Characterization of the helicase activity and substrate specificity of Mycobacterium tuberculosis UvrD

UvrD is a helicase that is widely conserved in gram-negative bacteria. A uvrD homologue was identified in Mycobacterium tuberculosis on the basis of the homology of its encoded protein with Escherichia coli UvrD, with which it shares 39% amino acid identity, distributed throughout the protein. The gene was cloned, and a histidine-tagged form of the protein was expressed and purified to homogeneity. The purified protein had in vitro ATPase activity that was dependent upon the presence of DNA. Oligonucleotides as short as four nucleotides were sufficient to promote the ATPase activity. The DNA helicase activity of the enzyme was only fueled by ATP and dATP. UvrD preferentially unwound 3'-single-stranded tailed duplex substrates over 5'-single-stranded ones, indicating that the protein had a duplex-unwinding activity with 3'-to-5' polarity. A 3' single-stranded DNA tail of 18 nucleotides was required for effective unwinding. By using a series of synthetic oligonucleotide substrates, we demonstrated that M. tuberculosis UvrD has an unwinding preference towards nicked DNA duplexes and stalled replication forks, representing the likely sites of action in vivo. The potential role of M. tuberculosis UvrD in maintenance of bacterial genomic integrity makes it a promising target for drug design against M. tuberculosis.     

DNA substrate specificity of DNA helicase E from calf thymus.

DNA helicase E from calf thymus has been characterized with respect to DNA substrate specificity. The helicase was capable of displacing DNA fragments up to 140 nucleotides in length, but was unable to displace a DNA fragment 322 nucleotides in length. DNA competition experiments revealed that helicase E was moderately processive for translocation on single strand M13mp18 DNA, and that the helicase would dissociate and rebind during a 15 minute reaction. Comparison of the rate of ATPase activity catalyzed by helicase E on single strand DNA substrates of different lengths, suggested a processivity consistent with the competition experiments. The helicase displayed a preference for displacing primers whose 5' terminus was fully annealed as opposed to primers with a 12 nucleotide 5' unannealed tail. The presence of a 12 nucleotide 3' tail had no effect on the rate of displacement. DNA helicase E was capable of displacing a primer downstream of either a four nucleotide gap, a one nucleotide gap or a nick in the DNA substrate. Helicase E was inactive on a fully duplex DNA 30 base pairs in length. Calf thymus RP-A stimulated the DNA displacement activity of helicase E. These properties are consistent with a role for DNA helicase E in chromosomal DNA repair.     

Biochemical characterization of the DNA substrate specificity of Werner syndrome helicase

Werner syndrome is a hereditary premature aging disorder characterized by genome instability. The product of the gene defective in WS, WRN, is a helicase/exonuclease that presumably functions in DNA metabolism. To understand the DNA structures WRN acts upon in vivo, we examined its substrate preferences for unwinding. WRN unwound a 3'-single-stranded (ss)DNA-tailed duplex substrate with streptavidin bound to the end of the 3'-ssDNA tail, suggesting that WRN does not require a free DNA end to unwind the duplex; however, WRN was completely blocked by streptavidin bound to the 3'-ssDNA tail 6 nucleotides upstream of the single-stranded/double-stranded DNA junction. WRN efficiently unwound the forked duplex with streptavidin bound just upstream of the junction, suggesting that WRN recognizes elements of the fork structure to initiate unwinding. WRN unwound two important intermediates of replication/repair, a 5'-ssDNA flap substrate and a synthetic replication fork. WRN was able to translocate on the lagging strand of the synthetic replication fork to unwind duplex ahead of the fork. For the 5'-flap structure, WRN specifically displaced the 5'-flap oligonucleotide, suggesting a role of WRN in Okazaki fragment processing. The ability of WRN to target DNA replication/repair intermediates may be relevant to its role in genome stability maintenance.     

XPB helicase regulates DNA incision by the Thermoplasma acidophilum endonuclease Bax1

Bax1 has recently been identified as a novel binding partner for the archaeal helicase XPB. We previously characterized Bax1 from Thermoplasma acidophilum as a Mg²⁺-dependent structure-specific endonuclease. Here we directly compare the endonuclease activity of Bax1 alone or in combination with XPB. Using several biochemical and biophysical approaches, we demonstrate regulation of Bax1 endonuclease activity by XPB. Interestingly, incision assays with Bax1 and XPB/Bax1 clearly demonstrate that Bax1 produces different incision patterns depending on the presence or absence of XPB. Using atomic force microscopy (AFM), we directly visualize and compare binding of Bax1 and XPB/Bax1 to different DNA substrates. Our AFM data support enhanced DNA binding affinity of Bax1 in the presence of XPB. Taken together, the DNA incision and binding results suggest that XPB is able to load and position Bax1 on the scissile DNA substrate, thus increasing the DNA substrate range of Bax1.     

