Auto-methylation of the mouse DNA-(cytosine C5)-methyltransferase Dnmt3a at its active site cysteine residue

The Dnmt3a DNA methyltransferase is responsible for establishing DNA methylation patterns during mammalian development. We show here that the mouse Dnmt3a DNA methyltransferase is able to transfer the methyl group from S-adenosyl-l-methionine (AdoMet) to a cysteine residue in its catalytic center. This reaction is irreversible and relatively slow. The yield of auto-methylation is increased by addition of Dnmt3L, which functions as a stimulator of Dnmt3a and enhances its AdoMet binding. Auto-methylation was observed in binary Dnmt3a AdoMet complexes. In the presence of CpG containing dsDNA, which is the natural substrate for Dnmt3a, the transfer of the methyl group from AdoMet to the flipped target base was preferred and auto-methylation was not detected. Therefore, this reaction might constitute a regulatory mechanism which could inactivate unused DNA methyltransferases in the cell, or it could simply be an aberrant side reaction caused by the high methyl group transfer potential of AdoMet. ENZYMES: Dnmt3a is a DNA-(cytosine C5)-methyltransferase, EC 2.1.1.37. STRUCTURED DIGITAL ABSTRACT: ? Dnmt3a methylates Dnmt3a by methyltransferase assay (View interaction) ? Dnmt3a and DNMT3L methylate Dnmt3a by methyltransferase assay (View interaction).     

DNA cytosine C5 methyltransferase Dnmt1: catalysis-dependent release of allosteric inhibition

We followed the cytosine C(5) exchange reaction with Dnmt1 to characterize its preference for different DNA substrates, its allosteric regulation, and to provide a basis for comparison with the bacterial enzymes. We determined that the methyl transfer is rate-limiting, and steps up to and including the cysteine-cytosine covalent intermediate are in rapid equilibrium. Changes in these rapid equilibrium steps account for many of the previously described features of Dnmt1 catalysis and specificity including faster reactions with premethylated DNA versus unmethylated DNA, faster reactions with DNA in which guanine is replaced with inosine [poly(dC-dG) vs poly(dI-dC)], and 10-100-fold slower catalytic rates with Dnmt1 relative to the bacterial enzyme M.HhaI. Dnmt1 interactions with the guanine within the CpG recognition site can prevent the premature release of the target base and solvent access to the active site that could lead to mutagenic deamination. Our results suggest that the beta-elimination step following methyl transfer is not mediated by free solvent. Dnmt1 shows a kinetic lag in product formation and allosteric inhibition with unmethylated DNA that is not observed with premethylated DNA. Thus, we suggest the enzyme undergoes a slow relief from allosteric inhibition upon initiation of catalysis on unmethylated DNA. Notably, this relief from allosteric inhibition is not caused by self-activation through the initial methylation reaction, as the same effect is observed during the cytosine C(5) exchange reaction in the absence of AdoMet. We describe limitations in the Michaelis-Menten kinetic analysis of Dnmt1 and suggest alternative approaches.     

Inactivation of the Heme Degrading Enzyme IsdI by an Active Site Substitution That Diminishes Heme Ruffling

IsdG and IsdI are paralogous heme degrading enzymes from the bacterium Staphylococcus aureus. Heme bound by these enzymes is extensively ruffled such that the meso-carbons at the sites of oxidation are distorted toward bound oxygen. In contrast, the canonical heme oxygenase family degrades heme that is bound with minimal distortion. Trp-66 is a conserved heme pocket residue in IsdI implicated in heme ruffling. IsdI variants with Trp-66 replaced with residues having less bulky aromatic and alkyl side chains were characterized with respect to catalytic activity, heme ruffling, and electrochemical properties. The heme degradation activity of the W66Y and W66F variants was approximately half that of the wild-type enzyme, whereas the W66L and W66A variants were inactive. A crystal structure and NMR spectroscopic analysis of the W66Y variant reveals that heme binds to this enzyme with less heme ruffling than observed for wild-type IsdI. The reduction potential of this variant (−96 ± 7 mV versus standard hydrogen electrode) is similar to that of wild-type IsdI (−89 ± 7 mV), so we attribute the diminished activity of this variant to the diminished heme ruffling observed for heme bound to this enzyme and conclude that Trp-66 is required for optimal catalytic activity.     

Identification of "buried" lysine residues in two variants of chloramphenicol acetyltransferase specified by R-factors.

