Probing the activity of NTHL1 orthologs by targeting conserved amino acid residues

The base excision repair DNA glycosylases, EcoNth and hNTHL1, are homologous, with reported overlapping yet different substrate specificities. The catalytic amino acid residues are known and are identical between the two enzymes although the exact structures of the substrate binding pockets remain to be determined. We sought to explore the sequence basis of substrate differences using a phylogeny-based design of site-directed mutations. Mutations were made for each enzyme in the vicinity of the active site and we examined these variants for glycosylase and lyase activity. Single turnover kinetics were done on a subgroup of these, comparing activity on two lesions, 5,6-dihydrouracil and 5,6-dihydrothymine, with different opposite bases. We report that wild type hNTHL1 and EcoNth are remarkably alike with respect to the specificity of the glycosylase reaction, and although hNTHL1 is a much slower enzyme than EcoNth, the tighter binding of hNTHL1 compensates, resulting in similar k_cat/K_d values for both enzymes with each of the substrates tested. For the hNTHL1 variant Gln287Ala, the specificity for substrates positioned opposite G is lost, but not that of substrates positioned opposite A, suggesting a discrimination role for this residue. The EcoNth Thr121 residue influences enzyme binding to DNA, as binding is significantly reduced with the Thr121Ala variant. Finally, we present evidence that hNTHL1 Asp144, unlike the analogous EcoNth residue Asp44, may be involved in resolving the glycosylase transition state.     

Functional changes in a novel uracil-DNA glycosylase determined by mutational analyses

Uracil-DNA glycosylase (UDG) is a ubiquitous enzyme found in bacteria and eukaryotes, which removes uracil residues from DNA strands. Methanococcus jannaschii UDG (MjUDG), a novel monofunctional glycosylase, contains a helix-hairpin-helix (HhH) motif and a Gly/Pro rich loop (GPD region), which is important for catalytic activity; it shares these features with other glycosylases, such as endonuclease III. First, to examine the role of two conserved amino acid residues (Asp150 and Tyr152) in the HhH-GPD region of MjUDG, mutant MjUDG proteins were constructed, in which Asp150 was replaced with either Glu or Trp (D150E and D150W), and Tyr152 was replaced with either Glu or Asn (Y152E and Y152N). Mutant D150W completely lacked DNA glycosylase activity, whereas D150E displayed reduced activity of about 70% of the wild type value. However, the mutants Y152E and Y152N retained unchanged levels of UDG activity. We also replaced Glu132 in the HhH motif with a lysine residue equivalent to Lys120 in endonuclease III. This mutation converted the enzyme into a bifunctional glycosylase/AP lyase capable of both removing uracil at a glycosylic bond and cleaving the phosphodiester backbone at an AP site. Mutant E132K catalyzes a β-elimination reaction at the AP site via uracil excision and forms a Schiff base intermediate in the form of a protein-DNA complex. This text was submitted by the authors in English.     

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.     

The catalytic mechanism of a pyrimidine dimer-specific glycosylase (pdg)/abasic lyase, Chlorella virus-pdg

The repair of UV light-induced cyclobutane pyrimidine dimers can proceed via the base excision repair pathway, in which the initial step is catalyzed by DNA glycosylase/abasic (AP) lyases. The prototypical enzyme studied for this pathway is endonuclease V from the bacteriophage T4 (T4 bacteriophage pyrimidine dimer glycosylase (T4-pdg)). The first homologue for T4-pdg has been found in a strain of Chlorella virus (strain Paramecium bursaria Chlorella virus-1), which contains a gene that predicts an amino acid sequence homology of 41% with T4-pdg. Because both the structure and critical catalytic residues are known for T4-pdg, homology modeling of the Chlorella virus pyrimidine dimer glycosylase (cv-pdg) predicted that a conserved glutamic acid residue (Glu-23) would be important for catalysis at pyrimidine dimers and abasic sites. Site-directed mutations were constructed at Glu-23 to assess the necessity of a negatively charged residue at that position (Gln-23) and the importance of the length of the negatively charged side chain (Asp-23). E23Q lost glycosylase activity completely but retained low levels of AP lyase activity. In contrast, E23D retained near wild type glycosylase and AP lyase activities on cis-syn dimers but completely lost its activity on the trans-syn II dimer, which is very efficiently cleaved by the wild type cv-pdg. As has been shown for other glyscosylases, the wild type cv-pdg catalyzes the cleavage at dimers or AP sites via formation of an imino intermediate, as evidenced by the ability of the enzyme to be covalently trapped on substrate DNA when the reactions are carried out in the presence of a strong reducing agent; in contrast, E23D was very poorly trapped on cis-syn dimers but was readily trapped on DNA containing AP sites. It is proposed that Glu-23 protonates the sugar ring, so that the imino intermediate can be formed.     

