An uncharacterized FMAG_01619 protein from Fusobacterium mortiferum ATCC 9817 demonstrates that some bacterial macrodomains can also act as poly-ADP-ribosylhydrolases.

Macrodomains constitute a conserved fold widely distributed that is not only able to bind ADP-ribose in its free and protein-linked forms but also can catalyse the hydrolysis of the latter. They are involved in the regulation of important cellular processes, such as signalling, differentiation, proliferation and apoptosis, and in host-virus response, and for this, they are considered as promising therapeutic targets to slow tumour progression and viral pathogenesis. Although extensive work has been carried out with them, including their classification into six distinct phylogenetically clades, little is known on bacterial macrodomains, especially if these latter are able to remove poly(ADP-ribose) polymer (PAR) from PARylated proteins, activity that only has been confirmed in human TARG1 (C6orf130) protein. To extend this limited knowledge, we demonstrate, after a comprehensive bioinformatic and phylogenetic analysis, that Fusobacterium mortiferum ATCC 9817 TARG1 (FmTARG1) is the first bacterial macrodomain shown to have high catalytic efficiency towards O-acyl-ADP-ribose, even more than hTARG1, and towards mono- and poly(ADPribosyl)ated proteins. Surprisingly, FmTARG1 gene is also inserted into a unique operonic context, only shared by the distantly related Fusobacterium perfoetens ATCC 29250 macrodomain, which include an immunity protein 51 domain, typical of bacterial polymorphic toxin systems.

Molecular cloning and expression analysis of the MTN gene during adventitious root development in IBA-induced tetraploid black locust.

5'-Methylthioadenosine (MTA) nucleosidase (MTN) plays a key role in the methionine (Met) recycling pathway of plants. Here, we report the isolation of the 1158 bp full-length, cDNA sequence encoding tetraploid black locust (Robinia pseudoacacia L.) MTN (TrbMTN), which contains an open reading frame of 810 bp that encodes a 269 amino acid protein. The amino acid sequence of TrbMTN has more than 88% sequence identity to the MTNs from other plants, with a closer phylogenetic relationship to MTNs from legumes than to MTNs from other plants. Subcellular localization analysis revealed that the TrbMTN gene localizes mainly to the cell membrane and cytoplasm of onion epidermal cells. Indole-3-butyric acid (IBA)-treated cuttings showed higher TrbMTN transcript levels than untreated control cuttings during root primordium and adventitious root formation. TrbMTN and key Met cycle genes showed differential expression in shoots, leaves, stems, and roots, with the highest expression observed in stems. IBA-treated cuttings also showed higher TrbMTN activity than control cuttings during root primordium and adventitious root formation. These results indicate that TrbMTN gene might play an important role in the regulation of IBA-induced adventitious root development in tetraploid black locust cuttings.

Structural insights into the substrate specificity and function of Escherichia coli K12 YgjK, a glucosidase belonging to the glycoside hydrolase family 63.

Proteins belonging to the glycoside hydrolase family 63 (GH63) are found in bacteria, archaea, and eukaryotes. Eukaryotic GH63 proteins are processing *-glucosidase I enzymes that hydrolyze an oligosaccharide precursor of eukaryotic N-linked glycoproteins. In contrast, the functions of the bacterial and archaeal GH63 proteins are unclear. Here we determined the crystal structure of a bacterial GH63 enzyme, Escherichia coli K12 YgjK, at 1.78 A resolution and investigated some properties of the enzyme. YgjK consists of the N-domain and the A-domain, joined by a linker region. The N-domain is composed of 18 antiparallel beta-strands and is classified as a super-beta-sandwich. The A-domain contains 16 *-helices, 12 of which form an (*/*)(6)-barrel; the remaining 4 *-helices are found in an extra structural unit that we designated as the A'-region. YgjK, a member of the glycoside hydrolase clan GH-G, shares structural similarity with glucoamylase (GH15) and chitobiose phosphorylase (GH94) [corrected] both of which belong to clan GH-L or GH-L-like [corrected] In crystal structures of YgjK in complex with glucose, mannose, and galactose, all of the glucose, mannose, and galactose units were located in the catalytic cleft. YgjK showed the highest activity for the *-1,3-glucosidic linkage of nigerose, but also hydrolyzed trehalose, kojibiose, and maltooligosaccharides from maltose to maltoheptaose, although the activities were low. These findings suggest that YgjK is a glucosidase with relaxed specificity for sugars.

Evidence for the importance of electrostatics in the function of two distinct families of ribosome inactivating toxins.