Two variants of chloramphenicol acetyltransferase which are specified by genes on plasmids found in Gram-negative bacteria were subjected to amidination with methyl acetimidate to determine the relative reactivity of surface lysine residues and to search for unreactive or "buried" amino groups which might contribute to stabilization of the native tetramers. Representative examples of the type-I and type-III variants of chloramphenicol acetyltransferase were found to have one lysine residue each in the native state which appears to be inaccessible to methyl acetimidate. The uniquely unreactive residue of the type-I protein is lysine-136, whereas the lysine that is "buried" in the type-III enzyme is provisonally assigned to residue 38 of the prototype sequence. It is suggested that the lysine residue in each case participates in the formation of an ion pair at the intersubunit interface and that the two amino groups in question occupy functionally equivalent positions in the quaternary structures of their respective enzyme variants. Lysine-136 of type-I enzyme is also uniquely unavailable for modification by citraconic anhydride, a reagent used to disrupt the quaternary structure of the native enzyme. Contrary to expectation, exhaustive citraconylation fails to dissociate the tetramer, but does destroy catalytic activity. Removal of citraconyl groups from modified chloramphenicol acetyltransferase is accompanied by a full region of catalytic activity. Analysis of the rate of hydrolysis of citraconyl groups from the modified tetramer by amidination of unblocked amino groups with methyl [14C]acetamidate reveals difference in lability for several of the ten modified lysine residues. Although the unique stability of the quaternary structure of chloramphenicol acetyltransferase may be due to strong hydrophobic interactions, it is argued that lysine-136 may contribute to stability via the formation of an ion pair at the subunit interface.     

Sequence-specific and mechanism-based crosslinking of Dcm DNA cytosine-C5 methyltransferase of E. coli K-12 to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine.

The product of the dcm gene is the only DNA cytosine-C5 methyltransferase of Escherichia coli K-12; it catalyses transfer of a methyl group from S-adenosyl methionine (SAM) to the C-5 position of the inner cytosine residue of the cognate sequence CCA/TGG. Sequence-specific, covalent crosslinking of the enzyme to synthetic oligonucleotides containing 5-fluoro-2'-deoxycytidine is demonstrated. This reaction is abolished if serine replaces the cysteine at residue #177 of the enzyme. These results lend strong support to a catalytic mechanism in which an enzyme sulfhydryl group undergoes Michael addition to the C5-C6 double bond, thus activating position C-5 of the substrate DNA cytosine residue for electrophilic attack by the methyl donor SAM. The enzyme is capable of self-methylation in a DNA-independent reaction requiring SAM and the presence of cysteine at position #177.     

Mutants that alter the covalent structure of catalase hydroperoxidase II from Escherichia coli.

The three-dimensional structures of two HPII variants, V169C and H392Q, have been determined at resolutions of 1.8 and 2.1 A, respectively. The V169C variant contains a new type of covalent bond between the sulfur atom of Cys(169) and a carbon atom on the imidazole ring of the essential His(128). This variant enzyme has only residual catalytic activity and contains heme b. The chain of water molecules visible in the main channel may reflect the organization of the hydrogen peroxide substrates in the active enzyme. Two alternative mechanisms, involving either compound I or free radical intermediates, are presented to explain the formation of the Cys-His covalent bond. The H392Q and H392E variants exhibit 75 and 25% of native catalytic activity, respectively. The Gln(392) variant contains only heme b, whereas the Glu(392) variant contains a mixture of heme b and cis and trans isomers of heme d, suggesting of a role for this residue in heme conversion. Replacement of either Gln(419) and Ser(414), both of which interact with the heme, affected the cis:trans ratio of spirolactone heme d. Implications for the heme oxidation mechanism and the His-Tyr bond formation in HPII are considered.     

Structural and Mechanistic Insights into the Pseudomonas fluorescens 2-Nitrobenzoate 2-Nitroreductase NbaA

The bacterial 2-nitroreductase NbaA is the primary enzyme initiating the degradation of 2-nitrobenzoate (2-NBA), and its activity is controlled by posttranslational modifications. To date, the structure of NbaA remains to be elucidated. In this study, the crystal structure of a Cys194Ala NbaA mutant was determined to a 1.7-Å resolution. The substrate analog 2-NBA methyl ester was used to decipher the substrate binding site by inhibition of the wild-type NbaA protein. Tandem mass spectrometry showed that 2-NBA methyl ester produced a 2-NBA ester bond at the Tyr193 residue in the wild-type NbaA but not residues in the Tyr193Phe mutant. Moreover, covalent binding of the 2-NBA methyl ester to Tyr193 reduced the reactivity of the Cys194 residue on the peptide link. The Tyr193 hydroxyl group was shown to be essential for enzyme catalysis, as a Tyr193Phe mutant resulted in fast dissociation of flavin mononucleotide (FMN) from the protein with the reduced reactivity of Cys194. FMN binding to NbaA varied with solution NaCl concentration, which was related to the catalytic activity but not to cysteine reactivity. These observations suggest that the Cys194 reactivity is negatively affected by a posttranslational modification of the adjacent Tyr193 residue, which interacts with FMN and the substrate in the NbaA catalytic site.     