19F-NMR reveals substrate specificity of CYP121A1 in Mycobacterium tuberculosis

Cytochromes P450 are versatile enzymes that function in endobiotic and xenobiotic metabolism and undergo meaningful structural changes that relate to their function. However, the way in which conformational changes inform the specific recognition of the substrate is often unknown. Here, we demonstrate the utility of fluorine (¹⁹F)-NMR spectroscopy to monitor structural changes in CYP121A1, an essential enzyme from Mycobacterium tuberculosis. CYP121A1 forms functional dimers that catalyze the phenol-coupling reaction of the dipeptide dicyclotyrosine. The thiol-reactive compound 3-bromo-1,1,1-trifluoroacetone was used to label an S171C mutation of the enzyme FG loop, which is located adjacent to the homodimer interface. Substrate titrations and inhibitor-bound ¹⁹F-NMR spectra indicate that ligand binding reduces conformational heterogeneity at the FG loop in both the dimer and in an engineered monomer of CYP121A1. However, only the dimer was found to promote a substrate-bound conformation that was preexisting in the substrate-free spectra, thus confirming a role for the dimer interface in dicyclotyrosine recognition. Moreover, ¹⁹F-NMR spectra in the presence of substrate analogs indicate the hydrogen-bonding feature of the dipeptide aromatic side chain as a dicyclotyrosine specificity criterion. This study demonstrates the utility of ¹⁹F-NMR as applied to a multimeric cytochrome P450, while also revealing mechanistic insights for an essential M. tuberculosis enzyme.     

Analysis of helicase activity and substrate specificity of Drosophila RECQ5

RecQ5 is one of five RecQ helicase homologs identified in humans. Three of the human RecQ homologs (BLM, WRN and RTS) have been linked to autosomal recessive human genetic disorders (Bloom syndrome, Werner syndrome and Rothmund–Thomson syndrome, respectively) that display increased genomic instability and cause elevated levels of cancers in addition to other symptoms. To understand the role of RecQ helicases in maintaining genomic stability, the WRN, BLM and Escherichia coli RecQ helicases have been characterized in terms of their DNA substrate specificity. However, little is known about other members of the RecQ family. Here we show that Drosophila RECQ5 helicase is a structure-specific DNA helicase like the other RecQ helicases biochemically characterized so far, although the substrate specificity is not identical to that of WRN and BLM helicases. Drosophila RECQ5 helicase is capable of unwinding 3′ Flap, three-way junction, fork and three-strand junction substrates at lower protein concentrations compared to 5′ Flap, 12 nt bubble and synthetic Holliday junction structures, which can be unwound efficiently by WRN and BLM.     

Analysis of the DNA substrate specificity of the human BACH1 helicase associated with breast cancer

We have investigated the DNA substrate specificity of BACH1 (BRCA1-associated C-terminal helicase). The importance of various DNA structural elements for efficient unwinding by purified recombinant BACH1 helicase was examined. The results indicated that BACH1 preferentially binds and unwinds a forked duplex substrate compared with a duplex flanked by only one single-stranded DNA (ssDNA) tail. In support of its DNA substrate preference, helicase sequestration studies revealed that BACH1 can be preferentially trapped by forked duplex molecules. BACH1 helicase requires a minimal 5 ' ssDNA tail of 15 nucleotides for unwinding of conventional duplex DNA substrates; however, the enzyme is able to catalytically release the third strand of the homologous recombination intermediate D-loop structure irrespective of DNA tail status. In contrast, BACH1 completely fails to unwind a synthetic Holliday junction structure. Moreover, BACH1 requires nucleic acid continuity in the 5 ' ssDNA tail of the forked duplex substrate within six nucleotides of the ssDNA-dsDNA junction to initiate efficiently DNA unwinding. These studies provide the first detailed information on the DNA substrate specificity of BACH1 helicase and provide insight to the types of DNA structures the enzyme is likely to act upon to perform its functions in DNA repair or recombination.     

