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Title: Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Authors:
; ; ; ;
Publication Date:
Sponsoring Org.:
USDOE
OSTI Identifier:
1398019
Grant/Contract Number:
AC02-06CH11357
Resource Type:
Journal Article: Publisher's Accepted Manuscript
Journal Name:
Bioorganic and Medicinal Chemistry
Additional Journal Information:
Journal Volume: 24; Journal Issue: 19; Related Information: CHORUS Timestamp: 2017-10-05 03:15:19; Journal ID: ISSN 0968-0896
Publisher:
Elsevier
Country of Publication:
United Kingdom
Language:
English

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 Kingdom: 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 Kingdom. doi:10.1016/j.bmc.2016.07.055.
Rice, Amy J., Lei, Hao, Santarsiero, Bernard D., Lee, Hyun, and Johnson, Michael E.. Sat . "Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)". United Kingdom. doi:10.1016/j.bmc.2016.07.055.
@article{osti_1398019,
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 = {},
doi = {10.1016/j.bmc.2016.07.055},
journal = {Bioorganic and Medicinal Chemistry},
number = 19,
volume = 24,
place = {United Kingdom},
year = {Sat Oct 01 00:00:00 EDT 2016},
month = {Sat Oct 01 00:00:00 EDT 2016}
}

Journal Article:
Free Publicly Available Full Text
Publisher's Version of Record at 10.1016/j.bmc.2016.07.055

Citation Metrics:
Cited by: 1work
Citation information provided by
Web of Science

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  • Anthrolysin O (ALO) is a pore-forming, cholesterol-dependent cytolysin (CDC) secreted by Bacillus anthracis, the etiologic agent for anthrax. Growing evidence suggests the involvement of ALO in anthrax pathogenesis. Here, we show that the apical application of ALO decreases the barrier function of human polarized epithelial cells as well as increases intracellular calcium and the internalization of the tight junction protein occludin. Using pharmacological agents, we also found that barrier function disruption requires increased intracellular calcium and protein degradation. We also report a crystal structure of the soluble state of ALO. Based on our analytical ultracentrifugation and light scattering studies, ALOmore » exists as a monomer. Our ALO structure provides the molecular basis as to how ALO is locked in a monomeric state, in contrast to other CDCs that undergo antiparallel dimerization or higher order oligomerization in solution. ALO has four domains and is globally similar to perfringolysin O (PFO) and intermedilysin (ILY), yet the highly conserved undecapeptide region in domain 4 (D4) adopts a completely different conformation in all three CDCs. Consistent with the differences within D4 and at the D2-D4 interface, we found that ALO D4 plays a key role in affecting the barrier function of C2BBE cells, whereas PFO domain 4 cannot substitute for this role. Novel structural elements and unique cellular functions of ALO revealed by our studies provide new insight into the molecular basis for the diverse nature of the CDC family.« less
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  • Biosynthesis of NAD(P) in bacteria occurs either de novo or through one of the salvage pathways that converge at the point where the reaction of nicotinate mononucleotide (NaMN) with ATP is coupled to the formation of nicotinate adenine dinucleotide (NaAD) and inorganic pyrophosphate. This reaction is catalyzed by nicotinate mononucleotide adenylyltransferase (NMAT), which is essential for bacterial growth, making it an attractive drug target for the development of new antibiotics. Steady-state kinetic and direct binding studies on NMAT from Bacillus anthracis suggest a random sequential Bi-Bi kinetic mechanism. Interestingly, the interactions of NaMN and ATP with NMAT were observed tomore » exhibit negative cooperativity, i.e. Hill coefficients <1.0. Negative cooperativity in binding is supported by the results of X-ray crystallographic studies. X-ray structures of the B. anthracis NMAT apoenzyme, and the NaMN- and NaAD-bound complexes were determined to resolutions of 2.50 A, 2.60 A and 1.75 A, respectively. The X-ray structure of the NMAT-NaMN complex revealed only one NaMN molecule bound in the biological dimer, supporting negative cooperativity in substrate binding. The kinetic, direct-binding, and X-ray structural studies support a model in which the binding affinity of substrates to the first monomer of NMAT is stronger than that to the second, and analysis of the three X-ray structures reveals significant conformational changes of NMAT along the enzymatic reaction coordinate. The negative cooperativity observed in B. anthracis NMAT substrate binding is a unique property that has not been observed in other prokaryotic NMAT enzymes. We propose that regulation of the NAD(P) biosynthetic pathway may occur, in part, at the reaction catalyzed by NMAT.« less
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