Enzymatic properties of recombinant Dnmt3a DNA methyltransferase from mouse: the enzyme modifies DNA in a non-processive manner and also methylates non-CpG [correction of non-CpA] sites

We present the first in vitro study investigating the catalytic properties of a mammalian de novo DNA methyltransferase. Dnmt3a from mouse was cloned and expressed in Escherichia coli. It was shown to be catalytically active in E. coli cells in vivo. The methylation activity of the purified protein was highest at pH 7.0 and 30 mM KCl. Our data show that recombinant Dnmt3a protein is indeed a de novo methyltransferase, as it catalyzes the transfer of methyl groups to unmethylated substrates with similar efficiency as to hemimethylated substrates. With oligonucleotide substrates, the catalytic activity of Dnmt3a is similar to that of Dnmt1: the K(m) values for the unmethylated and hemimethylated oligonucleotide substrates are 2.5 microM, and the k(cat) values are 0.05 h(-1) and 0.07 h(-1), respectively. The enzyme catalyzes the methylation of DNA in a distributive manner, suggesting that Dnmt3a and Dnmt1 may cooperate during de novo methylation of DNA. Further, we investigated the methylation activity of Dnmt3a at non-canonical sites. Even though the enzyme shows maximum activity at CpG sites, with oligonucleotide substrates, a high methylation activity was also found at CpA sites, which are modified only twofold slower than CpG sites. Therefore, the specificity of Dnmt3a is completely different from that of the maintenance methyltransferase Dnmt1, which shows a 40 to 50-fold preference for hemimethylated over unmethylated CpG sites and has almost no methylation activity at non-CpG sites.     

Changing the target base specificity of the EcoRV DNA methyltransferase by rational de novo protein-design

The EcoRV DNA-(adenine-N⁶)-methyltransferase (M.EcoRV) specifically modifies the first adenine residue within GATATC sequences. During catalysis, the enzyme flips its target base out of the DNA helix and binds it into a target base binding pocket which is formed in part by Lys16 and Tyr196. A cytosine residue is accepted by wild-type M.EcoRV as a substrate at a 31-fold reduced efficiency with respect to the k_cat/K_M values if it is located in a CT mismatch substrate (GCTATC/GATATC). Cytosine residues positioned in a CG base pair (GCTATC/GATAGC) are modified at much more reduced rates, because flipping out the target base is much more difficult in this case. We intended to change the target base specificity of M.EcoRV from adenine-N⁶ to cytosine-N⁴. To this end we generated, purified and characterized 15 variants of the enzyme, containing single, double and triple amino acid exchanges following different design approaches. One concept was to reduce the size of the target base binding pocket by site-directed mutagenesis. The K16R variant showed an altered specificity, with a 22-fold preference for cytosine as the target base in a mismatch substrate. This corresponds to a 680-fold change in specificity, which was accompanied by only a small loss in catalytic activity with the cytosine substrate. The K16R/Y196W variant no longer methylated adenine residues at all and its activity towards cytosine was reduced only 17-fold. Therefore, we have changed the target base specificity of M.EcoRV from adenine to cytosine by rational protein design. Because there are no natural paragons for the variants described here, a change of the target base specificity of a DNA interacting enzyme was possible by rational de novo design of its active site.     

Further studies on the catalytic mechanism of human liver alpha-L-fucosidase

Radiolabeling of human liver alpha-L-fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) with [1-3H]conduritol C trans-epoxide revealed that there are four active sites per tetrameric enzyme complex. Solvent isotope effect experiments give evidence for a proton transfer at the rate-limiting step in catalysis. Transglycosylase activity was observed using methanol as an alternative glycone acceptor to produce methyl alpha-L-fucoside, suggesting that alpha-L-fucose is formed when water is the acceptor. Initial burst kinetics experiments suggest that a glycosyl-enzyme intermediate is formed, although the magnitude of the burst is not stoichiometric with the number of active sites. These data, along with previous results, suggest a general acid-general base catalytic mechanism involving double inversion of stereochemistry at C-1 of fucose, as well as the formation of either a covalent glycosyl-enzyme intermediate or a tight ion pair between a charged active-site residue and a hypothetical fucosyl oxocarbonium ion intermediate.     

