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Crystal structure, steady-state and pre-steady-state kinetics of Acinetobacter baumannii ATP phosphoribosyltransferase
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dc.contributor.author | Read, Benjamin | |
dc.contributor.author | Cadzow, Andrew | |
dc.contributor.author | Alphey, Magnus Stephen | |
dc.contributor.author | Mitchell, John B. O. | |
dc.contributor.author | da Silva, R.G. | |
dc.date.accessioned | 2024-01-23T11:30:01Z | |
dc.date.available | 2024-01-23T11:30:01Z | |
dc.date.issued | 2024-01-16 | |
dc.identifier | 297365641 | |
dc.identifier | 91346c02-6a92-438f-8128-0ac44a100df0 | |
dc.identifier | 85181565746 | |
dc.identifier.citation | Read , B , Cadzow , A , Alphey , M S , Mitchell , J B O & da Silva , R G 2024 , ' Crystal structure, steady-state and pre-steady-state kinetics of Acinetobacter baumannii ATP phosphoribosyltransferase ' , Biochemistry , vol. 63 , no. 2 , pp. 230-240 . https://doi.org/10.1021/acs.biochem.3c00551 | en |
dc.identifier.issn | 0006-2960 | |
dc.identifier.other | ORCID: /0000-0002-1308-8190/work/151191212 | |
dc.identifier.other | ORCID: /0000-0002-0379-6097/work/151191220 | |
dc.identifier.other | ORCID: /0000-0002-9353-3716/work/151191351 | |
dc.identifier.uri | https://hdl.handle.net/10023/29060 | |
dc.description | Funding: This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (grant BB/M010996/1) via an EASTBIO Doctoral Training Partnership studentship to B.J.R. | en |
dc.description.abstract | The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N1-(5-phospho-β-d-ribosyl)-ATP (PRATP) and pyrophosphate, is catalyzed by the hetero-octameric enzyme ATP phosphoribosyltransferase, a promising target for antibiotic design. The catalytic subunit, HisGS, is allosterically activated upon binding of the regulatory subunit, HisZ, to form the hetero-octameric holoenzyme (ATPPRT), leading to a large increase in kcat. Here, we present the crystal structure of ATPPRT, along with kinetic investigations of the rate-limiting steps governing catalysis in the nonactivated (HisGS) and activated (ATPPRT) forms of the enzyme. A pH-rate profile showed that maximum catalysis is achieved above pH 8.0. Surprisingly, at 25 °C, kcat is higher when ADP replaces ATP as substrate for ATPPRT but not for HisGS. The HisGS-catalyzed reaction is limited by the chemical step, as suggested by the enhancement of kcat when Mg2+ was replaced by Mn2+, and by the lack of a pre-steady-state burst of product formation. Conversely, the ATPPRT-catalyzed reaction rate is determined by PRATP diffusion from the active site, as gleaned from a substantial solvent viscosity effect. A burst of product formation could be inferred from pre-steady-state kinetics, but the first turnover was too fast to be directly observed. Lowering the temperature to 5 °C allowed observation of the PRATP formation burst by ATPPRT. At this temperature, the single-turnover rate constant was significantly higher than kcat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step. | |
dc.format.extent | 11 | |
dc.format.extent | 4198230 | |
dc.language.iso | eng | |
dc.relation.ispartof | Biochemistry | en |
dc.subject | QR Microbiology | en |
dc.subject | DAS | en |
dc.subject | MCC | en |
dc.subject.lcc | QR | en |
dc.title | Crystal structure, steady-state and pre-steady-state kinetics of Acinetobacter baumannii ATP phosphoribosyltransferase | en |
dc.type | Journal article | en |
dc.contributor.institution | University of St Andrews. Biomedical Sciences Research Complex | en |
dc.contributor.institution | University of St Andrews. School of Biology | en |
dc.contributor.institution | University of St Andrews. EaSTCHEM | en |
dc.contributor.institution | University of St Andrews. School of Chemistry | en |
dc.contributor.institution | University of St Andrews. Institute of Behavioural and Neural Sciences | en |
dc.identifier.doi | https://doi.org/10.1021/acs.biochem.3c00551 | |
dc.description.status | Peer reviewed | en |
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