Kinetic analysis of the active site of an intracellular β-glucosidase fromCellulomonas biazotea

Purified β-glucosidase fromCellulomonas biazotea had an apparentK _m andV for 2-nitrophenyl β-d-glucopyranoside (oNPG) of 0.416 mmol/L and 0.22 U/mg protein, respectively. The activation energy for the hydrolysis of pNPG of β-glucosidase was 65 kJ/mol. The inhibition by Mn²⁺ vs. oNPG of parental β-glucosidase was of mixed type with apparent inhibition constants of 0.19 and 0.60 μmol/L for the enzyme and enzyme-substrate complex, respectively. Ethanol at lower concentrations activated while at higher concentrations it inhibited the enzyme. The determination of apparent pK _a’s at different temperatures and in the presence of 30 % dioxane indicated two carboxyl groups which control theV value. The thermal stability of β-glucosidase decreased in the presence of 10 % ethanol. The half-life of β-glucosidase in 1.75 mol/L urea at 35 °C was 145 min, as determined by 0–9 mol/L transverse urea gradient-PAGE. This work was financed in part by a grant made by theUS Agency for International Development under PSTC proposal 6-163,USAID grant no. 9365542-G-00-89-42-00, and PAEC.     

Wheat DNA polymerase CI: a homologue of rat DNA polymerase β

We have previously described a low-molecular-weight DNA polymerase (52 kDa) from wheat embryo: DNA polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA polymerase (pol ). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol -type DNA polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol . Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a -type DNA polymerase in plants.     

Protected immobilization of Taq DNA polymerase by active site masking on self-assembled monolayers of ω-functionalized thiols

A new efficient immobilization method that enables oriented immobilization of biologically active proteins was developed based on concepts of active site masking and kinetic control. Taq DNA polymerase was immobilized covalently on mixed self-assembled monolayers (SAMs) of ω-carboxylated thiol and ω-hydroxylated thiol through amide bonds between the protein and the carboxyl group in SAMs. Activity of the immobilized enzyme as large as 70% of solution-phase enzyme was achieved by masking the active site of the Taq DNA polymerase prior to the immobilization. In addition, the number of immobilization bonds was controlled by optimizing the carboxyl group concentration in the mixed monolayer. The maximum activity of immobilized Taq DNA polymerase was achieved at 5% of 12-mercaptododecanoic acid. The activity observed with protected immobilized enzyme was approximately 20 times higher than that observed with randomly immobilized enzyme. The maximum activity was acquired at a 1:1 DNA/enzyme masking ratio, immobilization pH 8.3, and within 10 min of reaction time. This concept of the active site masking and kinetic control of the number of covalent bonds between proteins and the surface can be generally applicable to a broad range of proteins to be immobilized on the solid surface with higher activity.     

Human DNA polymerase η is pre-aligned for dNTP binding and catalysis

Pre-steady-state kinetic studies on Y-family DNA polymerase η (Polη) have suggested that the polymerase undergoes a rate-limiting conformational change step before the phosphoryl transfer of the incoming nucleotide to the primer terminus. However, the nature of this rate-limiting conformational change step has been unclear, due in part to the lack of structural information on the Polη binary complex. We present here for the first time a crystal structure of human Polη (hPolη) in binary complex with its DNA substrate. We show that the hPolη domains move only slightly on dNTP binding and that the polymerase by and large is pre-aligned for dNTP binding and catalysis. We also show that there is no major reorientation of the DNA from a nonproductive to a productive configuration and that the active site is devoid of metals in the absence of dNTP. Together, these observations lead us to suggest that the rate-limiting conformational change step in the Polη replication cycle likely corresponds to a rate-limiting entry of catalytic metals in the active site.     