Alpha-sarcin and ricin represent two structurally and mechanistically distinct families of site-specific enzymes that block translation by irreversibly modifying the sarcin/ricin loop (SRL) of 23S-28S rRNA. alpha-Sarcin family enzymes are designated as ribotoxins and act as endonucleases. Ricin family enzymes are designated as ribosome inactivating proteins (RIP) and act as N-glycosidases. Recently, we demonstrated that basic surface residues of the ribotoxin restrictocin promote rapid and specific ribosome targeting by this endonuclease. Here, we report that three RIP: ricin A, saporin, and gypsophilin depurinate the ribosome with strong salt sensitivity and achieve unusually fast kcat/Km approximately 10(9)-10(10) M(-1) s(-1), implying that RIP share with ribotoxins a common mechanism of electrostatically facilitated ribosome targeting. Bioinformatics analysis of RIP revealed that surface charge properties correlate with the presence of the transport chain in the RIP molecule, suggesting a second role for the surface charge in RIP transport. These findings put forward surface electrostatics as an important determinant of RIP activity.

Iris bulbs express type 1 and type 2 ribosome-inactivating proteins with unusual properties.

Two closely related lectins from bulbs of the Dutch iris (Iris hollandica var. Professor Blaauw) have been isolated and cloned. Both lectins, called Iris agglutinin b and Iris agglutinin r, possess N-glycosidase activity and share a high sequence similarity with previously described type 2 ribosome-inactivating proteins (RIP). However, these lectins show only 57% to 59% sequence identity to a previously characterized type 1 RIP from iris, called IRIP. The identification of the iris lectins as type 2 RIP provides unequivocal evidence for the simultaneous occurrence of type 1 and type 2 RIP in iris bulbs and allowed a detailed comparison of type 1 and type 2 RIP from a single plant, which provides further insight into the molecular evolution of RIP. Binding studies and docking experiments revealed that the lectins exhibit binding activity not only toward Gal/N-acetylgalactosamine, but also toward mannose, demonstrating for the first time that RIP-binding sites can accommodate mannose.

Involvement of phylogenetically conserved acidic amino acid residues in catalysis by an oxidative DNA damage enzyme formamidopyrimidine glycosylase.

Formamidopyrimidine glycosylase (Fpg) is an important bacterial base excision repair enzyme, which initiates removal of damaged purines such as the highly mutagenic 8-oxoguanine. Similar to other glycosylase/AP lyases, catalysis by Fpg is known to proceed by a nucleophilic attack by an amino group (the secondary amine of its N-terminal proline) on C1' of the deoxyribose sugar at a damaged base, which results in the departure of the base from the DNA and removal of the sugar ring by beta/delta-elimination. However, in contrast to other enzymes in this class, in which acidic amino acids have been shown to be essential for glycosyl and phosphodiester bond scission, the catalytically essential acidic residues have not been documented for Fpg. Multiple sequence alignments of conserved acidic residues in all known bacterial Fpg-like proteins revealed six conserved glutamic and aspartic acid residues. Site-directed mutagenesis was used to change glutamic and aspartic acid residues to glutamines and asparagines, respectively. While the Asp to Asn mutants had no effect on the incision activity on 8-oxoguanine-containing DNA, several of the substitutions at glutamates reduced Fpg activity on the 8-oxoguanosine DNA, with the E3Q and E174Q mutants being essentially devoid of activity. The AP lyase activity of all of the glutamic acid mutants was slightly reduced as compared to the wild-type enzyme. Sodium borohydride trapping of wild-type Fpg and its E3Q and E174Q mutants on 8-oxoguanosine or AP site containing DNA correlated with the relative activity of the mutants on either of these substrates.

A single engineered point mutation in the adenine glycosylase MutY confers bifunctional glycosylase/AP lyase activity.

