibiotics bialaphos and dehydrophos
Author(s):Lee, Jin-Hee
Director of Research:van der Donk, Wilfred A.
Doctoral Committee Chair(s):van der Donk, Wilfred A.
Doctoral Committee Member(s):Zimmerman, Steven C.; Silverman, Scott K.; Kelleher, Neil L.
Department / Program:Chemistry
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):phosphonate antibiotics
nonribosomal peptide synthetase
Abstract:Phosphonate and phosphinate natural products possess a range of biological activities as a result of their ability to mimic phosphate esters or tetrahedral intermediates formed in enzymatic reactions involved in carboxyl group metabolism. The widespread use in medicine and agriculture of these compounds has resulted in active researches, revealing novel biochemistry behind their metabolic pathways. One of such compounds, PTT, contains unusual amino acid phosphinothricin attached to two alanine residues. The peptide bond formation and methylation on phosphorus in PTT are key steps in PTT biosynthesis, however, the exact timing of these transformations was not known. To provide insights into these questions, a heterologous expression system was developed for PhsA, the first of three NRPS proteins involved in PTT biosynthesis, and kcat/Km value for the ATP-pyrophosphate exchange activities of the purified protein with putative substrates were determined. The kcat/Km value for L-N-AcPT was 7-fold higher than for D,L-N-AcDMPT, suggesting the former might be the physiological substrate. Both substrates were loaded onto the thiolation domain of PhsA as demonstrated by FTMS. The possible implications of these findings for the timing of the methylation reaction are discussed. Another phosphonate presented here is antibiotic dehydrophos, which contains a unique methyl ester of a phosphonodehydroalanine group. Since the dianionic form of phosphonates at pH 7 poses a drawback with respect to their ability to mimic carboxylates and tetrahedral intermediates, esterification of a phosphonate group by SAM-dependent methyltransferase, DhpI would provide a solution to alleviating the high charge state, and thus improve the bioavailability of phosphonates. This study describes expression, purification and reconstitution of DhpI in vitro. Also, substrate scope and X-ray crystal structure of DhpI are also discussed. The enzyme utilizes S-adenosylmethionine to methylate a variety of phosphonates including 1-HEP, 1,2-DHEP, and Ac-1-AEP. Kinetic analysis showed that the best substrates are tripeptides containing as C-terminal residue a phosphonate analog of alanine suggesting the enzyme acts late in the biosynthesis of dehydrophos. These conclusions are corroborated by the X-ray structure that reveals an active site that can accommodate a tripeptide substrate. Furthermore, the structural studies demonstrate a novel conformational change brought about by substrate or product binding. Interestingly, the enzyme has low substrate specificity and was used to methylate the clinical antibiotic fosfomycin and the clinical anti-malaria candidate fosmidomycin, showing its promise for applications in bioengineering. Overall, the findings in this thesis provide valuable insights in Nature’s strategy for making novel phosphonates which can be further developed into applications in novel drug discovery.
Issue Date:2010-08-31
Rights Information:2010 by Jin Hee Lee
Date Available in IDEALS:2010-08-31
Date Deposited:2010-08

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