Unique subunit packing in mycobacterial nanoRNase leads to alternate substrate recognitions in DHH phosphodiesterases

Abstract

DHH superfamily includes RecJ, nanoRNases (NrnA), cyclic nucleotide phosphodiesterases and pyrophosphatases. In this study, we have carried out in vitro and in vivo investigations on the bifunctional NrnA-homolog from Mycobacterium smegmatis, MSMEG_2630. The crystal structure of MSMEG_2630 was determined to 2.2-Å resolution and reveals a dimer consisting of two identical subunits with each subunit folding into an N-terminal DHH domain and a C-terminal DHHA1 domain. The overall structure and fold of the individual domains is similar to other members of DHH superfamily. However, MSMEG_2630 exhibits a distinct quaternary structure in contrast to other DHH phosphodiesterases. This novel mode of subunit packing and variations in the linker region that enlarge the domain interface are responsible for alternate recognitions of substrates in the bifunctional nanoRNases. MSMEG_2630 exhibits bifunctional 3′-5′ exonuclease [on both deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) substrates] as well as CysQ-like phosphatase activity (on pAp) in vitro with a preference for nanoRNA substrates over single-stranded DNA of equivalent lengths. A transposon disruption of MSMEG_2630 in M. smegmatis causes growth impairment in the presence of various DNA-damaging agents. Further phylogenetic analysis and genome organization reveals clustering of bacterial nanoRNases into two distinct subfamilies with possible role in transcriptional and translational events during stress.

Regulation of the CRISPR-Associated Genes by Rv2837c (CnpB) via an Orn-Like Activity in Tuberculosis Complex Mycobacteria

“…The CRISPR-Cas systems in TB complex mycobacteria have been recognized based on sequence analyses (16,17) but have not been investigated experimentally. DHH superfamily proteins include RecJ, nanoRNases (NrnA), c-di-AMP phosphodiesterases, and pyrophosphatases (28). According to our current knowledge, M. tuberculosis CnpB is a DHH superfamily protein that cleaves c-di-AMP, c-di-GMP, and nanoRNA (19,23).…”

“…For example, the end products of degradation catalyzed by RNase II and RNase R range from 1-mers to 6-mers, which vary in size with different RNases (41). M. tuberculosis CnpB and its homolog in Mycobacterium smegmatis have been shown to cleave 2-mer nanoRNAs (28). This process likely provides a source for nucleotide recycling.…”

Too much of a good thing: regulated depletion of c-di-AMP in the bacterial cytoplasm

“…A Phyre2 structural model of the GdpP DHH-DHHA1 domain shows remarkable resemblance to the NrnA crystal structures and the DhhP structure of M. smegmatis ( Fig. 2A ) [4,5]. Among these proteins, the amino-terminal DHH subunit and the carboxy-terminal DHHA1 subunit are connected by a flexible loop to form a cleft.…”

“…Nevertheless, in M. smegmatis , the catalytic efficiency (k cat /k M ) is approximately 200-fold higher for c-di-AMP than for c-di-GMP, indicating that c-di-AMP is the much preferred substrate [10]. Additionally, at least the M. tuberculosis homolog has also been characterized as an NrnA with exonuclease activity on short single stranded nucleic acids, as well as phosphatase activity on pAp [5]. …”

The DNA Repair Repertoire of Mycobacterium smegmatis FenA Includes the Incision of DNA 5′ Flaps and the Removal of 5′ Adenylylated Products of Aborted Nick Ligation

“…M. smegmatis HR in the absence of the AdnAB nucleases requires RecO and RecR (4). Whereas the M. smegmatis proteome includes a DHH protein (MSMEG_2630), it has been characterized biochemically as an oligoribonuclease with 3=-to-5= exonuclease activity (8), i.e., it is not RecJ-like. To our knowledge, there has been no report to date of a mycobacterial DNA single-strand 5=-to-3= exonuclease enzyme.…”