In Vitro Characterization of Guanylyl Cyclase BdPepR2 from Brachypodium distachyon Identified through a Motif-Based Approach.

Maria Duszyn,Brygida Świeżawska-Boniecka,Krzysztof Jaworski,Aloysius Wong,Adriana Szmidt-Jaworska

doi:10.3390/ijms22126243

Maria Duszyn, Brygida Świeżawska-Boniecka + Show 3 more

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https://doi.org/10.3390/ijms22126243

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Abstract

In recent years, cyclic guanosine 3′,5′-cyclic monophosphate (cGMP) and guanylyl cyclases (GCs), which catalyze the formation of cGMP, were implicated in a growing number of plant processes, including plant growth and development and the responses to various stresses. To identify novel GCs in plants, an amino acid sequence of a catalytic motif with a conserved core was designed through bioinformatic analysis. In this report, we describe the performed analyses and consider the changes caused by the introduced modification within the GC catalytic motif, which eventually led to the description of a plasma membrane receptor of peptide signaling molecules—BdPepR2 in Brachypodium distachyon. Both in vitro GC activity studies and structural and docking analyses demonstrated that the protein could act as a GC and contains a highly conserved 14-aa GC catalytic center. However, we observed that in the case of BdPepR2, this catalytic center is altered where a methionine instead of the conserved lysine or arginine residues at position 14 of the motif, conferring higher catalytic activity than arginine and alanine, as confirmed through mutagenesis studies. This leads us to propose the expansion of the GC motif to cater for the identification of GCs in monocots. Additionally, we show that BdPepR2 also has in vitro kinase activity, which is modulated by cGMP.

Highlights

Guanosine 30,50 -cyclic monophosphate is a key signaling molecule that is involved in signal transduction and controls different physiological responses and processes in numerous prokaryotic and eukaryotic cells
We investigated PepR2 from Brachypodium distachyon (BdPepR2), which belongs to the leucine-rich repeat receptor-like protein kinase family and is the orthologs of the PepR1 from A. thaliana and Hippeastrum hybridum, which were experimentally confirmed to be functional guanylyl cyclases (GCs) [12,13]
A. thaliana is a dicotyledonous plant; divergences in the amino acid sequences of these proteins may result from their separate evolutionary development

Summary

Introduction

Guanosine 30 ,50 -cyclic monophosphate (cGMP) is a key signaling molecule that is involved in signal transduction and controls different physiological responses and processes in numerous prokaryotic and eukaryotic cells. The role and importance of cGMP and GC in mammals and prokaryotes was known for a long time, while their presence in plants was questionable, as they show less activity in vitro than in animal cells [1,2]. In recent years, an increasing number of reports on proteins with GC activity have appeared, which indicates that they play important roles in signal transduction and in several physiological processes in plants [3,4]. The cGMP-dependent signaling pathway is involved in root gravitropism of Glycine max [5], phytochrome-controlled induction of flowering of Pharbitis nil [6], and regulation of Arabidopsis thaliana seed germination [7].

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