Liu, Yiquan (2015-12). Development of Chemical Strategies for Specifically Probing and Identifying Sulfur Carrier Proteins and Vitamin B6-Dependent Enzymes in Bacteria. Doctoral Dissertation. Thesis uri icon

abstract

  • Activity based protein profiling (ABPP) is a functional proteomic technology that uses chemical probes to detect mechanistically related classes of enzymes. Chemically probing a certain class of proteins helps to understand their biological function as a group, and discover new biosynthetic pathways for drug design. This research describes two activity based proteomic methods that have been developed to probe and identify sulfur carrier proteins and vitamin B6 dependent proteins, respectively. Sulfur carrier proteins are small proteins (<10 kDa) involved in pathways for efficient sulfur delivery. A chemical probe with sulfonyl-azide functional group was designed to label and identify the sulfur carrier proteins through a thioacid-azide reaction. This method identified a new sulfur carrier protein in Streptomyces coelicolor. Further study of its biological function led to the discovery and characterization of a new pathway of homocysteine formation, which is probably another direct sulfurylation of methionine biosynthesis. Vitamin B6 dependent proteins are a class of enzymes that cover a wide range of cellular functions such as transamination, racemization, and decarboxylation. Also, vitamin B6 dependent proteins have a critical role in human disease and the metabolic pathways of pathogens and plants. We used Escherichia coli as a model system to develop both radioactive and nonradioactive based methods to probe and identify vitamin B6 containing proteins in the bacterial proteome. This technique was then used to study how vitamin B6 proteins are regulated in response to cellular stress.
  • Activity based protein profiling (ABPP) is a functional proteomic technology that uses chemical probes to detect mechanistically related classes of enzymes. Chemically probing a certain class of proteins helps to understand their biological function as a group, and discover new biosynthetic pathways for drug design.
    This research describes two activity based proteomic methods that have been developed to probe and identify sulfur carrier proteins and vitamin B6 dependent proteins, respectively. Sulfur carrier proteins are small proteins (<10 kDa) involved in pathways for efficient sulfur delivery. A chemical probe with sulfonyl-azide functional group was designed to label and identify the sulfur carrier proteins through a thioacid-azide reaction. This method identified a new sulfur carrier protein in Streptomyces coelicolor. Further study of its biological function led to the discovery and characterization of a new pathway of homocysteine formation, which is probably another direct sulfurylation of methionine biosynthesis.
    Vitamin B6 dependent proteins are a class of enzymes that cover a wide range of cellular functions such as transamination, racemization, and decarboxylation. Also, vitamin B6 dependent proteins have a critical role in human disease and the metabolic pathways of pathogens and plants. We used Escherichia coli as a model system to develop both radioactive and nonradioactive based methods to probe and identify vitamin B6 containing proteins in the bacterial proteome. This technique was then used to study how vitamin B6 proteins are regulated in response to cellular stress.

publication date

  • December 2015