Kinetic analysis of the unique error signature of human DNA polymerase ν

The fidelity of DNA synthesis by A-family DNA polymerases ranges from very accurate for bacterial, bacteriophage, and mitochondrial family members to very low for certain eukaryotic homologues. The latter include DNA polymerase ν (Pol ν) which, among all A-family polymerases, is uniquely prone to misincorporating dTTP opposite template G in a highly sequence-dependent manner. Here we present a kinetic analysis of this unusual error specificity, in four different sequence contexts and in comparison to Pol ν's more accurate A-family homologue, the Klenow fragment of Escherichia coli DNA polymerase I. The kinetic data strongly correlate with rates of stable misincorporation during gap-filling DNA synthesis. The lower fidelity of Pol ν compared to that of Klenow fragment can be attributed primarily to a much lower catalytic efficiency for correct dNTP incorporation, whereas both enzymes have similar kinetic parameters for G-dTTP misinsertion. The major contributor to sequence-dependent differences in Pol ν error rates is the reaction rate, k(pol). In the sequence context where fidelity is highest, k(pol) for correct G-dCTP incorporation by Pol ν is ~15-fold faster than k(pol) for G-dTTP misinsertion. However, in sequence contexts where the error rate is higher, k(pol) is the same for both correct and mismatched dNTPs, implying that the transition state does not provide additional discrimination against misinsertion. The results suggest that Pol ν may be fine-tuned to function when high enzyme activity is not a priority and may even be disadvantageous and that the relaxed active-site specificity toward the G-dTTP mispair may be associated with its cellular function(s).     

Contributions of active site residues to cofactor binding and catalysis of 3α-hydroxysteroid dehydrogenase/carbonyl reductase

3α-Hydroxysteroid dehydrogenase/carbonyl reductase reversely catalyzes the oxidation of androsterone with NAD⁺ to form androstanedione and NADH. In this study, we investigated the function of active site residues N86, Y155, and K159 in NADH binding and catalysis in the reduction of androstanedione, using site-directed mutagenesis, steady-state kinetics, fluorescence quenching, and anisotropy measurements. The N86A, Y155F, and K159A mutant enzymes decreased the catalytic constant by 37- to 220-fold and increased the dissociation constant by 3- to 75-fold, respectively. Binding of NADH with wild-type and mutant enzymes caused different levels of fluorescence resonance energy transfer, implying a different orientation of nicotinamide ring versus W173. In addition, the enzyme-bound NADH decreased the fluorescence anisotropy value in the order WT > N86A > Y155F > K159A, indicating an increase in the mobility of the bound NADH for the mutants. Data suggest that hydrogen bonding with the hydroxyl group of nicotinamide ribose by K159 and Y155 is important to maintain the orientation of NADH and contributes greatly to the transition-state binding energy to facilitate the catalysis. N86 is important for stabilizing the position of K159. Substitution of alanine for N86 has a minor effect on NADH binding through K159, resulting in a slight increase in the mobility of the bound NADH and decreases in affinity and catalytic constant.     

Design and characterization of new β-glucuronidase active site variants with altered substrate specificity

Objective

To isolate and characterize the kinetics of variants of E. coli β-glucuronidase (GUS) having altered substrate specificity.

Results

Two small combinatorial libraries of E. coli GUS variants were constructed and screened for improved activities towards the substrate p-nitrophenyl-β-d-galactoside (pNP-gal). Nine of the most active variants were purified and their kinetic parameters were determined. These variants show up to 134-fold improved k_cat/K_M value towards pNP-gal compared to wild-type GUS, up to 9 × 10⁸-fold shift in specificity from p-nitrophenyl-β-d-glucuronide (pNP-glu) to pNP-gal compared to wild-type, and 10³-fold increase in specificity shift compared to a previously evolved GUS variant.

Conclusions

The kinetic data collected for nine new GUS variants is invaluable for training computational protein design models that better predict amino acid substitutions which improve activity of enzyme variants having altered substrate specificity.

