Biosynthesis of Prodiginine and Tambjamine Natural Products in Pseudoalteromonas Sp.
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Authors
Picott, Katherine
Date
Type
thesis
Language
eng
Keyword
Biosynthesis , Natural Products , Enzymology , Biosynthetic Enzymes , Prodiginine , Tambjamine , PigC , TamQ , Pseudoalteromonas
Alternative Title
Abstract
Prodiginines and tambjamines are two classes of pigmented natural products that display structural variety and hold many useful bioactivities, making them a desirable target for pharmaceutical agents. Enzymes PigC and TamQ are responsible for the final step in prodiginine and tambjamine biosynthesis respectively, involving the coupling of a product specific intermediate to the shared intermediate 4-methoxy-2,2’-bipyrrole-5-carbaldehyde. Previous investigations have focused solely on PigC and have not successfully purified the enzyme, inciting the use of crude cell lysate to test enzyme activity. The objectives of this research were to directly compare the kinetic characteristics and substrate selectivity of PigC and TamQ. PigC was cloned from Pseudoalteromonas rubra, a known producer of several prodiginine analogues. TamQ was cloned from Pseudoalteromonas citrea, which is predicted to produce tambjamine YP1 based on genomic mining. PigC and TamQ were successfully heterologously expressed and purified, and enzymatic activities of both PigC and TamQ were reconstituted with their native and pseudo-native substrates. The true kinetic parameters of two substrates for PigC were determined, allowing for prediction of the reaction sequence and a proposed Ter Bi Ping Pong enzyme system. PigC also demonstrated some substrate flexibility by accepting a synthetic halogenated analogue in addition to its natural substrate 2-methyl-3-amyl-pyrrole (MAP). Apparent kinetic values for TamQ were obtained using a close analogue of its native substrate. Additionally, the enzyme could accept and turnover the PigC substrate MAP, though, the structure of the reaction product has yet to be determined. Three other biosynthetic enzymes involved in tambjamine YP1 (TamA, TamT, and TamH) construction were cloned from P. citrea and expressed for future functionality studies. Lastly, a novel cyclized variant of tambjamine YP1 was isolated from P. citrea. The complete structure of this compound was determined by NMR and X-ray crystallography analysis.
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Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada
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Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.
Queen's University's Thesis/Dissertation Non-Exclusive License for Deposit to QSpace and Library and Archives Canada
ProQuest PhD and Master's Theses International Dissemination Agreement
Intellectual Property Guidelines at Queen's University
Copying and Preserving Your Thesis
This publication is made available by the authority of the copyright owner solely for the purpose of private study and research and may not be copied or reproduced except as permitted by the copyright laws without written authority from the copyright owner.