Issue 13, 2024

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase

Abstract

The conversion of CO2 by enzymes such as carbonic anhydrase or carboxylases plays a crucial role in many biological processes. However, in situ methods following the microscopic details of CO2 conversion at the active site are limited. Here, we used infrared spectroscopy to study the interaction of CO2, water, bicarbonate, and other reactants with β-carbonic anhydrase from Escherichia coli (EcCA) and crotonyl-CoA carboxylase/reductase from Kitasatospora setae (KsCcr), two of the fastest CO2-converting enzymes in nature. Our data reveal that KsCcr possesses a so far unknown metal-independent CA-like activity. Site-directed mutagenesis of conserved active site residues combined with molecular dynamics simulations tracing CO2 distributions in the active site of KsCCr identify an ‘activated’ water molecule forming the hydroxyl anion that attacks CO2 and yields bicarbonate (HCO3). Computer simulations also explain why substrate binding inhibits the anhydrase activity. Altogether, we demonstrate how in situ infrared spectroscopy combined with molecular dynamics simulations provides a simple yet powerful new approach to investigate the atomistic reaction mechanisms of different enzymes with CO2.

Graphical abstract: Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase

Supplementary files

Article information

Article type
Edge Article
Submitted
11 Aug 2023
Accepted
27 Feb 2024
First published
29 Feb 2024
This article is Open Access

All publication charges for this article have been paid for by the Royal Society of Chemistry
Creative Commons BY license

Chem. Sci., 2024,15, 4960-4968

Infrared spectroscopy reveals metal-independent carbonic anhydrase activity in crotonyl-CoA carboxylase/reductase

A. Gomez, M. Tinzl, G. Stoffel, H. Westedt, H. Grubmüller, T. J. Erb, E. Vöhringer-Martinez and S. T. Stripp, Chem. Sci., 2024, 15, 4960 DOI: 10.1039/D3SC04208A

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

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