Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)
- Univ. of Illinois, Chicago, IL (United States)
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.
- Research Organization:
- Argonne National Laboratory (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
- Sponsoring Organization:
- USDOE Office of Science (SC); Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor; National Institute of General Medical Sciences (NIGMS)
- Grant/Contract Number:
- AC02-06CH11357; 085P1000817
- OSTI ID:
- 1329433
- Alternate ID(s):
- OSTI ID: 1398019
- Journal Information:
- Bioorganic and Medicinal Chemistry, Vol. 24, Issue 19; ISSN 0968-0896
- Publisher:
- ElsevierCopyright Statement
- Country of Publication:
- United States
- Language:
- ENGLISH
Web of Science
Characterization of the catalytic flexible loop in the dihydroorotase domain of the human multi-enzymatic protein CAD
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journal | December 2018 |
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