Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)
Abstract
Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. In this paper, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlightmore »
- Authors:
-
- Univ. of Illinois, Chicago, IL (United States)
- Publication Date:
- Research Org.:
- Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Org.:
- USDOE Office of Science (SC); Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor; National Institute of General Medical Sciences (NIGMS)
- OSTI Identifier:
- 1329433
- Alternate Identifier(s):
- OSTI ID: 1398019
- Grant/Contract Number:
- AC02-06CH11357; 085P1000817
- Resource Type:
- Accepted Manuscript
- Journal Name:
- Bioorganic and Medicinal Chemistry
- Additional Journal Information:
- Journal Volume: 24; Journal Issue: 19; Journal ID: ISSN 0968-0896
- Publisher:
- Elsevier
- Country of Publication:
- United States
- Language:
- ENGLISH
- Subject:
- 60 APPLIED LIFE SCIENCES; Dihydroorotase (DHOase); Bacillus anthracis; High-throughput screening (HTS); Surface plasmon resonance (SPR)
Citation Formats
Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, and Johnson, Michael E. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase). United States: N. p., 2016.
Web. doi:10.1016/j.bmc.2016.07.055.
Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, & Johnson, Michael E. Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase). United States. https://doi.org/10.1016/j.bmc.2016.07.055
Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, and Johnson, Michael E. Fri .
"Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)". United States. https://doi.org/10.1016/j.bmc.2016.07.055. https://www.osti.gov/servlets/purl/1329433.
@article{osti_1329433,
title = {Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)},
author = {Rice, Amy J. and Lei, Hao and Santarsiero, Bernard D. and Lee, Hyun and Johnson, Michael E.},
abstractNote = {Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. In this paper, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (KD) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.},
doi = {10.1016/j.bmc.2016.07.055},
journal = {Bioorganic and Medicinal Chemistry},
number = 19,
volume = 24,
place = {United States},
year = {Fri Jul 29 00:00:00 EDT 2016},
month = {Fri Jul 29 00:00:00 EDT 2016}
}
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Works referencing / citing this record:
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