Our studies have been steadily revealing the deep evolutionary connections between systems involved in cofactor, amino acid and secondary metabolite biosynthesis, and those involved in modifications of proteins and nucleic acids. For example, the origin of several eukaryotic enzymes that add or remove a methyl group on lysines and arginines in histones and other proteins can be directly traced to bacterial pathways involved in synthesizing peptide-derived antibiotics and siderophores (Click on numbers to read various papers : [1] [2]). In a similar vein, multiple components of the peptide ligation and deubiquitination pathways in the eukaryotic ubiquitin system show evolutionary relationships to enzymes involved in diverse bacterial biosynthetic systems for cofactors (thiamine and molybdopterin), siderophores, antibiotics and the amino acid cysteine (click numbers to read papers : [3] [4] [5]). Enzymes catalyzing other major forms of peptide tagging of proteins in eukaryotes, e.g. protein polyglutamylation, polyglycination and tyrosinylation also display evolutionary connections to peptide ligases involved in diverse prokaryotic pathways for the biosynthesis of various antibiotics, the amino acid lysine and cofactors like peptidylated tetrahydrosarcinapterin (a folate-like pterin derivative) and F420 (a flavin-like molecule) (Click to access paper). The generality of this theme is further reinforced by the evolutionary links between enzymes catalyzing other forms of peptide tagging of proteins, such as pupylation and protein arginylation/leucylation, and enzymes mediating peptide-bond formation, respectively, in the synthesis of the peptide cofactor glutathione, and a variety of compounds, such as peptidoglycan and peptide-modified lipids (Click numbers to access papers: [7] [8]). Thus, the ultimate origin of numerous enzymes involved in covalent modifications of proteins and nucleic acids, particularly in eukaryotic regulatory systems, can be linked to enzymes catalyzing similar reactions in bacterial biosynthetic systems specializing in the production of cofactors, amino acids and metabolites such as antibiotics, siderophores and cell-cell communication molecules.
We now consider the links between the biosynthetic and regulatory pathways centered on the ancient and ubiquitous metabolite, nicotinamide adenine dinucleotide (NAD) or its phosphorylated derivative NADP. NAD fits particularly well into the above-discussed patterns because it is both a cofactor for numerous enzymes as well as substrate for numerous protein- and nucleic acid-modifying reactions. As a cofactor it functions as one of the central redox molecules or hydrogen-carriers in the cell for reactions catalyzed by several diverse oxidoreductases, usually of the Rossmann fold. As a substrate in protein and nucleic acid modification it supplies the ADP ribose moiety for modification of side chains of amino acids such as glutamate, glutamine, lysine, asparagine, cysteine and diphthamide (a modified histidine) and arginine and guanine in DNA. The most common superfamily of enzymes that catalyze such reactions unites the ADP ribosyltransferases (ARTs), which catalyze the transfer of a single ADP ribose moiety to a target molecule, and polyADP ribose polymerases/polyADP ribose transferases (PARPs/PARTs) that transfer multiple such moieties to form branched or straight chain ADP ribose polymers. A nucleic acid-modifying ART is the RNA 2’phosphotransferase KptA/Tpt1, a RNA-repair enzyme that transfers the 2’ phosphate, which is generated as a result of tRNA splicing and RNA ligase action, to NAD, resulting in the generation of ADP-ribose 1”-2” cyclic diphosphate (Appr>p) and release of nicotinamide. The rifamycin ART, which is related to above RNA-processing enzyme, instead inactivates the antibiotic by ADP ribosylation of a hydroxyl group on its carbon.
In recent years there has been tremendous progress in terms of structural and biochemical understanding of ARTs, PARTs, sirtuins, MACROs and several NAD biosynthesis enzymes. There have also been several efforts in terms of sequence analysis leading to the discovery of novel ART superfamily enzymes and tremendous interest in the connections between NAD metabolism and the dynamics of heterochromatin formation, especially in the context of organismal aging. Our comparative genomic and sequence analyses of NAD-utilizing and synthesizing enzymes has led to the identification of a novel enzymatic fold that appears to have supplied multiple distinct families of proteins implicated in NAD/ADP ribose metabolism in diverse contexts. Using contextual analysis we show that some of these proteins potentially act in the context of RNA repair, where NAD is used to remove 2'-3' cyclic phosphodiester linkages. Likewise, we uncover novel NAD-dependent proteins ADP-ribosylation systems involving novel ADP-ribosyltransferases. Some of these are type-II toxin-antitoxin like systems with ART and different ribosylglycohydrolase enzymes analogous to the DraG-DraT system. We present evidence that some of these TA-like systems are likely to regulate certain restriction-modification enzymes in bacteria. We also show that eukaryotic relatives of such ARTs constitute a novel family typified by NEURL4. This leads to a key prediction that ADP-ribosylation of specific proteins in conjunction with ubiquitination might be a critical step in centrosomal assembly. Other ARTs represent a novel group of bacterial polymorphic toxins deployed by contact, T6SS and T7SS/Esx. The ADP-ribosyltransferases found in these, the bacterial polymorphic toxin and host-directed toxin systems of bacteria such Waddlia also throw light on the evolution of this fold and the origin of eukaryotic polyADP-ribosyltransferases. We also infer a novel biosynthetic pathway that might be involved in the synthesis of a nicotinate-derived compound in conjunction with an asparagine synthetase and AMPylating peptide ligase. This work has also yielded some additional novel domains involved in NAD metabolism. To read the paper, click here.