Post-translational phosphorylation of serine 74 of human deoxycytidine kinase favors the enzyme adopting the open conformation making it competent for nucleoside binding and release

Deoxycytidine kinase (dCK) uses either ATP or UTP as a phosphoryl donor to catalyze the phosphorylation of nucleoside acceptors. The kinetic properties of human dCK are modulated in vivo by phosphorylation of serine 74. This residue is a part of the insert region and is distant from the active site. Replacing the serine with a glutamic acid (S74E variant) can mimic phosphorylation of Ser74. To understand how phosphorylation affects the catalytic properties of dCK, we examined the S74E variant of dCK both structurally and kinetically. We observe that the presence of a glutamic acid at position 74 favors the adoption by the enzyme of the open conformation. Glu74 stabilizes the open conformation by directly interacting with the indole side chain of Trp58, a residue that is in the proximity of the base of the nucleoside substrate. The open dCK conformation is competent for the binding of nucleoside but not for phosphoryl transfer. In contrast, the closed conformation is competent for phosphoryl transfer but not for product release. Thus, dCK must make the transition between the open and closed states during the catalytic cycle. We propose a reaction scheme for dCK that incorporates the transition between the open and closed states, and this serves to rationalize the observed kinetic differences between wild-type dCK and the S74E variant.     

The role of arginine 310 in catalysis and substrate specificity in xanthine dehydrogenase from Rhodobacter capsulatus

The rapid reaction kinetics of wild-type xanthine dehydrogenase from Rhodobacter capsulatus and variants at Arg-310 in the active site have been characterized for a variety of purine substrates. With xanthine as substrate, k(red) (the limiting rate of enzyme reduction by substrate at high [S]) decreased approximately 20-fold in an R310K variant and 2 x 10(4)-fold in an R310M variant. Although Arg-310 lies on the opposite end of the substrate from the C-8 position that becomes hydroxylated, its interaction with substrate still contributed approximately 4.5 kcal/mol toward transition state stabilization. The other purines examined fell into two distinct groups: members of the first were effectively hydroxylated by the wild-type enzyme but were strongly affected by the exchange of Arg-310 to methionine (with a reduction in k(red) greater than 10(3)), whereas members of the second were much less effectively hydroxylated by wild-type enzyme but also much less significantly affected by the amino acid exchanges (with a reduction in k(red) less than 50-fold). The effect was such that the 4000-fold range in k(red) seen with wild-type enzyme was reduced to a mere 4-fold in the R310M variant. The data are consistent with a model in which "good" substrates are bound "correctly" in the active site in an orientation that allows Arg-310 to stabilize the transition state for the first step of the overall reaction via an electrostatic interaction at the C-6 position, thereby accelerating the reaction rate. On the other hand, "poor" substrates bound upside down relative to this "correct" orientation. In so doing, they are unable to avail themselves of the additional catalytic power provided by Arg-310 in wild-type enzyme but, for this reason, are significantly less affected by mutations at this position. The kinetic data thus provide a picture of the specific manner in which the physiological substrate xanthine is oriented in the active site relative to Arg-310 and how this residue is used catalytically to accelerate the reaction rate (rather than simply bind substrate) despite being remote from the position that is hydroxylated.     

Direct observation of cytosine flipping and covalent catalysis in a DNA methyltransferase

Methylation of the five position of cytosine in DNA plays important roles in epigenetic regulation in diverse organisms including humans. The transfer of methyl groups from the cofactor S-adenosyl-L-methionine is carried out by methyltransferase enzymes. Using the paradigm bacterial methyltransferase M.HhaI we demonstrate, in a chemically unperturbed system, the first direct real-time analysis of the key mechanistic events-the flipping of the target cytosine base and its covalent activation; these changes were followed by monitoring the hyperchromicity in the DNA and the loss of the cytosine chromophore in the target nucleotide, respectively. Combined with studies of M.HhaI variants containing redesigned tryptophan fluorophores, we find that the target base flipping and the closure of the mobile catalytic loop occur simultaneously, and the rate of this concerted motion inversely correlates with the stability of the target base pair. Subsequently, the covalent activation of the target cytosine is closely followed by but is not coincident with the methyl group transfer from the bound cofactor. These findings provide new insights into the temporal mechanism of this physiologically important reaction and pave the way to in-depth studies of other base-flipping systems.     

