Biochemical effects of the carbamylation post-translational modification on three distinct Arabidopsis thaliana proteins: Ubiquitin, Nucleoside diphosphate kinase 1 and Rubisco

Abstract

As atmospheric CO2 rises at an unprecedented rate, understanding the biochemical interactions between autotrophic plants and CO2 has never been more important. The carbamylation post-translational modification is a potentially wide-spread CO2-signalling mechanism, in which CO2 interacts directly with privileged neutral amino groups on proteins, to induce biochemical changes. This thesis is focused on the physiologically relevant carbamylation of three Arabidopsis thaliana proteins: ubiquitin, nucleoside diphosphate kinase 1 (NDK1) and Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). These enzymes are functionally distinct but carbamylated residues were identified on all three proteins by triethyloxonium-mediated carbamate trapping and 13C NMR. Important sites included the K6 and K48 residues of ubiquitin, the K9 residue of AtNDK1, and the K183 residue of Rubisco. Ubiquitination determines the fate of proteins via a complex multi-enzyme mechanism. A. thaliana ubiquitin conjugating enzyme 5 (AtUBC5)-catalysed diubiquitin formation and cognate AtUBC5-ubiquitin conjugate formation were both upregulated in vitro by increasing near-physiological concentrations of CO2. The former was ubiquitin K6-dependent suggesting it might be driven by ubiquitin carbamylation, while the latter provided a potential mechanism. The implications of ubiquitin’s CO2-sensitivity are far-reaching due to the essential nature of ubiquitin signalling. NDKs are responsible for the homeostasis of nucleotide pools, in turn regulating many cellular processes. AtNDK1 kinase activity was insensitive to carbamate formation at K9, a mechanistically and structurally essential active site residue. K9-carbamylation is conserved across orthologous NDKs and might represent a novel example of how substrate binding can render carbamylation non-functional to canonical enzyme activity. AtNDK1 K9 was sensitive to acetylation, and intriguingly, this process was upregulated in a K9-dependent, Ci-mediated fashion. Finally, in vitro analyses of Rubisco’s carboxylation activity indicated an important interface residue (K183), susceptible to transient carbamylation, increased the affinity of the enzyme for CO2. Biochemical effects of transient carbamylation appear to be protein-specific and diverse, and the elucidation of further plant protein-CO2 interactions could be of significant agricultural value.