Characterization of the active site of group B streptococcal hyaluronan lyase

Hyaluronan lyase is secreted by most strains of the human pathogen, group B streptococcus. Site-directed mutagenesis of the enzyme identified three amino acid residues important for enzyme activity, H479, Y488, and R542. These three residues are in close proximity in the putative active site of a homology model of group B streptococcal hyaluronan lyase. The homology model was based on the crystal structure of another related glycosaminoglycan lyase, chondroitin AC lyase, which exhibits different substrate specificity. Two asparagine residues in the active site groove, N429 and N660, were also found to be essential for enzyme activity. In addition, conversion of two adjacent tryptophan residues in the groove to alanines abolished activity. All amino acids found to be essential in GBS hyaluronan lyase are conserved in both enzymes. However, several amino acids in the active site groove of the two enzymes are not conserved. In the 18 cases in which one of these amino acids in GBS hyaluronan lyase was replaced with its corresponding amino acid in chondroitin AC lyase, no major loss of activity or change in substrate specificity was observed.     

Topological role of cytochrome P450 2D6 active site residues

Recent reports have identified Phe120, Asp301, Thr309, and Glu216 as important residues in cytochrome P450 2D6 (CYP2D6) substrate binding and catalysis. Complementary homology models have located these amino acids within the binding pocket of CYP2D6 and in the present study we have used aryldiazenes to test these models and gain further insight in the role these amino acids have in maintaining the integrity of the active site cavity. When Phe120 was replaced to alanine, there was a significant increase in probe migration to pyrrole nitrogens C and D, in agreement with homology models which have located the phenyl side-chain of Phe120 above these two pyrrole rings. No changes in topology were observed with the D301Q mutant, supporting claims that in this mutant the electrostatic interactions with the B/C-loop are largely maintained and the loop retains its native orientation. The T309V mutation resulted in significant topological alteration suggesting that, in addition to its potential role in dioxygen activation, Thr309 plays an important structural role within the active site crevice. Replacement of Ile106 with Glu, engineered to cause electrostatic repulsion with Glu216, had a profound topological effect in the higher region within the active site cavity and impaired the catalytic activity towards CYP2D6 probe substrates.     

Determination of active site residues in Escherichia coli endonuclease VIII.

Endonuclease VIII from Escherichia coli is a DNA glycosylase/lyase that removes oxidatively damaged bases. EndoVIII is a functional homologue of endonuclease III, but a sequence homologue of formamidopyrimidine-DNA glycosylase (Fpg). Using multiple sequence alignments, we have identified six target residues in endoVIII that may be involved in the enzyme's glycosylase and/or lyase functions: the N-terminal proline, and five acidic residues that are completely conserved in the endoVIII-Fpg proteins. To investigate the contribution of these residues, site-directed mutagenesis was used to create seven mutants: P2T, E3D, E3Q, E6Q, D129N, D160N, and E174Q. Each mutant was assayed both for lyase activity on abasic (AP) sites and for glycosylase/lyase activity on 5-hydroxyuracil, thymine glycol, and gamma-irradiated DNA with multiple lesions. The P2T mutant did not have lyase or glycosylase/lyase activity but could efficiently form Schiff base intermediates on AP sites. E6Q, D129N, and D160N behaved essentially as endoVIII in all assays. E3D, E3Q, and E174Q retained significant AP lyase activity but had severely diminished or abolished glycosylase/lyase activities on the DNA lesions tested. These studies provide detailed predictions concerning the active site of endoVIII.     

