# Long-Range Allosteric Communication Modulated by Active Site Mn(II) Coordination Drives Catalysis in Xanthobacter autotrophicus Acetone Carboxylase

## TL;DR

This study explores how a large enzyme uses long-range communication to coordinate chemical reactions at different sites during catalysis.

## Contribution

The study reveals a novel carboxylate shift mechanism linking local and long-range structural changes in a multi-subunit enzyme.

## Key findings

- A single carboxylic acid residue mediates both catalysis and communication across 40 Å in the enzyme complex.
- Allosteric communication is modulated by changes in Mn(II) coordination during the catalytic cycle.
- Combining structural and biophysical methods provides new insights into multi-subunit enzyme function.

## Abstract

Acetone carboxylase (AC) from Xanthobacter autotrophicus is a 360 KDa α2β2γ2 heterohexamer that catalyzes the ATP-dependent formation of phosphorylated acetone and bicarbonate intermediates that react at Mn(II) metal active sites to form acetoacetate. Structural models of X. autotrophicus AC (XaAC) with and without nucleotides reveal that the binding and phosphorylation of the two substrates occurs ~40 Å from the Mn(II) active sites where acetoacetate is formed. Based on the crystal structures, a significant conformational change was proposed to open and close a tunnel that facilitates the passage of reaction intermediates between the sites for nucleotide binding and phosphorylation of substrates and Mn(II) sites of acetoacetate formation. We have employed electron paramagnetic resonance (EPR), kinetic assays, and hydrogen/deuterium exchange mass spectrometry (HDX-MS) of poised ligand-bound states and site-specific amino acid variants to complete an in-depth analysis of Mn(II) coordination and allosteric communication throughout the catalytic cycle. In contrast with the established paradigms for carboxylation, our analyses of XaAC suggested a carboxylate shift that couples both local and long-range structural transitions. Shifts in the coordination mode of a single carboxylic acid residue (αE89) mediate both catalysis proximal to a Mn(II) center and communication with an ATP active site in a separate subunit of a 180 kDa α2β2γ2 complex at a distance of 40 Å. This work demonstrates the power of combining structural models from X-ray crystallography with solution-phase spectroscopy and biophysical techniques to elucidate functional aspects of a multi-subunit enzyme.