The crystal structure of the B subtilis Maf BSU
The crystal structure of the B. subtilis Maf (BSU28050) was published in 2000, and, to date, it represents the only experimental work with a purified Maf protein (Minasov et al., 2000). The structure revealed a structural fold similar to that found in ITPases and YjjX proteins, which are nucleotide pyrophosphatases hydrolyzing ITP, dITP, and XTP (Hwang et al., 1999, Savchenko et al., 2007, Zheng et al., 2005). Based on the subsequent analysis of BSU28050 structure, Maf proteins have been proposed to belong to a group of “house-cleaning” nucleotide hydrolyzing enzymes, which also include Nudix hydrolases, ITPases, dUTPases, and all-α nucleoside triphosphate pyrophosphatases (Bessman et al., 1996, Galperin et al., 2006, Moroz et al., 2005). The house-cleaning nucleotide pyrophosphatases hydrolyze various noncanonical nucleotides (dUTP, dITP, 8-oxo-dGTP, and 2-oxo-dATP), which can cause mispairing and mutation if incorporated into DNA. Thus, house-cleaning enzymes prevent the incorporation of noncanonical nucleotides into cellular DNA working in parallel with DNA repair proteins. The structural similarity of ITPases and B. subtilis Maf suggests that these proteins have a common evolutionary origin and that Maf proteins are likely to catalyze a similar chemical reaction; however, no nucleotide hydrolysis was demonstrated for BSU28050 (Minasov et al., 2000). The structure of BSU28050 in complex with dUTP (Protein Data Bank [PDB] code 1EXC) also revealed that the dUTP Sitafloxacin Hydrate what is located on the protein surface and makes limited contacts with the protein (Minasov et al., 2000). However, a subsequent analysis of this structure pointed to a large pocket equivalent to the base recognition site of ITPases, suggesting that Maf proteins can bind and hydrolyze the nucleotide substrates like ITPases (Galperin et al., 2006). Interest in Maf proteins was revived by a recent genetic work on the B. subtilis BSU28050, which indicated that it is involved in the cell division arrest associated with DNA transformation and repair (Briley et al., 2011). Following DNA damage or DNA transformation, cells need to inhibit cell division to allow time for DNA repair or integration of transforming DNA. In competent B. subtilis cells, cell division is blocked because the traffic ATPase ComGA inhibits the polymerization of the tubulin-like protein FtsZ (Haijema et al., 2001). In B. subtilis, maf is a competence-induced gene whose product (1) is localized near the cell poles; (2) interacts with ComGA, DivIVA, and FtsW; and (3) blocks septation during the escape from competence (when the transformed cells resume growth) (Briley et al., 2011). The traffic ATPase ComGA is essential for the binding of transforming DNA to the competent cell surface and its uptake inside the cell (Chung and Dubnau, 1998), whereas DivIVA is a scaffold protein that helps to localize other proteins (e.g., the division inhibitor MinC/MinD) to cell division sites or polar regions (Lenarcic et al., 2009). This work demonstrated that Maf is responsible for cell division inhibition in the absence of ComGA, suggesting that Maf functions as an additional (backup) system in the delay of cell division (Briley et al., 2011). What is intriguing about Maf is that this biochemically uncharacterized protein is conserved in all kingdoms of life, suggesting that it represents a general molecular mechanism of the inhibition of cell division (Hamoen, 2011). Here, we present the results of the biochemical, structural, and mutational studies of six Maf proteins from prokaryotic and eukaryotic organisms, including E. coli, Salmonella typhimurium, B. subtilis, Saccharomyces cerevisiae, and humans. We have demonstrated that the two subfamilies of Maf proteins (YhdE and YceF) have nucleoside triphosphate pyrophosphatase activity against both canonical (dTTP, UTP, and CTP) and modified (5-methyl-UTP, pseudo-UTP, 5-methyl-CTP, and 7-methyl-GTP) nucleotides. Overexpression of the E. coli Maf protein YhdE increased the intracellular concentration of dTMP and UMP. Crystal structures of the human ASMTL-Maf domain, BSU28050, and the E. coli YceF revealed their active site, which was further characterized by site-directed mutagenesis and substrate docking. We propose that nucleoside triphosphate pyrophosphatase activity of Maf proteins against canonical and noncanonical nucleotides might represent a molecular mechanism for a dual role of these proteins in cell division arrest and in preventing the incorporation of modified nucleotides into cellular nucleic acids (house cleaning).