Crystal Structure, Steady-State, and Pre-Steady-State Kinetics of Acinetobacter baumannii ATP Phosphoribosyltransferase

• Published in: Biochemistry (Easton) 2024-01, Vol.63 (2), p.230-240
• Main Authors: Read, Benjamin J., Cadzow, Andrew F., Alphey, Magnus S., Mitchell, John B. O., da Silva, Rafael G.
• Format: Article
• Language: English
• Subjects: Acinetobacter baumannii - metabolism; Adenosine Triphosphate - metabolism; ATP Phosphoribosyltransferase - chemistry; Catalysis; Catalytic Domain; Diphosphates; Kinetics
• ISSN: 0006-2960; 1520-4995; 1520-4995
• DOI: 10.1021/acs.biochem.3c00551

The first step of histidine biosynthesis in Acinetobacter baumannii, the condensation of ATP and 5-phospho-α-d-ribosyl-1-pyrophosphate to produce N 1-(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 k cat. 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, k cat 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 k cat 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 k cat, providing additional evidence for a step after chemistry limiting catalysis by ATPPRT. This demonstrates allosteric activation by HisZ accelerates the chemical step.