     

DNA polymerase δ and ζ switch by sharing accessory subunits of DNA polymerase δ

Translesion DNA synthesis is an important branch of the DNA damage tolerance pathway that assures genomic integrity of living organisms. The mechanisms of DNA polymerase (Pol) switches during lesion bypass are not known. Here, we show that the C-terminal domain of the Pol ζ catalytic subunit interacts with accessory subunits of replicative DNA Pol δ. We also show that, unlike other members of the human B-family of DNA polymerases, the highly conserved and similar C-terminal domains of Pol δ and Pol ζ contain a [4Fe-4S] cluster coordinated by four cysteines. Amino acid changes in Pol ζ that prevent the assembly of the [4Fe-4S] cluster abrogate Pol ζ function in UV mutagenesis. On the basis of these data, we propose that Pol switches at replication-blocking lesions occur by the exchange of the Pol δ and Pol ζ catalytic subunits on a preassembled complex of accessory proteins retained on DNA during translesion DNA synthesis.     

Consecutive incorporation of fluorophore-labeled nucleotides by mammalian DNA polymerase β

In the present study, we investigated mammalian polymerases that consecutively incorporate various fluorophore-labeled nucleotides. We found that rat DNA polymerase β (pol β) consecutively incorporated fluorophore-labeled nucleotides to a greater extent than four bacterial polymerases, Sequenase Version 2.0, Vent_R (exo-), DNA polymerase IIIα and the Klenow fragment, and the mammalian polymerases DNA polymerase α and human DNA polymerase δ, under mesophilic conditions. Furthermore, we investigated the kinetics of correct or mismatched incorporation with labeled nucleotides during synthesis by rat pol β. The kinetic parameters K_m and k_cat were measured and used for evaluating: (i) the discrimination against correct pair incorporation of labeled nucleotides relative to unlabeled nucleotides; and (ii) the fidelity for all nucleotide combinations of mismatched pairs in the presence of labeled or unlabeled nucleotides. We also investigated the effect of fluorophore-labeled nucleotides on terminal deoxynucleotidyl transferase activity of rat pol β. We have demonstrated for the first time that mammalian pol β can consecutively incorporate various fluorophore-labeled dNTPs. These findings suggest that pol β is useful for high-density labeling of DNA probes and single-molecule sequencing for high-speed genome analysis.     

The spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β

To provide molecular-level insights into the spontaneous replication error and the mismatch discrimination mechanisms of human DNA polymerase β (polβ), we report four crystal structures of polβ complexed with dG•dTTP and dA•dCTP mismatches in the presence of Mg²⁺ or Mn²⁺. The Mg²⁺-bound ground-state structures show that the dA•dCTP-Mg²⁺ complex adopts an ‘intermediate’ protein conformation while the dG•dTTP-Mg²⁺ complex adopts an open protein conformation. The Mn²⁺-bound ‘pre-chemistry-state’ structures show that the dA•dCTP-Mn²⁺ complex is structurally very similar to the dA•dCTP-Mg²⁺ complex, whereas the dG•dTTP-Mn²⁺ complex undergoes a large-scale conformational change to adopt a Watson–Crick-like dG•dTTP base pair and a closed protein conformation. These structural differences, together with our molecular dynamics simulation studies, suggest that polβ increases replication fidelity via a two-stage mismatch discrimination mechanism, where one is in the ground state and the other in the closed conformation state. In the closed conformation state, polβ appears to allow only a Watson–Crick-like conformation for purine•pyrimidine base pairs, thereby discriminating the mismatched base pairs based on their ability to form the Watson–Crick-like conformation. Overall, the present studies provide new insights into the spontaneous replication error and the replication fidelity mechanisms of polβ.     