Mycobacterium tuberculosis antigen 85A and 85C structures confirm binding orientation and conserved substrate specificity

The maintenance of the highly hydrophobic cell wall is central to the survival of Mycobacterium tuberculosis within its host environment. The antigen 85 proteins (85A, 85B, and 85C) of M. tuberculosis help maintain the integrity of the cell wall 1) by catalyzing the transfer of mycolic acids to the cell wall arabinogalactan and 2) through the synthesis of trehalose dimycolate (cord factor). Additionally, these secreted proteins allow for rapid invasion of alveolar macrophages via direct interactions between the host immune system and the invading bacillus. Here we describe two crystal structures: the structure of antigen 85C co-crystallized with octylthioglucoside as substrate, resolved to 2.0 A, and the crystal structure of antigen 85A, which was solved at a resolution of 2.7 A. The structure of 85C with the substrate analog identifies residues directly involved in substrate binding. Elucidation of the antigen 85A structure, the last of the three antigen 85 homologs to be solved, shows that the active sites of the three antigen 85 proteins are virtually identical, indicating that these share the same substrate. However, in contrast to the high level of conservation within the substrate-binding site and the active site, surface residues disparate from the active site are quite variable, indicating that three antigen 85 enzymes are needed to evade the host immune system.     

Characterization of DNA substrate specificities of apurinic/apyrimidinic endonucleases from Mycobacterium tuberculosis

Apurinic/apyrimidinic (AP) endonucleases are key enzymes involved in the repair of abasic sites and DNA strand breaks. Pathogenic bacteria Mycobacterium tuberculosis contains two AP endonucleases: MtbXthA and MtbNfo members of the exonuclease III and endonuclease IV families, which are exemplified by Escherichia coli Xth and Nfo, respectively. It has been shown that both MtbXthA and MtbNfo contain AP endonuclease and 3′ → 5′ exonuclease activities. However, it remains unclear whether these enzymes hold 3′-repair phosphodiesterase and nucleotide incision repair (NIR) activities. Here, we report that both mycobacterial enzymes have 3′-repair phosphodiesterase and 3′-phosphatase, and MtbNfo contains in addition a very weak NIR activity. Interestingly, depending on pH, both enzymes require different concentrations of divalent cations: 0.5 mM MnCl₂ at pH 7.6 and 10 mM at pH 6.5. MtbXthA requires a low ionic strength and 37°C, while MtbNfo requires high ionic strength (200 mM KCl) and has a temperature optimum at 60 °C. Point mutation analysis showed that D180 and N182 in MtbXthA and H206 and E129 in MtbNfo are critical for enzymes activities. The steady-state kinetic parameters indicate that MtbXthA removes 3′-blocking sugar-phosphate and 3′-phosphate moieties at DNA strand breaks with an extremely high efficiency (k_cat/K_M = 440 and 1280  μM^-1∙min⁻¹, respectively), while MtbNfo exhibits much lower 3′-repair activities (k_cat/K_M = 0.26 and 0.65 μM^-1∙min⁻¹, respectively). Surprisingly, both MtbXthA and MtbNfo exhibited very weak AP site cleavage activities, with kinetic parameters 100- and 300-fold lower, respectively, as compared with the results reported previously. Expression of MtbXthA and MtbNfo reduced the sensitivity of AP endonuclease-deficient E. coli xth nfo strain to methylmethanesulfonate and H₂O₂ to various degrees. Taken together, these data establish the DNA substrate specificity of M. tuberculosis AP endonucleases and suggest their possible role in the repair of oxidative DNA damage generated by endogenous and host- imposed factors.     

Inhibition of DNA helicase II unwinding and ATPase activities by DNA-interacting ligands. Kinetics and specificity.

Although DNA helicases play important roles in the processing of DNA, little is known about the effects of DNA-interacting ligands on these helicases. Therefore, the effects of a wide variety of DNA-binding ligands on the unwinding and ATPase reactions catalyzed by Escherichia coli DNA helicase II were examined. DNA minor groove binders and simple DNA intercalators did not inhibit helicase II. However, DNA intercalators, such as mitoxantrone and nogalamycin, which position functionalities in the major groove upon binding duplex DNA, were potent inhibitors of helicase II. To determine the mechanism by which mitoxantrone inhibited helicase II, the unwinding and DNA-dependent ATPase activities of helicase II were measured using a spectrum of double- and single-stranded DNA substrates. Using either a 71-base pair (bp) M13mp7 partially duplexed DNA substrate or a 245-bp bluntended, fully duplexed DNA substrate, the apparent Ki value for inhibition by mitoxantrone of both the unwinding and ATPase reactions was approximately 1 microM for both substrates, suggesting that the mechanism of inhibition of helicase II by mitoxantrone is the same for both substrates and requires the presence of double-stranded structure. To strengthen this conclusion, the ability of mitoxantrone to inhibit the DNA-dependent ATPase activity of helicase II was determined using two single-stranded substrates, poly(dT) and the 245-bp substrate after heat denaturation. Using either substrate, mitoxantrone inhibited the ATPase activity of helicase II far less effectively. Thus, these results indicate that the intercalation of mitoxantrone into double-stranded DNA, with accompanying placement of functionalities in the major groove, generates a complex that impedes helicase II, resulting in both inhibition of ATP hydrolysis and unwinding activity. Furthermore, we report here that DNA-binding ligands inhibit the unwinding activity of helicases I and IV and Rep protein from E. coli, demonstrating that the inhibition observed for helicase II is not unique to this enzyme.     