Mutational analysis of metallo-beta-lactamase CcrA from Bacteroides fragilis

In an effort to evaluate the roles of Lys184, Asn193, and Asp103 in the binding and catalysis of metallo-beta-lactamase CcrA from Bacteroides fragilis, site-directed mutants of CcrA were generated and characterized using metal analyses, CD spectroscopy, and kinetic studies. Three Lys184 mutants were generated where the lysine was replaced with alanine, leucine, and glutamate, and the analysis of these mutants indicates that Lys184 is not greatly involved in binding of cephalosporins to CcrA; however, this residue does have a significant role in binding of penicillin G. Three Asn193 mutants were generated where the asparagine was replaced with alanine, leucine, and aspartate, and these mutants exhibited <4-fold decrease in k(cat), suggesting that Asn193 does not play a large role in catalysis. However, stopped-flow visible kinetic studies showed that the Asn193 mutants exhibit a slower substrate decay rate and no change in the product formation rate as compared with wild-type CcrA. These results support the proposed role of Asn193 in interacting with and activating substrate during catalysis. Two Asp103 mutants were generated where the aspartate was replaced with serine and cysteine. The D103C and D103S mutants bind the same amount of Zn(II) as wild-type CcrA and exhibited a 10(2)-fold and 10(5)-fold decrease in activity, respectively. Results from solvent isotope, proton inventory, and rapid-scanning visible studies suggest that Asp103 plays a role in generating the enzyme intermediate but does not donate a proton to the enzyme intermediate during the rate-limiting step of the catalytic mechanism.     

Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase.

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T-G and U-G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a beta-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this beta-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.     

Deduced amino-acid sequence and possible catalytic residues of a novel pectate lyase from an alkaliphilic strain of Bacillus.

The nucleotide sequence of the gene for a highly alkaline, low-molecular-mass pectate lyase (Pel-15) from an alkaliphilic Bacillus isolate was determined. It harbored an open reading frame of 672 bp encoding the mature enzyme of 197 amino acids with a predicted molecular mass of 20 924 Da. The deduced amino-acid sequence of the mature enzyme showed very low homology (< 20.4% identity) to those of known pectinolytic enzymes in the large pectate lyase superfamily (the polysaccharide lyase family 1). In an integrally conserved region designated the BF domain, Pel-15 showed a high degree of identity (40.5% to 79.4%) with pectate lyases in the polysaccharide lyase family 3, such as PelA, PelB, PelC, and PelD from Fusarium solani f. sp. pisi, PelB from Erwinia carotovora ssp. carotovora, PelI from E. chrysanthemi, and PelA from a Bacillus strain. By site-directed mutagenesis of the Pel-15 gene, we replaced Lys20 in the N-terminal region, Glu38, Lys41, Glu47, Asp63, His66, Trp78, Asp80, Glu83, Asp84, Lys89, Asp106, Lys107, Asp126, Lys129, and Arg132 in the BF domain, and Arg152, Tyr174, Lys182, and Lys185 in the C-terminal region of the enzyme individually with Ala and/or other amino acids. Consequently, some carboxylate and basic residues selected from Glu38, Asp63, Glu83, Asp106, Lys107, Lys129, and Arg132 were suggested to be involved in catalysis and/or calcium binding. We constructed a chimeric enzyme composed of Ala1 to Tyr105 of Pel-15 in the N-terminal regions, Asp133 to Arg159 of FsPelB in the internal regions, and Gln133 to Tyr197 of Pel-15 in the C-terminal regions. The substituted PelB segment could also express beta-elimination activity in the chimeric molecule, confirming that Pel-15 and PelB share a similar active-site topology.     