Targeting human DNA polymerase α for the inhibition of keratinocyte proliferation. Part 1. Homology model,active site architecture and ligand binding

In order to understand the binding modes of human DNA polymerase α (pol α) inhibitors on a molecular level, a 3D homology model of the active site of the enzyme was proposed based on the application of molecular modelling methods and molecular dynamic simulations using available crystal coordinates of pol α relatives. Docking results for a series of known nucleotide analogue inhibitors were consistent with reported experimental binding data and offered the possibility to elucidate structure-activity relationships via investigations of active site-inhibitor interactions. Furthermore, the study could explain, at least partially, the inhibitory effect of aphidicolin on pol α. In molecular dynamics simulations, aphidicolin occupied the catalytic centre, but acted in a not truly competitive manner with respect to nucleotides. It destabilized the replicating “closed” form of the pol α and transferred the enzyme into the inactive “open” conformation. This result is consistent with recent experiments on the binding mode of aphidicolin.     

Study of the inhibitory effect of fatty acids on the interaction between DNA and polymerase β

The binding of human DNA polymerase β (pol β) to DNA template-primer duplex and single-stranded DNA in the absence or presence of pol β inhibitors has been studied using a surface plasmon resonance biosensor. Two fatty acids, linoleic acid and nervonic acid, were used as potent pol β inhibitors. In the interaction between pol β and DNA, pol β could bind to ssDNA in a single binding mode, but bound to DNA template-primer duplexes in a parallel mode. Both pol β inhibitors prevented the binding of pol β to the single strand overhang and changed the binding from parallel to single mode. The affinities of pol β to the template-primer duplex region in the presence of nervonic acid or linoleic acid were decreased by 20 and 5 times, respectively. The significant inhibitory effect of nervonic acid on the pol β-duplex interaction was due to both a 2-fold decrease in the association rate and a 9-fold increase in the dissociation rate. In the presence of linoleic acid, no significant change of association rate was observed, and the decrease in binding affinity of pol β to DNA was mainly due to 7-fold increase in the dissociation rate. Published in Russian in Biokhimiya, 2009, Vol. 74, No. 7, pp. 1000–1006. These authors contributed equally.     

Synthesis and assembly of soybean β-conglycinin in vitro

The construction of SP6-derived expression plasmids that encode normal and modified -conglycinin subunits is described. With the exception of an additional methionine at their NH₂-terminal ends and the lack of glycans, the normal subunits synthesized at the direction of these plasmids coresponded to mature and subunits isolated from soybean seeds. The subunits assembled into trimers in vitro that were equivalent in size to those formed in vivo. This result shows that the glycans are not required either for protein folding or oligomer assembly. Subunits produced from other plasmids, which had modifications in a highly conserved hydrophobic region in the COOH-terminal end of the subunits, either did not assemble or assembled at an extremely low rate compared to unmodified subunits. Structural changes at the more hydrophilic NH₂-terminal end had mixed effects. Several subunits modified in this region assembled into trimers at rates that were either equal or greater than those for normal subunits. Others assembled less completely than the normal subunits. Our results indicate that the in vitro synthesis and assembly assay will be useful in evaluating structure-function relationships in modified -conglycinin subunits. The results also show that structural changes at the NH₂-terminal end of the subunits are tolerated to a greater extent than modifications in the hydrophobic conserved region in the COOH-terminal half of the subunits, and this information will be useful in efforts to improve soybean quality.     

On the active site of β-hexosaminidase from latex of Ficus glabrata

N-Acetyl-β-d-hexosaminidase (EC 3.2.1.52), active against both p-nitrophenyl-N-acetyl-β-d-glucosaminide and p-nitrophenyl-N-acetyl-β-d-galactosaminide, is present in latex of Ficus glabrata. The final specific activity was increased 150-fold from crude extract after ammonium sulphate fractionation and affinity chromatography on Concanavalin A-Sepharose. The activity ratio β-N-acetylglucosaminidase-β-N-acetylgalactosaminidase remained constant. Substrate competition, competitive inhibition studies and Arrhenius plots confirm that, in the hexosamidase, only one kind of active site is responsible for both activities. Acetate and acetamide are more effective competitive inhibitors than iodoacetamide, N-acetylglucosamine and N-acetylgalactosamine more than glucosamine and galactosamine, and α-methylmannoside more than mannose, suggesting that the active site binds the N-acetyl moiety of the substrate and a hydrophobic interaction of the methyl group is involved. The difference between the strength of the inhibition by mannosamine with respect to glucosamine and galactosamine, that do not inhibit, seems to be due to the position at C-2 of the amino group in the pyranose ring.     

