New Perspectives on Plant Adenylyl Cyclases.

Oziniel Ruzvidzo,Aloysius Wong,Chris Gehring

doi:10.3389/fmolb.2019.00136

Oziniel Ruzvidzo, Aloysius Wong + Show 1 more

Open Access

https://doi.org/10.3389/fmolb.2019.00136

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Abstract

It is increasingly clear that plant genomes encode numerous complex multidomain proteins that harbor functional adenylyl cyclase (AC) centers. These AC containing proteins have well-documented roles in development and responses to the environment. However, it is only for a few of these proteins that we are beginning to understand the intramolecular mechanisms that govern their cellular and biological functions, as detailed characterizations are biochemically and structurally challenging given that these poorly conserved AC centers typically constitute only a small fraction (<10%) of complex plant proteins. Here, we offer fresh perspectives on their seemingly cryptic activities specifically showing evidence for the presence of multiple functional AC centers in a single protein and linking their catalytic strengths to the Mg2+/Mn2+-binding amino acids. We used a previously described computational approach to identify candidate multidomain proteins from Arabidopsis thaliana that contain multiple AC centers and show, using an Arabidopsis leucine-rich repeat containing protein (TAIR ID: At3g14460; AtLRRAC1) as example, biochemical evidence for multienzymatic activities. Importantly, all AC-containing fragments of this protein can complement the AC-deficient mutant cyaA in Escherichia coli, while structural modeling coupled with molecular docking simulations supports catalytic feasibility albeit to varying degrees as determined by the frequency of suitable substrate binding poses predicted for the AC sites. This statistic correlates well with the enzymatic assays, which implied that the greatly reduced AC activities is due to the absence of the negatively charged [DE] amino acids previously assigned to cation-, in particular Mg2+/Mn2+-binding roles in ACs.

Highlights

It has been established that cyclic nucleotide monophosphates, cyclic guanosine monophosphate, and cyclic adenosine monophosphate, and their generating enzymes, guanylyl cyclases (GCs) and adenylyl cyclases (ACs), play critical roles in many diverse biological processes of living organisms ranging from prokaryotes (e.g., Escherichia coli) to the complex multicellular Homo sapiens (Moutinho et al, 2001; Newton and Smith, 2004; Schaap, 2005; Meier and Gehring, 2006; Lomovatskaya et al, 2008)
Initial efforts to identify ACs in higher plants were based on the construction of a 14-amino-acid search motif derived from annotated and experimentally tested GCs and ACs catalytic centers whereby the amino acid at position 1 forms hydrogen bonds with purine, amino acid at position 3 confers substrate specificity, and amino acid in position 14 stabilizes the transition from ATP to cyclic adenosine monophosphate (cAMP)
AC activity was only detected when the recombinant protein was raised to an unphysiologically high amount of 400 μg. This protein has a nucleotide cyclase center with the [DE] Mg2+/Mn2+-binding residue, and it has three AC centers (Figure 1A), they all lacked the [DE] amino acids which might account for the weakened AC activity

Summary

INTRODUCTION

It has been established that cyclic nucleotide monophosphates, cyclic guanosine monophosphate, and cyclic adenosine monophosphate (cAMP), and their generating enzymes, guanylyl cyclases (GCs) and adenylyl cyclases (ACs), play critical roles in many diverse biological processes of living organisms ranging from prokaryotes (e.g., Escherichia coli) to the complex multicellular Homo sapiens (Moutinho et al, 2001; Newton and Smith, 2004; Schaap, 2005; Meier and Gehring, 2006; Lomovatskaya et al, 2008). We define a “correct binding pose” as ATP orientation with its adenine head pointing toward the amino acid at position 1 and its phosphate tail pointing toward the positively charged amino acid at position 14 of the AC motif (Figure 1A inset; Supplementary Figure 2)–a definition that is consistent with that of previous works on plant ACs and GCs (Wong and Gehring, 2013b; Wong et al, 2015) This orientation of nucleotides has been deemed favorable for catalysis in previously characterized ACs and GCs (Al-Younis et al, 2015, 2018; Wheeler et al, 2017; Chatukuta et al, 2018; Bianchet et al, 2019) where, notably, mutagenesis of these residues have yielded reduced catalytic activities (Wheeler et al, 2017; Al-Younis et al, 2018). We showed for the first time that AC centers missing the [DE] residues have significantly reduced activities, and these preliminary data could explain past results, e.g., AtDGK4 by Dias et al (2019) and/or guide future characterization works

DISCUSSION AND OUTLOOK

MATERIALS AND METHODS

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