The E. coli adenine glycosylase MutY is a member of the base excision repair (BER) superfamily of DNA repair enzymes. MutY plays an important role in preventing mutations caused by 7, 8-dihydro-8-oxo-2'-deoxyguanosine (OG) by removing adenine from OG:A base pairs. Some enzymes of the BER superfamily catalyze a strand scission even concomitant with base removal. These bifunctional glycosylase/AP lyases bear a conserved lysine group in the active site region, which is believed to be the species performing the initial nucleophilic attack at C1' in the catalysis of base removal. Monofunctional glycosylases such as MutY are thought to perform this C1' nucleophilic displacement by a base-activated water molecule, and, indeed, the conservation of amine functionality positioning has not been observed in protein sequence alignments. Bifunctional glycosylase/AP lyase activity was successfully engineered into MutY by replacing serine 120 with lysine. MutY S120K is capable of catalyzing DNA strand scission at a rate equivalent to that of adenine excision for both G:A and OG:A mispair substrates. The extent of DNA backbone cleavage is independent of treating reaction aliquots with 0.1 M NaOH. Importantly, the replacement of the serine with lysine results in a catalytic rate that is compromised by at least 20-fold. The reduced efficiency in the glycosylase activity is also reflected in a reduced ability of S120K MutY to prevent DNA mutations in vivo. These results illustrate that the mechanisms of action of the two classes of these enzymes are quite similar, such that a single amino acid change is sufficient, in the case of MutY, to convert a monofunctional glycosylase to a bifunctional glycosylase/AP lyase.

Structure and function in the uracil-DNA glycosylase superfamily.

Deamination of cytosine to uracil is one of the major pro-mutagenic events in DNA, causing G:C-->A:T transition mutations if not repaired before replication. Repair of uracil-DNA is achieved in a base-excision pathway initiated by a uracil-DNA glycosylase (UDG) enzyme of which four families have so far been identified. Family-1 enzymes are active against uracil in ssDNA and dsDNA, and recognise uracil explicitly in an extrahelical conformation via a combination of protein and bound-water interactions. Extrahelical recognition requires an efficient process of substrate location by 'base-sampling' probably by hopping or gliding along the DNA. Family-2 enzymes are mismatch specific and explicitly recognise the widowed guanine on the complementary strand rather than the extrahelical scissile pyrimidine. This allows a broader specificity so that some Family-2 enzymes can excise uracil and 3, N(4)-ethenocytosine from mismatches with guanine. Although structures are not yet available for Family-3 (SMUG) and Family-4 enzymes, sequence analysis suggests similar overall folds, and identifies common active site motifs but with a surprising lack of conservation of catalytic residues between members of the super-family.

A novel 3-methyladenine DNA glycosylase from Helicobacter pylori defines a new class within the endonuclease III family of base excision repair glycosylases.

The cloning, purification, and characterization of MagIII, a 3-methyladenine DNA glycosylase from Helicobacter pylori, is presented in this paper. Sequence analysis of the genome of this pathogen failed to identify open reading frames potentially coding for proteins with a 3-methyladenine DNA glycosylase activity. The putative product of the HP602 open reading frame, reported as an endonuclease III, shares extensive amino acid sequence homology with some bacterial members of this family and has the canonic active site helix-hairpin-helix-GPD motif. Surprisingly, this predicted H. pylori endonuclease III encodes a 25,220-Da protein able to release 3-methyladenine, but not oxidized bases, from modified DNA. MagIII has no abasic site lyase activity and displays the substrate specificity of the 3-methyladenine-DNA glycosylase type I of Escherichia coli (Tag) because it is not able to recognize 7-methylguanine or hypoxanthine as substrates. The expression of the magIII open reading frame in null 3-methyladenine glycosylase E. coli (tag alkA) restores to this mutant partial resistance to alkylating agents. MagIII-deficient H. pylori cells show an alkylation-sensitive phenotype. H. pylori wild type cells exposed to alkylating agents present an adaptive response by inducing the expression of magIII. MagIII is thus a novel bacterial member of the endonuclease III family, which displays biochemical properties not described for any of the members of this group until now.

The alpha/beta fold uracil DNA glycosylases: a common origin with diverse fates.

BACKGROUND: Uracil DNA glycosylases (UDGs) are major repair enzymes that protect DNA from mutational damage caused by uracil incorporated as a result of a polymerase error or deamination of cytosine. Four distinct families of UDGs have been identified, which show very limited sequence similarity to each other, although two of them have been shown to possess the same structural fold. The structural and evolutionary relationships between the rest of the UDGs remain uncertain. RESULTS: Using sequence profile searches, multiple alignment analysis and protein structure comparisons, we show here that all known UDGs possess the same fold and must have evolved from a common ancestor. Although all UDGs catalyze essentially the same reaction, significant changes in the configuration of the catalytic residues were detected within their common fold, which probably results in differences in the biochemistry of these enzymes. The extreme sequence divergence of the UDGs, which is unusual for enzymes with the same principal activity, is probably due to the major role of the uracil-flipping caused by the conformational strain enacted by the enzyme on uracil-containing DNA, as compared with the catalytic action of individual polar residues. We predict two previously undetected families of UDGs and delineate a hypothetical scenario for their evolution. CONCLUSIONS: UDGs form a single protein superfamily with a distinct structural fold and a common evolutionary origin. Differences in the catalytic mechanism of the different families combined with the construction of the catalytic pocket have, however, resulted in extreme sequence divergence of these enzymes.

