Abstract
In Staphylococcus aureus, the branched-chain amino acid biosynthetic pathway provides essential intermediates for membrane biosynthesis. Threonine deaminase (IlvA) is the first enzyme in the pathway, and isoleucine feedback regulates the enzyme in Escherichia coli. These studies on E. coli IlvA (EcIlvA) introduced the concept of allosteric regulation. To investigate the regulation of S. aureus IlvA (SaIlvA), we first conducted additional studies on EcIlvA. The previously determined crystal structure of EcIlvA revealed a tetrameric assembly of protomers, each with catalytic and regulatory domains, but the structural basis of isoleucine regulation was not characterized. Here, we present the crystal structure of the EcIlvA regulatory domain bound to isoleucine, which reveals the isoleucine binding site and conformational changes that initiate at Phe352 and propagate 23 Å across the domain. This suggests an allosteric pathway that extends to the active site of the adjacent protomer, mediating regulation across the protomer-protomer interface. The EcIlvA(F352A) mutant binds isoleucine but is feedback-resistant due to the absence of the initiating Phe352. In contrast, SaIlvA is not feedback-regulated by isoleucine and does not bind it. The structure of the SaIlvA regulatory domain reveals a different organization that lacks the isoleucine binding site. Other potential allosteric inhibitors of SaIlvA, including phospholipid intermediates, do not affect enzyme activity. We propose that the absence of feedback inhibition in SaIlvA is due to its role in membrane biosynthesis. These findings enhance our understanding of IlvA’s allosteric regulation and offer opportunities for engineering feedback-resistant IlvA variants for biotechnological use.
Original language | English |
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Pages (from-to) | 2793-2810 |
Number of pages | 18 |
Journal | Biochemistry |
Volume | 64 |
Issue number | 13 |
DOIs | |
State | Published - Jul 1 2025 |
Bibliographical note
Publisher Copyright:© 2025 The Authors.
Funding
We thank the St. Jude Children’s Research Hospital (SJCRH) Biomolecular X-ray Crystallography Center for support with X-ray data collection, Dr. Amanda Nourse for the Sedimentation Velocity-Analytical Ultracentrifugation (SV-AUC) analysis, and the SJCRH Hartwell Center DNA Sequencing Shared Resource for DNA sequencing. We particularly thank Dr. Stephen W. White for thoughtful suggestions and valuable input in developing this manuscript.The X-ray data were collected at three shared resources. (1) The Advanced Photon Source (APS) is a U.S. Department of Energy (DOE) Office of Science user facility operated for the DOE Office of Science by Argonne National Laboratory under Contract No. DE-AC02-06CH11357. Diffraction data were collected at the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline. SER-CAT is supported by its member institutions ( https://www3.ser.aps.anl.gov/contact-us#TITLE_SER_CAT_Memberships ), equipment grants (S10_RR25528, S10_RR028976 and S10_OD027000) from the National Institutes of Health, and funding from the Georgia Research Alliance. SAXS data were collected at BioCAT (18-ID beamline), which is supported by grant P30 GM138395 from the National Institute of General Medical Sciences of the National Institutes of Health. (2) Diffraction data were collected at the Center for BioMolecular Structure (CBMS) at the National Synchrotron Light Source II at Brookhaven National Laboratory and is primarily supported by the National Institutes of Health, National Institute of General Medical Sciences (NIGMS) through a Center Core P30 Grant (P30GM133893), and by the DOE Office of Biological and Environmental Research (KP1607011). The National Synchrotron Light Source II is operated under Contract No. DE-SC0012704 for the U.S. Department of Energy (DOE), Office of Science. (3) Diffraction data were collected at beamline 5.0.2 of the Advanced Light Source, a DOE Office of Science User Facility at Lawrence Berkeley Laboratory under Contract No. DE-AC02-05CH11231, and is supported in part by the ALS-ENABLE program funded by the National Institutes of Health, National Institute of General Medical Sciences, grant P30 GM124169-01. This work was supported by National Institutes of Health grants AI166116 (C.D.R.), GM034496 (C.O.R.), Cancer Center Support Grant CA21765, and ALSAC, St. Jude Children’s Research Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funders | Funder number |
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Office of Science Programs | |
U.S. Department of Energy Oak Ridge National Laboratory U.S. Department of Energy National Science Foundation National Energy Research Scientific Computing Center | |
US DOE Office of Science | |
TUNL DOE | |
St. Jude Children's Research Hospital | |
American Lebanese Syrian Associated Charities | |
National Institute of General Medical Sciences DP2GM119177 Sophie Dumont National Institute of General Medical Sciences | P30GM133893 |
DOE Office of Biological and Environmental Research | KP1607011, DE-AC02-05CH11231, P30 GM124169-01, DE-SC0012704 |
Markey Cancer Center's Cancer Center Support | CA21765 |
Georgia Research Alliance | P30 GM138395 |
National Institutes of Health (NIH) | GM034496, AI166116 |
Argonne National Laboratory | S10_RR028976, S10_RR25528, DE-AC02-06CH11357, S10_OD027000 |
ASJC Scopus subject areas
- Biochemistry