Activity and substrate specificity of lytic polysaccharide monooxygenases: An ATR FTIR-based sensitive assay tested on a novel species from Pseudomonas putida.

Ilenia Serra,Marco Sola,Paul H Walton,Carlo Augusto Bortolotti,Marzia Bellei,Daniele Piccinini,Alessandro Paradisi,Gianantonio Battistuzzi,Giulia Di Rocco,Luisa Ciano

doi:10.1002/pro.4255

Abstract

Pseudomonas putida W619 is a soil Gram-negative bacterium commonly used in environmental studies thanks to its ability in degrading many aromatic compounds. Its genome contains several putative carbohydrate-active enzymes such as glycoside hydrolases and lytic polysaccharide monooxygenases (PMOs). In this study, we have heterologously produced in Escherichia coli and characterized a new enzyme belonging to the AA10 family, named PpAA10 (Uniprot: B1J2U9), which contains a chitin-binding type-4 module and showed activity toward β-chitin. The active form of the enzyme was produced in E. coli exploiting the addition of a cleavable N-terminal His tag which ensured the presence of the copper-coordinating His as the first residue. Electron paramagnetic resonance spectroscopy showed signal signatures similar to those observed for the copper-binding site of chitin-cleaving PMOs. The protein was used to develop a versatile, highly sensitive, cost-effective and easy-to-apply method to detect PMO's activity exploiting attenuated total reflection-Fourier transform infrared spectroscopy and able to easily discriminate between different substrates.

Highlights

Activity measurements rely on the identification of soluble oligosaccharides which are released following the Polysaccharide monooxygenases (PMOs) activity on the complex biomasses, such as in the case of the kinetic analysis made on a 14C-labelled substrate,[17] while other methods are based on the spectroscopic quantitation of a colored pyrocatechol–Ni2+ complex[17,20,21] or a dimer product named coerulignone.[22,23]
We describe here a simple, versatile, and sensitive assay for the assessment of PMO activity and substrate specificity using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy
We characterized a novel chitin-active PMO enzyme belonging to the AA10 family from the bacterium P. putida

Summary

| INTRODUCTION

Polysaccharide monooxygenases (PMOs) are copperdependent redox enzymes currently divided into eight families of auxiliary activity (AA) enzymes (AA9–AA11 and AA13–AA17) in the Carbohydrate-Active EnZYmes. The use of the natural substrate is fundamental to prevent protein deactivation, which has been shown to affect the results of the assay.[25,26,27] Despite the availability of numerous studies focused on enzyme production processes, sensitive, cost-effective and easy-toapply method to detect PMO activity and to discriminate between substrates has yet to be developed. In this context, we describe here a simple, versatile, and sensitive assay for the assessment of PMO activity and substrate specificity using attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. A new bacterial enzyme active on chitin, that is, the monomeric putative PMO from P. putida W619 (PpAA10 hereafter), was produced in Escherichia coli, characterized with spectroscopic and mass spectrometry techniques and used as model system in order to develop and test the above-mentioned activity assay

| RESULTS AND DISCUSSION

| CONCLUSIONS

| MATERIALS AND METHODS