Characterization of NrnA homologs from Mycobacterium tuberculosis and Mycoplasma pneumoniae

Abstract

Processive RNases are unable to degrade efficiently very short oligonucleotides, and they are complemented by specific enzymes, nanoRNases, that assist in this process. We previously identified NrnA (YtqI) from Bacillus subtilis as a bifunctional protein with the ability to degrade nanoRNA (RNA oligos #5 nucleotides) and to dephosphorylate 39-phosphoadenosine 59-phosphate (pAp) to AMP. While the former activity is analogous to that of oligoribonuclease (Orn) from Escherichia coli, the latter corresponds to CysQ. NrnA homologs are widely present in bacterial and archaeal genomes. They are found preferably in genomes that lack Orn or CysQ homologs. Here, we characterize NrnA homologs from important human pathogens, Mpn140 from Mycoplasma pneumoniae, and Rv2837c from Mycobacterium tuberculosis. Like NrnA, these enzymes degrade nanoRNA and dephosphorylate pAp in vitro. However, they show dissimilar preferences for specific nanoRNA substrate lengths. Whereas NrnA prefers RNA 3-mers with a 10-fold higher specific activity compared to 5-mers, Rv2837c shows a preference for nanoRNA of a different length, namely, 2-mers. Mpn140 degrades Cy5-labeled nanoRNA substrates in vitro with activities varying within one order of magnitude as follows: 5-mer>4-mer>3-mer>2-mer. In agreement with these in vitro activities, both Rv2837c and Mpn140 can complement the lack of their functional counterparts in E. coli: CysQ and Orn. The NrnA homolog from Streptococcus mutans, SMU.1297, was previously shown to hydrolyze pAp and to complement an E. coli cysQ mutant. Here, we show that SMU.1297 can complement an E. coli orn À mutant, suggesting that having both pAp-phosphatase and nanoRNase activity is a common feature of NrnA homologs.

Identification of a novel nanoRNase in Bartonella

“…In addition to its activity on nanoRNA, NrnA can degrade pAp (Mechold et al, 2007). In contrast to an earlier report on the NrnA homologue of Streptococcus mutans (Zhang & Biswas, 2009), bifunctionality seems to be common among NrnA homologues (Postic et al, 2012). NanoRNases from Gammaproteobacteria and A+T-rich Firmicutes, in particular, have widely divergent structures while displaying the same activity (Fang et al, 2009).…”

New Insights into the Cyclic Di-adenosine Monophosphate (c-di-AMP) Degradation Pathway and the Requirement of the Cyclic Dinucleotide for Acid Stress Resistance in Staphylococcus aureus

“…The S. aureus Pde2 enzyme shares a high degree of sequence similarity with the B. subtilis nanoRNase A NrnA (also referred to as YtqI), M. tuberculosis Rv2837c (also referred to as MtbPDE or CnpB), and S. pneumoniae Pde2 (also referred to as PapP). The latter two enzymes have been shown to degrade c-di-AMP and pApA to AMP, but all three enzymes also have confirmed nanoRNase and pAp-phosphatase activities (24, 29, 30, 32, 33, 39, 41). Based on these findings, it seems likely that S. aureus Pde2 may also degrade pAp and function as a general nanoRNA ribonuclease in the cell.…”

“…Through recent studies it has become apparent that stand-alone DHH/DHHA1-domain enzymes can act on a variety of different nucleotide substrates ranging from nanoRNAs (RNA molecules ≤5 nucleotides), pApA, pGpG, cyclic-di-nucleotides, and pAp, which is a byproduct of sulfur assimilation and of acyl carrier protein production (39, 40). The S. aureus Pde2 enzyme shares a high degree of sequence similarity with the B. subtilis nanoRNase A NrnA (also referred to as YtqI), M. tuberculosis Rv2837c (also referred to as MtbPDE or CnpB), and S. pneumoniae Pde2 (also referred to as PapP).…”

An HD-domain phosphodiesterase mediates cooperative hydrolysis of c-di-AMP to affect bacterial growth and virulence

“…Stand-alone DHH-DHHA1 domain proteins in the Spirochete Borrelia burgdorferi and the Actinobacterium Mycobacterium tuberculosis have been reported to modulate c-di-AMP nucleotide levels within these species, illustrating that GdpP homologs are not the only c-di-AMP phosphodiestereases that act via the DHH-DHHA1 domain. However, stand-alone DHH-DHHA1 proteins also hydrolyze substrates other than c-di-AMP, such as nanoRNA and pAp (39,40). Furthermore, they are widely conserved in organisms that do not synthesize c-di-AMP, including all classes of Proteobacteria and several Eukaryotes.…”