Sulfur metabolic pathways are essential for the virulence and survival of human pathogens. In microbial cysteine biosynthesis, sulfonucleotide reductases (SRs) catalyze the reduction of 5'-phosphosulfoadenosine (APS) or 3'-phospho-5'phosphosulfoadenosine (PAPS) to sulfite using reducing equivalents from a protein cofactor, thioredoxin (Trx). In later stages, sulfite is further reduced to sulfide, which is used for the production of essential sulfur-containing metabolites including cysteine, methionine, coenzymes, iron-sulfur clusters and antioxidants. SRs are excellent new targets for antibiotic development because of their critical role in bacterial survival and the lack of analogous enzymes in humans. This class of enzymes is particularly intriguing due to the nature of the chemical reaction they catalyze. In addition, our preliminary results suggest that a highly unusual iron-sulfur cluster in APS reductase may play an important catalytic role. However, many fundamental questions about their mechanism and structure remain unknown. Because the chemistry and biology of bacterial SRs is not well understood, scientists have not been able to explore the potential of these enzymes as anti-infective targets. To this end, the broad goal of this project is directed towards obtaining detailed mechanistic and structural information on bacterial SRs, and on identifying small molecule inhibitors of SRs. The proposed research has three Specific Aims: (1) To elucidate the function of the [4Fe-4S] cluster in APS reductase, (2) To investigate large-scale conformational dynamics in the SR catalytic cycle, and (3) To discover SR inhibitors using library screening and virtual docking approaches. This work may lead to the development of antibiotics that can be used to combat drug-resistant bacteria, which would have a major impact on human health. Furthermore, we anticipate that these experiments will lead to important new fundamental insights into the (bio)chemistry of protein-associated iron-sulfur clusters and bacterial sulfur metabolism.

Public Health Relevance

Bacterial must assimilate sulfate from their environment in order to survive and initiate human infections. The discovery of methods to block this process could have a profound impact of public health. There is an urgent, global need for new antimicrobial therapies;the ability to interfere with bacterial virulence by intercepting sulfate metabolism represents a completely new therapeutic approach and is clinically timely.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Macromolecular Structure and Function A Study Section (MSFA)
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Anderson, Vernon
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Scripps Florida
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Paritala, Hanumantharao; Palde, Prakash B; Carroll, Kate S (2016) Functional Site Discovery in a Sulfur Metabolism Enzyme by Using Directed Evolution. Chembiochem 17:1873-1878
Palde, Prakash B; Bhaskar, Ashima; PedrĂ³ Rosa, Laura E et al. (2016) First-in-Class Inhibitors of Sulfur Metabolism with Bactericidal Activity against Non-Replicating M. tuberculosis. ACS Chem Biol 11:172-84
Palde, Prakash B; Carroll, Kate S (2015) A universal entropy-driven mechanism for thioredoxin-target recognition. Proc Natl Acad Sci U S A 112:7960-5
Paritala, Hanumantharao; Suzuki, Yuta; Carroll, Kate S (2015) Design, synthesis and evaluation of Fe-S targeted adenosine 5'-phosphosulfate reductase inhibitors. Nucleosides Nucleotides Nucleic Acids 34:199-220
Bhaskar, Ashima; Chawla, Manbeena; Mehta, Mansi et al. (2014) Reengineering redox sensitive GFP to measure mycothiol redox potential of Mycobacterium tuberculosis during infection. PLoS Pathog 10:e1003902
Paritala, Hanumantharao; Carroll, Kate S (2013) New targets and inhibitors of mycobacterial sulfur metabolism. Infect Disord Drug Targets 13:85-115
Paritala, Hanumantharao; Suzuki, Yuta; Carroll, Kate S (2013) Efficient microwave-assisted solid phase coupling of nucleosides, small library generation and mild conditions for release of nucleoside derivatives. Tetrahedron Lett 54:1869-1872
Paritala, Hanumantharao; Carroll, Kate S (2013) A continuous spectrophotometric assay for adenosine 5'-phosphosulfate reductase activity with sulfite-selective probes. Anal Biochem 440:32-9
Bhave, Devayani P; Hong, Jiyoung A; Keller, Rebecca L et al. (2012) Iron-sulfur cluster engineering provides insight into the evolution of substrate specificity among sulfonucleotide reductases. ACS Chem Biol 7:306-15
Holsclaw, Cynthia M; Muse 3rd, Wilson B; Carroll, Kate S et al. (2011) Mass Spectrometric Analysis of Mycothiol levels in Wild-Type and Mycothiol Disulfide Reductase Mutant Mycobacterium smegmatis. Int J Mass Spectrom 305:151-156

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