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|Title: ||Investigating the Biosynthesis of Thio-Quinolobactin and the Development of a Proteomics Probe for Thiamin Utilizing Enzymes|
|Authors: ||Godert, Amy|
|Issue Date: ||19-Jul-2006|
|Abstract: ||Quinolobactin, 8-hydroxy-4-methoxy-quinaldic acid, is a siderophore produced by P. fluorescens ATCC 17400. A tryptophan catabolite, quinolobactin is isolated as the thio-carboxylate, referred to as thio-quinolobactin. The biosynthesis of thio-quinolobactin appears to combine two pathways that have been studied previously in the Begley laboratory. The first half of the pathway catabolizes tryptophan to 3-hydroxykynurenine and appears to require similar enzymes to those found in the biosynthesis of nicotinamide. The latter half of the pathway incorporates sulfur into the quinolobactin molecule as a thio-carboxylate. The enzymes responsible for the sulfur transfer are highly similar to those found in sulfur transfer in thiamin in B. subtilis and cysteine in M. tuberculosis.
We have identified activities for the proteins in the pathway that are responsible for the transformation of 3-hydroxykynurenine, the point at which this pathway diverges from that of nicotinamide biosynthesis, to quinolobactin. QbsB, a hydroxykynurenine aminotransferase, is responsible for the deamination and cyclization of 3-hydroxykynurenine to xanthurenic acid. QbsL then methylates the 4-hydroxy position of xanthurenic acid, and also activates the carboxylate as the acyl adenylate. Although we have not characterized sulfur transfer to quinolobactin to form thio-quinolobactin, we have identified roles for the putative sulfur transfer proteins QbsC, QbsD, and QbsE. QbsE is a small sulfur carrier protein that likely delivers sulfur to quinolobactin. Before the C-terminus of QbsE is activated as the thiocarboxylate, the two amino acids at the C-terminus following the diglycine are hydrolyzed by QbsD. This diglycine C-terminus can be adenylated and sulfurylated by QbsC, forming the thio-carboxylate.
In another project, we have synthesized and tested thiamin analogs incorporating a photo-labile substituent as proteomics probes. Thiamin pyrophosphate is an essential cofactor utilized by proteins in key prokaryotic and eukaryotic metabolic pathways. The ability to observe and compare relative amounts of these proteins when cells are grown under different conditions gives valuable insight into how cells respond to stressors. A promising probe was successfully synthesized and did exhibit the desirable properties of photo-lability and inhibition of thiamin pyrophosphate utilizing enzymes. Unfortunately, a high amount of non-specific labeling prevented it from being a useful proteome probe.|
|Appears in Collections:||Theses and Dissertations (OPEN)|
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