Mutation at active site lysine 212 to arginine uncouples the glycosylase activity from the lyase activity of human endonuclease III.

The human endonuclease III (hNTH1) is an important DNA glycosylase with associated abasic lyase activity. We previously demonstrated that the K212Q mutant was totally inactive, while the K212R mutant had reduced DNA glycosylase/lyase activity and could form a covalent complex with the substrate DNA upon reduction. We further characterized the biochemical properties of this K212R mutant protein. NH2- (N-) terminal sequencing in combination with mass spectrometry of the peptide-DNA adduct suggested that "opportunistic" lysine(s) in the lysine-rich N-terminal tail formed a Schiff base which might result in beta-elimination. However, simultaneous substitution of Lys-75 with Gln and deletion of first 72 residues in the N-terminal tail could not cause further alteration in the glycosylase reaction or beta-elimination event. Nonetheless, the time kinetics of K212R and its subsequent mutants showed glycosylase activity without any detectable AP-lyase activity during the first 10 min of the reaction. These results suggest that a single point mutation at the active site (K212R) uncoupled the glycosylase activity from the lyase activity. We propose that the uncoupled reaction carried out by K212R is a result of direct attack either by the nonionized form of the guanidino group of arginine which forms an unstable Schiff base that hydrolyzes prior to the beta-elimination event or by hydroxide ion to cleave the glycosylic bond. In either case this reaction is followed by a secondary beta-elimination event performed by random lysine residues primarily from the N-terminal tail region.     

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.     

Identification of the catalytic residues involved in the carboxyl transfer of pyruvate carboxylase

To clarify the mechanism of carboxyl transfer from carboxylbiotin to pyruvate, the following conserved amino acid residues present in the carboxyl transferase domain of Bacillus thermodenitrificans pyruvate carboxylase were converted to homologous amino acids: Asp543, Glu576, Glu592, Asp649, Lys712, Asp713, and Asp762. The carboxylase activity of the resulting mutants, D543E, E576D, E576Q, E592Q, D649N, K712R, K712Q, D713E, D713N, D762E, and D762N, was generally less than that of the wild type from mutation, but it decreased the most to 5% or even less than that of the wild type with D543E, D576Q, D649N, K712R, and K712Q. The decrease in activity observed for Asp543, Asp649, and Lys712 mutants was not for structural reasons because their structures seemed to remain intact as assessed by gel filtration and circular dichroism. On the basis of these data, a mechanism is proposed where Lys712 and Asp543 serve as the key acid and base catalyst, respectively.     

Role of the N-terminal proline residue in the catalytic activities of the Escherichia coli Fpg protein

The Escherichia coli Fpg protein is a DNA glycosylase/AP lyase. It removes, in DNA, oxidized purine residues, including the highly mutagenic C8-oxo-guanine (8-oxoG). The catalytic mechanism is believed to involve the formation of a transient Schiff base intermediate formed between DNA containing an oxidized residue and the N-terminal proline of the Fpg protein. The importance and the role of this proline upon the various catalytic activities of the Fpg protein was examined by targeted mutagenesis, resulting in the construction of three mutant Fpg proteins: Pro-2 --> Gly (FpgP2G), Pro-2 --> Thr (FpgP2T), and Pro-2 --> Glu (FpgP2E). The formamidopyrimidine DNA glycosylase activities of FpgP2G and FpgP2T were comparable and accounted for 10% of the wild-type activity. FpgP2G and FpgP2T had barely detectable 8-oxoG-DNA glycosylase activity and produced minute Schiff base complex with 8-oxoG/C DNA. FpgP2G and FpgP2T mutants did not cleave a DNA containing preformed AP site but readily produced Schiff base complex with this substrate. FpgP2E was completely inactive in all the assays. The binding constants of the different mutants when challenged with a duplex DNA containing a tetrahydrofuran residue were comparable. The mutant Fpg proteins barely or did not complement in vivo the spontaneous transitions G/C --> T/A in E. coli BH990 (fpg mutY) cells. These results show the mandatory role of N-terminal proline in the 8-oxoG-DNA glycosylase activity of the Fpg protein in vitro and in vivo as well as in its AP lyase activity upon preformed AP site but less in the 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine-DNA glycosylase activity.     