Single-nucleotide base excision repair DNA polymerase activity in C. elegans in the absence of DNA polymerase β

The base excision DNA repair (BER) pathway known to occur in Caenorhabditis elegans has not been well characterized. Even less is known about the DNA polymerase (pol) requirement for the gap-filling step during BER. We now report on characterization of in vitro uracil-DNA initiated BER in C. elegans. The results revealed single-nucleotide (SN) gap-filling DNA polymerase activity and complete BER. The gap-filling polymerase activity was not due to a DNA polymerase β (pol β) homolog, or to another X-family polymerase, since computer-based sequence analyses of the C. elegans genome failed to show a match for a pol β-like gene or other X-family polymerases. Activity gel analysis confirmed the absence of pol β in the C. elegans extract. BER gap-filling polymerase activity was partially inhibited by both dideoxynucleotide and aphidicolin. The results are consistent with a combination of both replicative polymerase(s) and lesion bypass/BER polymerase pol θ contributing to the BER gap-filling synthesis. Involvement of pol θ was confirmed in experiments with extract from pol θ null animals. The presence of the SN BER in C. elegans is supported by these results, despite the absence of a pol β-like enzyme or other X-family polymerase.     

DNA polymerase β-dependent cell survival independent of XRCC1 expression

Base excision repair (BER) is a primary mechanism for repair of base lesions in DNA such as those formed by exposure to the DNA methylating agent methyl methanesulfonate (MMS). Both DNA polymerase β (pol β)- and XRCC1-deficient mouse fibroblasts are hypersensitive to MMS. This is linked to a repair deficiency as measured by accumulation of strand breaks and poly(ADP-ribose) (PAR). The interaction between pol β and XRCC1 is important for recruitment of pol β to sites of DNA damage. Endogenous DNA damage can substitute for MMS-induced damage such that BER deficiency as a result of either pol β- or XRCC1-deletion is associated with sensitivity to PARP inhibitors. Pol β shRNA was used to knock down pol β in Xrcc1^+/+ and Xrcc1^−/− mouse fibroblasts. We determined whether pol β-mediated cellular resistance to MMS and PARP inhibitors resulted entirely from coordination with XRCC1 within the same BER sub-pathway. We find evidence for pol β-dependent cell survival independent of XRCC1 expression for both types of agents. The results suggest a role for pol β-dependent, XRCC1-independent repair. PAR immunofluorescence data are consistent with the hypothesis of a decrease in repair in both pol β knock down cell variants.     

Effects of active site residues of 3α-hydroxysteroid dehydrogenase from pseudomonas sp. b-0831 on its catalysis and cofactor binding

We overexpressed and purified 3α-hydroxysteroid dehydrogenase from Pseudomonas sp. B-0831 (Ps3αHSD) and its mutants where the active site residues known as the SYK triad, Ser114, Tyr153, and Lys157, were mutated. Ps3αHSD catalyzes the reaction by using a nucleotide cofactor. The NADH binding affinity of K157A mutant was much lower than that of the wild-type, mainly due to loss of a hydrogen bond. The decreased affinity would result in decreased k_cat. Compared to the wild-type, the mutants S114A and Y153F showed higher K_m and lower k_cat values in both oxidation and reduction reactions. Simultaneous mutation of S114A and Y153F resulted in a significant decrease in k_cat relative to the single mutant. These results are supported by the notion that Tyr153 is a catalytic base and Ser114 would be a substitute. Loss of hydrogen bonding with NADH upon the Y153F mutation resulted in increased enthalpy change, partially compensated by increased entropy change.