Robb Nicewonger

Overexpression, purification and characterization of two pyrimidine kinases involved in the biosynthesis of thiamin: 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate kinase

Abstract The overexpression, purification and characterization of 4-amino-5-hydroxymethyl-2-methylpyrimidine kinase (HMP kinase) and 4-amino-5-hydroxymethyl-2-methylpyrimidine monophosphate kinase (HMP-P kinase) are described. Surprisingly HMP-P kinase also shows HMP kinase activity. These enzymes are useful reagents for the preparation of intermediates on the thiamin biosynthetic pathway.

Identification of Modification Sites in Large Biomolecules by Stable Isotope Labeling and Tandem High Resolution Mass Spectrometry THE ACTIVE SITE NUCLEOPHILE OF THIAMINASE I

A widely used procedure for site localization of covalent protein modifications involves proteolysis, partial chromatographic separation of the resulting complex mixture, and tandem mass spectrometry (MS/MS) to identify peptides whose molecular weight (M r) has been increased appropriately by the modification. As found previously for MS of small molecules, this study shows that protein fragment identification can be greatly simplified by labeling the modification with stable isotopes. Further, the high resolution capabilities of Fourier transform MS make possible the direct identification of CH3/CD3-labeled peptides without chromatographic separation. Although separate Asp-N, Lys-C, and α-chymotrypsin digests of thiaminase I (42 kDa) yielded as many as 70 peptides, FTMS identification of the labeled peptide localized the modification site of a mechanism-based inhibitor to Arg101-Lys121, Asp90-Gly122, and Gly107-Tyr119, respectively. The measured mass difference values of the two labels agreed with that expected for CH3/CD3, 3.019 Da, with a standard deviation of 0.005 Da, providing persuasive identity verification. MS/MS fragmentation narrowed the site to Pro109-Phe118 and also caused loss of the derivative with a sulfur atom, uniquely identifying Cys113as the thiaminase I active-site nucleophile among the 379 amino acids.

Cofactor biosynthesis : a mechanistic perspective

The chemistry of the cofactors has provided a fertile area of overlap between organic chemistry and biochemistry, and the organic chemistry of the cofactors is now a thoroughly studied area. In contrast, the chemistry of cofactor biosynthesis is still relatively underdeveloped. In this review the biosynthesis of nicotinamide adenine dinucleotide, riboflavin, folate, molyb-dopterin, thiamin, biotin, lipoic acid, pantothenic acid, coenzyme A, S-adenosylmethionine, pyridoxal phosphate, ubiquinone and menaquinone in E. coli will be described with a focus on unsolved mechanistic problems.

Synthesis of the spore photoproduct

Abstract The spore photoproduct was synthesized in seven steps from dihydrothymine and 5-formyluracil using a mixed Aldol coupling as the key bond forming step.

Microwave-assisted acylation of 7-amino-5-aryl-6-cyanopyrido[2,3-d]pyrimidines.

A microwave-assisted method is described for monoacylating 7-amino-5-aryl-6-cyanopyrido[2,3-d]pyrimidines using excess acid chlorides in pyridine. A diacylated intermediate is effectively deacylated to the product amide by a macroporous-Tris resin. A small library of 17 amides was prepared to validate the method. The integration of commercial microwave technology into the ArQule chemistry platform is also discussed.