Structural and functional properties of CiNTH, an endonuclease III homologue of the ascidian Ciona intestinalis: critical role of N-terminal region

Oxidatively damaged bases in DNA can cause cell death, mutation and/or cancer induction. To overcome such deleterious effects of DNA base oxidation, cells are equipped with base excision repair (BER) initiated by DNA glycosylases. Endonuclease III (Nth), a major DNA glycosylase, mainly excises oxidatively damaged pyrimidines from DNA. The aims of this study were to obtain an overview of the repair mechanism of oxidatively damaged bases and to elucidate the function of BER in maintaining genome stability during embryogenesis and development. In this study, we used the ascidian Ciona intestinalis because at every developmental stage it is possible to observe the phenotype of individuals with DNA damage or mutations. Sequence alignment analysis revealed that the amino acid sequence of Ciona intestinalis Nth homologue (CiNTH) had high homology with those of Escherichia coli, Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans and human Nth homologues. It was evident that two domains, the Helix-hairpin-Helix and 4Fe-4S cluster domains that are critical regions for the Nth activity, are well conserved in CiNTH. CiNTH efficiently complemented the sensitivity of E. coli nth nei mutant to H(2)O(2). CiNTH was bifunctional, with DNA glycosylase and AP lyase activities. It removed thymine glycol, 5-formyluracil and 8-oxoguanine paired with G from DNA via a β-elimination reaction. Interestingly, the N-terminal 44 amino acids were essential for the DNA glycosylase activity of CiNTH.     

Human endonuclease VIII-like (NEIL) proteins in the giant DNA Mimivirus

Endonuclease VIII (Nei), which recognizes and repairs oxidized pyrimidines in the base excision repair (BER) pathway, is sparsely distributed among both the prokaryotes and eukaryotes. Recently, we and others identified three homologs of Escherichia coli endonuclease VIII-like (NEIL) proteins in humans. Here, we report identification of human NEIL homologs in Mimivirus, a giant DNA virus that infects Acanthamoeba. Characterization of the two mimiviral homologs, MvNei1 and MvNei2, showed that they share not only sequence homology but also substrate specificity with the human NEIL proteins, that is, they recognize oxidized pyrimidines in duplex DNA and in bubble substrates and as well show 5'2-deoxyribose-5-phosphate lyase (dRP lyase) activity. However, unlike MvNei1 and the human NEIL proteins, MvNei2 preferentially cleaves oxidized pyrimidines in single stranded DNA forming products with a different end chemistry. Interestingly, opposite base specificity of MvNei1 resembles human NEIL proteins for pyrimidine base damages whereas it resembles E. coli formamidopyrimidine DNA glycosylase (Fpg) for guanidinohydantoin (Gh), an oxidation product of 8-oxoguanine. Finally, a conserved arginine residue in the "zincless finger" motif, previously identified in human NEIL1, is required for the DNA glycosylase activity of MvNei1. Thus, Mimivirus represents the first example of a virus to carry oxidative DNA glycosylases with substrate specificities that resemble human NEIL proteins. Based on the sequence homology to the human NEIL homologs and novel bacterial NEIL homologs identified here, we predict that Mimivirus may have acquired the DNA glycosylases through the host-mediated lateral transfer from either a bacterium or from vertebrates.     

Functional identification of an 8-oxoguanine specific endonuclease from Thermotoga maritima

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of NaBH4, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.     