A new member of the endonuclease III family of DNA repair enzymes that removes methylated purines from DNA.

DNA is constantly exposed to endogenous andexogenous alkylating agents that can modify its bases,resulting in mutagenesis in the absence of DNA repair [1,2]. Alkylation damage is removed by the action of DNA glycosylases, which initiate the base excision repair pathway and protect the sequence information of the genome [3-5]. We have identified a new class of methylpurine DNA glycosylase, designated MpgII, that is a member of the endonuclease III family of DNA repair enzymes. We expressed and purified MpgII from Thermotoga maritima and found that the enzyme releases both 7-methylguanine and 3-methyladenine from DNA. We cloned the MpgII genes from T. maritima and from Aquifex aeolicus and found that both genes could restore methylmethanesulfonate (MMS) resistance to Escherichia coli alkA tagA double mutants, which are deficient in the repair of alkylated bases. Analogous genes are found in other Bacteria and Archaea and appear to be the only genes coding for methylpurine DNA glycosylase activity in these organisms. MpgII is the fifth member of the endonuclease III family of DNA repair enzymes, suggesting that the endonuclease III protein scaffold has been modified during evolution to recognize and repair a variety of DNA damage.

A new class of uracil-DNA glycosylases related to human thymine-DNA glycosylase.

Mispairs in DNA of guanine with uracil and thymine can arise as a result of deamination of cytosine and 5-methylcytosine, respectively. In humans such mispairs are removed by thymine-DNA glycosylase (TDG). By deleting the carboxy and amino termini of this enzyme we have identified a core region capable of processing G/U but not G/T mispairs. We have further identified two bacterial proteins with strong sequence homology to this core and shown that the homologue from Escherichia coli (dsUDG) can remove uracil from G/U mispairs. This enzyme is likely to act as a back-up to the highly efficient and abundant enzyme uracil-DNA glycosylase (UDG) which is found in most organisms. Pupating insects have been reported to lack UDG activity, but we have identified an enzyme similar to dsUDG in cell lines from three different insect species. These data imply the existence of a family of double-strand-specific uracil-DNA glycosylases which, although they are subservient to UDG in most organisms, may constitute the first line of defence against the mutagenic effects of cytosine deamination in insects.

DNA glycosylase enzymes induced during chemical adaptation of M. luteus.

Five peaks of DNA glycosylase activity showing a preference for MNNG alkylated DNA have been identified from extracts of adapted M. luteus. They are numerically designated as GI to GV in order of their decreasing molecular weights. The first two of these peaks have been highly purified. GI, is a constitutive heat labile protein, 35% stimulated by the presence of 50 mM NaCl, acts exclusively on 3 MeA residues in alkylated DNA, 60-70% inhibited by the presence of 2 mM free 3MeA and has been designated as 3MeA DNA glycosylase enzyme. GII, which is an inducible protein, is heat stable, 28% inhibited by the presence of 50 mM NaCl, removes 3MeA, 3MeG, 7MeA & 7MeG with different efficiency, and has been designated as 3,7 methylpurine DNA glycosylase enzyme. The rate of release of 3 methylpurines is 30 times that of 7MeG. There is no activity of either enzyme on O2-MeC, O2-MeT, O4-MeT or O6-MeG. The apparent molecular weights of GI and GII proteins are 28 Kd and 22 Kd respectively.

DNA glycosylases.

Various DNA glycosylases exist, which initiate the first step in base-excision repair. A summary of the kinetic and physical characteristics of three classes of DNA glycosylases are presented here. The first class discussed, include glycosylases which recognize alkylated DNA. Various data from enzymes derived from both prokaryotic and eukaryotic sources is discussed. The second class deals with a glycosylase that recognizes and initiates the excision of pyrimidine dimers in DNA. To date, this enzyme has only been uncovered from two sources, Micrococcus luteus and the T4 bacteriophage of E. coli. The third class consists of the most studied of the glycosylases, the uracil-DNA glycosylase enzymes. Various characteristics are presented for the uracil-DNA glycosylases derived from various sources. Recent information from our laboratory is presented implicating that herpes simplex virus may mediate a uracil-DNA glycosylase activity in productivity infected cells.