Structural and Functional Characterization of an RNase HI Domain from the Bifunctional Protein Rv2228c from Mycobacterium tuberculosis

The open reading frame Rv2228c from Mycobacterium tuberculosis is predicted to encode a protein composed of two domains, each with individual functions, annotated through sequence similarity searches. The N-terminal domain is homologous with prokaryotic and eukaryotic RNase H domains and the C-terminal domain with α-ribazole phosphatase (CobC). The N-terminal domain of Rv2228c (Rv2228c/N) and the full-length protein were expressed as fusions with maltose binding protein (MBP). Rv2228c/N was shown to have RNase H activity with a hybrid RNA/DNA substrate as well as double-stranded RNase activity. The full-length protein was shown to have additional CobC activity. The crystal structure of the MBP-Rv2228c/N fusion protein was solved by molecular replacement and refined at 2.25-Å resolution (R = 0.182; R_free = 0.238). The protein is monomeric in solution but associates in the crystal to form a dimer. The Rv2228c/N domain has the classic RNase H fold and catalytic machinery but lacks several surface features that play important roles in the cleavage of RNA/DNA hybrids by other RNases H. The absence of either the basic protrusion of some RNases H or the hybrid binding domain of others appears to be compensated by the C-terminal CobC domain in full-length Rv2228c. The double-stranded-RNase activity of Rv2228c/N contrasts with classical RNases H and is attributed to the absence in Rv2228c/N of a key phosphate binding pocket.The bacterium Mycobacterium tuberculosis is the causative agent of the disease tuberculosis (TB), which kills 2 million to 3 million people worldwide every year. One-third of the world''s population has latent infection, and 10% of these will develop the active form of the disease. The evolution of multidrug-resistant strains and the increase in HIV-related immunocompromisation have led to serious reemergence of the disease. The sequencing and annotation of the M. tuberculosis genome (9) have enabled a fuller evaluation of the biology of this important human pathogen and the identification of new potential targets for anti-TB drug discovery, although annotations are potentially compromised by the absence of direct structural or functional data (5). Some examples of misannotations have already been noted (6, 20, 46).An area of direct relevance to the emergence of drug-resistant strains of M. tuberculosis is that of DNA replication and repair (3). Although many genes homologous to the DNA repair machinery of other organisms can be recognized, some apparent absences have been noted (29). Here, we focus on an unusual gene product, Rv2228c, which is annotated as a bifunctional, two-domain protein, comprising an N-terminal RNase H domain and a C-terminal domain homologous with α-ribazole phosphatase (CobC), presumed to act in vitamin B₁₂ biosynthesis.The RNases H are a family of endonucleases that specifically degrade the RNA of RNA/DNA hybrids (43). These enzymes are found in eukaryotes, bacteria, archaea, and retroviruses, where they have essential roles in DNA replication and repair (11, 17, 19, 22, 32). They are highly variable in size, sequence, and specificity, making classification difficult. Most commonly, they are divided into two classes: type 1 and type 2. The classical type 1 RNase H enzymes are encoded by the rnhA gene and are typically less than 20 kDa in size, although N-terminal and C-terminal extensions frequently provide additional domains that modulate function (8, 44). Eukaryotic RNase HI enzymes, for example, have N-terminal hybrid binding domains that precede the C-terminal catalytic domain (7). The type 2 RNase H enzymes, encoded by the rnhB or rnhC gene, are typically larger and more diverse in sequence but nevertheless have in common a similar RNase H catalytic domain (7).The M. tuberculosis genome contains no classical rnhA gene, although one rnhB gene, encoding Rv2902c, is present. BLAST searches do, however, identify the N-terminal domain of the open reading frame Rv2228c (Rv2228c/N) as having 31% sequence identity with RNase HI from Escherichia coli (EcRNaseH) and 23% identity with human RNase HI (HsRnaseH). This leads to the hypothesis that this domain provides the essential RNase HI activity in M. tuberculosis. The C-terminal domain of Rv2228c presents a puzzle, however. It has 34% sequence identity with the α-ribazole phosphatase CobC of Synechococcus sp., but it is also homologous with PhoE from Bacillus subtilis (34% identity) and Rv3214 from M. tuberculosis (28% identity), both of which have acid phosphatase activity (39, 46). Bifunctional proteins similar to Rv2228c are encoded by the genomes of other Actinomycetales bacteria, including those of the Mycobacterium, Streptomyces, Corynebacterium, and Nocardia genera, and one of these bifunctional proteins, SCO2299 from Streptomyces coelicolor, has RNase HI activity in its N-terminal domain and acid phosphatase activity in its C-terminal domain (34).We undertook the structural and functional characterization of Rv2228c/N in order to establish the function of this domain and the possible significance of its associated C-terminal domain. The crystal structure of Rv2228c/N, determined at 2.25-Å resolution as a maltose binding protein (MBP) fusion protein, reveals a classic RNase H fold, but with structural and functional characteristics that make it most like the archaeal RNase H from Sulfolobus tokodaii and differentiate it from classical RNases H. Functional studies confirm the RNase H activity of Rv2228c/N and show that the C-terminal domain has both acid phosphatase and CobC activity, together with a role in enhancing the RNase H activity of the N-terminal domain.