Mass spectrometric analysis of the reactions catalyzed by -2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of -2-haloalkanoic acids to produce the corresponding -2-hydroxyalkanoic acids. Asp10 of -2-haloacid dehalogenase from Pseudomonas sp. YL nucleophilically attacks the α-carbon atom of the substrate to form an ester intermediate, which is subsequently hydrolyzed by an activated water molecule. We previously showed that the replacement of Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177, and Asp180 causes significant loss in the enzyme activity, indicating the involvement of these residues in catalysis. In the present study, we tried to determine which process these residues are involved in by monitoring the formation of the ester intermediate by measuring the molecular masses of the mutant enzymes using ionspray mass spectrometry. When the wild-type enzyme and the T14A, S118D, K151R, Y157F, S175A, and N177D mutant enzymes were mixed with the substrate, the ester intermediate was immediately produced. In contrast, the R41K, D180N, and D180A mutants formed the intermediate much more slowly than the wild-type enzyme, indicating that Arg41 and Asp180 participate in the formation of the ester intermediate. This study presents a new method to analyze the roles of amino acid residues in catalysis.     

The Carboxy-Terminal Domain of ROS1 Is Essential for 5-Methylcytosine DNA Glycosylase Activity

Arabidopsis thaliana repressor of silencing 1 (ROS1) is a multi-domain bifunctional DNA glycosylase/lyase, which excises 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) as well as thymine and 5-hydroxymethyluracil (i.e., the deamination products of 5mC and 5hmC) when paired with a guanine, leaving an apyrimidinic (AP) site that is subsequently incised by the lyase activity. ROS1 is slow in base excision and fast in AP lyase activity, indicating that the recognition of pyrimidine modifications might be a rate-limiting step. In the C-terminal half, the enzyme harbors a helix–hairpin–helix DNA glycosylase domain followed by a unique C-terminal domain. We show that the isolated glycosylase domain is inactive for base excision but retains partial AP lyase activity. Addition of the C-terminal domain restores the base excision activity and increases the AP lyase activity as well. Furthermore, the two domains remain tightly associated and can be co-purified by chromatography. We suggest that the C-terminal domain of ROS1 is indispensable for the 5mC DNA glycosylase activity of ROS1.     

Role of the charge interaction between Arg(70) and Asp(120) in the Tn10-encoded metal-tetracycline/H(+) antiporter of Escherichia coli

We reported that the positive charge of Arg(70) is mandatory for tetracycline transport activity of Tn10-encoded metal-tetracycline/H(+) antiporter (TetA(B)) (Someya, Y., and Yamaguchi, A. (1996) Biochemistry 35, 9385-9391). Arg(70) may function through a charge-pairing with a negatively charged residue in close proximity. Therefore, we mutated Asp(66) and Asp(120), which are only two negatively charged residues located close to Arg(70) in putative secondary structure of TetA(B) and highly conserved throughout transporters of the major facilitator superfamily. Site-directed mutagenesis studies revealed that Asp(66) is essential, but Asp(120) is important for TetA(B) function. Surprisingly, when Asp(120) was replaced by a neutral residue, the R70A mutant recovered tetracycline resistance and transport activity. There was no such effect in the Asp(66) mutation. The charge-exchanged mutant, R70D/D120R, also showed significant drug resistance and transport activity (about 50% of the wild type), although the R70D mutant had absolutely no activity, and the D120R mutant retained very low activity (about 10% of the wild type). Both the R70C and D120C mutants were inactivated by N-ethylmaleimide. Mercuric ion (Hg(2+)), which gives a positive charge to a SH group of a Cys residue through mercaptide formation, had an opposite effect on the R70C and D120C mutants. The activity of the R70C mutant was stimulated by Hg(2+); however, on the contrary, the D120C mutant was partially inhibited. On the other hand, the R70C/D120C double mutant was almost completely inactivated by Hg(2+), probably because the side chains at positions 70 and 120 are bridged with Hg(2+). The close proximity of positions 70 and 120 were confirmed by disulfide cross-linking formation of the R70C/D120C double mutant when it was oxidized by copper-(1,10-phenanthroline). These results indicate that the positive charge of Arg(70) requires the negative charge of Asp(120) for neutralization, probably for properly positioning transmembrane segments in the membrane.