Enhancing Transport of Hydrogenophagaflava ENV735 for Bioaugmentation of Aquifers Contaminated with Methyl tert-Butyl Ether

The gasoline oxygenate methyl tert-butyl ether (MTBE) has become a widespread contaminant in groundwater throughout the United States. Bioaugmentation of aquifers with MTBE-degrading cultures may be necessary to enhance degradation of the oxygenate in some locations. However, poor cell transport has sometimes limited bioaugmentation efforts in the past. The objective of this study was to evaluate the transport characteristics of Hydrogenophaga flava ENV735, a pure culture capable of growth on MTBE, and to improve movement of the strain through aquifer solids. The wild-type culture moved only a few centimeters in columns of aquifer sediment. An adhesion-deficient variant (H. flava ENV735:24) of the wild-type strain that moved more readily through sediments was obtained by sequential passage of cells through columns of sterile sediment. Hydrophobic and electrostatic interaction chromatography revealed that the wild-type strain is much more hydrophobic than the adhesion-deficient variant. Electrophoretic mobility assays and transmission electron microscopy showed that the wild-type bacterium contains two distinct subpopulations, whereas the adhesion-deficient strain has only a single, homogeneous population. Both the wild-type strain and adhesion-deficient variant degraded MTBE, and both were identified by 16S rRNA analysis as pure cultures of H. flava. The effectiveness of surfactants for enhancing transport of the wild-type strain was also evaluated. Many of the surfactants tested were toxic to ENV735; however, one nonionic surfactant, Tween 20, enhanced cell transport in sand columns. Improving microbial transport may lead to a more effective bioaugmentation strategy for MTBE-contaminated sites where indigenous oxygenate degraders are absent.     

Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co(2+). The triple-site variant produced 213 g/liter l-ribose from 300 g/liter L-ribulose for 60 min, with a volumetric productivity of 213 g liter(-1) h(-1), which was 4.5-fold higher than that of the wild-type enzyme. The k(cat)/K(m) and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.     

The role of the site 342 in catalytic efficiency and pH optima of endoglucanase II from Trichoderma reesei as probed by saturation mutagenesis

The role of site 342 of endoglucanase II from Trichoderma reesei in catalytic efficiency and pH optima was investigated by site saturation mutagenesis. The mutations identified in this study can be divided into three separate classes according to their amino acid features. When Asn342 was substituted by hydrophobic and non-polar amino acids, most variants exhibited an up-shift in pH optimum and their catalytic efficiency was similar to that of the wild-type at their optimal pH. N342R variant had a pH optimum at 6.2. N342K variant did not give an up-shift in pH optimum, although K and R are both amino acids carrying positive charges. Molecular modelling indicated that residue 342 was located at the C-terminus of one of the α-helices near two catalytic residues. Hydrophobic side chains and more H-bonds would make the helix more rigid, which might affect the stability and activity of the enzyme at higher pH.     

Mutability of an HNH nuclease imidazole general base and exchange of a deprotonation mechanism

Several unique protein folds that catalyze the hydrolysis of phosphodiester bonds have arisen independently in nature, including the PD(D/E)XK superfamily (typified by type II restriction endonucleases and many recombination and repair enzymes) and the HNH superfamily (found in an equally wide array of enzymes, including bacterial colicins and homing endonucleases). Whereas the identity and position of catalytic residues within the PD(D/E)XK superfamily are highly variable, the active sites of HNH nucleases are much more strongly conserved. In this study, the ability of an HNH nuclease to tolerate a mutation of its most conserved catalytic residue (its histidine general base), and the mechanism of the most active enzyme variant, were characterized. Conversion of this residue into several altered chemistries, glutamine, lysine, or glutamate, resulted in measurable activity. The histidine to glutamine mutant displays the highest residual activity and a pH profile similar to that of the wild-type enzyme. This activity is dependent on the presence of a neighboring imidazole ring, which has taken over as a less efficient general base for the reaction. This result implies that mutational pathways to alternative HNH-derived catalytic sites do exist but are not as extensively or successfully diverged or reoptimized in nature as variants of the PD(D/E)XK nuclease superfamily. This is possibly due to multiple steric constraints placed on the compact HNH motif, which is simultaneously involved in protein folding, DNA binding, and catalysis, as well as the use of a planar, aromatic imidazole group as a general base.     

AdoMet-dependent methyl-transfer: Glu119 is essential for DNA C5-cytosine methyltransferase M.HhaI

The role of Glu119 in S-adenosyl-L-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 10(2)-10(6) fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 10(3)-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 A and 2.75 A, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding.