Work

A binuclear metallohydrolase model for RelA/SpoT-Homolog (RSH) hydrolases

Year: 2024

Type: article

Abstract: When challenged by starvation, bacterial organisms synthesize guanosine pentaphosphate and tetraphosphate, collectively denoted as (p)ppGpp, as second messengers to reprogram metabolism toward slower growth and enhanced stress tolerance. When starvation is alleviated, the R elA- S poT H omolog (RSH) hydrolases downregulate (p)ppGpp, cleaving the 3′-diphosphate to produce GTP or GDP. Metazoan RSH hydrolases possess phosphatase activity responsible for converting cytoplasmic NADPH to NADH in mammalian cells. Inhibitor development for this family may therefore provide therapies to combat bacterial infection or metabolic dysregulation. Despite the availability of dozens of high-resolution structures, catalytic mechanisms of RSH hydrolases have remained poorly understood. All RSH hydrolases tightly bind a Mn 2+ near its active center, which is believed sufficient for hydrolase activity. In contrast to this notion, we demonstrate, using the (p)ppGpp hydrolase SpoT from Acinetobacter baumannii , that a second divalent cation, presumably a Mg 2+ under physiological conditions, is required for efficient catalysis. We also show that SpoT preferentially cleaves 3′-diphosphate over 3′-phosphate substrates, likely due to a key coordination between the β-phosphate and the second metal center. Metazoan RSH hydrolase replaces this β-phosphate with the side chain of an aspartate residue, thereby functioning as a phosphatase. We propose a binuclear metallohydrolase model where an invariant ED (Glu-Asp) diad, previously believed to activate the water nucleophile, instead coordinates to a Mg 2+ center. The refined molecular and evolutionary blueprint of RSH hydrolases will provide a more reliable foundation for the development of small-molecule inhibitors of this important enzyme family.

Source: Journal of Biological Chemistry

DOI: 10.1016/j.jbc.2024.107841

Authors: Rich W. Zhou, Berti Manisa, Boyuan Wang

Institution: The University of Texas Southwestern Medical Center

Language: English

Topic: Biochemical and Molecular Research

Subfield: Molecular Biology

Field: Biochemistry, Genetics and Molecular Biology

Domain: Life Sciences

Open Access status: gold

Estimated APC paid: $2,800

Funders: Welch Foundation, University of Texas Southwestern Medical Center, University of Texas at Dallas, National Institute of General Medical Sciences