Rv2607 from Mycobacterium tuberculosis is a pyridoxine 5'-phosphate oxidase with unusual substrate specificity

Despite intensive effort, the majority of the annotated Mycobacterium tuberculosis genome consists of genes encoding proteins of unknown or poorly understood function. For example, there are seven conserved hypothetical proteins annotated as homologs of pyridoxine 5'-phosphate oxidase (PNPOx), an enzyme that oxidizes pyridoxine 5'-phosphate (PNP) or pyridoxamine 5'-phosphate (PMP) to form pyridoxal 5'-phosphate (PLP). We have characterized the function of Rv2607 from Mycobacterium tuberculosis H37Rv and shown that it encodes a PNPOx that oxidizes PNP to PLP. The k(cat) and K(M) for this reaction were 0.01 s(-1) and 360 μM, respectively. Unlike many PNPOx enzymes, Rv2607 does not recognize PMP as a substrate.     

Oxidizing substrate specificity of Mycobacterium tuberculosis alkyl hydroperoxide reductase E: kinetics and mechanisms of oxidation and overoxidation

Alkyl hydroperoxide reductase E (AhpE), a novel subgroup of the peroxiredoxin family, comprises Mycobacterium tuberculosis AhpE (MtAhpE) and AhpE-like proteins present in many bacteria and archaea, for which functional characterization is scarce. We previously reported that MtAhpE reacted ~ 10³ times faster with peroxynitrite than with hydrogen peroxide, but the molecular reasons for that remained unknown. Herein, we investigated the oxidizing substrate specificity and the oxidative inactivation of the enzyme. In most cases, both peroxidatic thiol oxidation and sulfenic acid overoxidation followed a trend in which those peroxides with the lower leaving-group pK_a reacted faster than others. These data are in agreement with the accepted mechanisms of thiol oxidation and support that overoxidation occurs through sulfenate anion reaction with the protonated peroxide. However, MtAhpE oxidation and overoxidation by fatty acid-derived hydroperoxides (~ 10⁸ and 10⁵ M^− 1 s^− 1, respectively, at pH 7.4 and 25 °C) were much faster than expected according to the Brønsted relationship with leaving-group pK_a. A stoichiometric reduction of the arachidonic acid hydroperoxide 15-HpETE to its corresponding alcohol was confirmed. Interactions of fatty acid hydroperoxides with a hydrophobic groove present on the reduced MtAhpE surface could be the basis of their surprisingly fast reactivity.     

Enzymatic methylation of the Mycobacterium tuberculosis heparin-binding haemagglutinin

Heparin-binding haemagglutinin (HBHA) is an important Mycobacterium tuberculosis virulence factor. It displays a complex methylation pattern in its C-terminal, functional domain, which protects this domain against proteolysis. Here, it is shown that HBHA methylation is catalysed by mycobacterial enzymes and a radio-enzymatic and a nonradioactive enzyme assay are described, based on the recognition of methylated HBHA by monoclonal antibodies. MS analysis of in vitro methylated HBHA shows a complex methylation pattern similar to that of naturally methylated HBHA. Using recombinant hybrid molecules as acceptor substrates, it was found that the N-terminal domain of HBHA is not required for recognition by the HBHA-methyltransferase(s), although it is required for in vivo methylation.     

Expression,purification and characterization of UvrD2 helicase from Mycobacterium tuberculosis

Helicases appear to be important for genome stability of dormant Mycobacterium tuberculosis responsible for latent tuberculosis infection and big proportion of active disease cases caused by reactivation. It was demonstrated that in both M. tuberculosis and Mycobacterium smegmatis, a helicase termed UvrD2 is essential for bacterial growth making it a promising target to fight tuberculosis. In many cases expression of soluble and active mycobacterial proteins in Escherichia coli is a complicated issue. In this work we for the first time report a non-trivial expression procedure in E. coli, leading to soluble UvrD2 from M. tuberculosis which possesses DNA-dependent ATP-ase activity.     

DNA binding by the substrate specificity (wedge) domain of RecG helicase suggests a role in processivity

RecG differs from most helicases acting on branched DNA in that it is thought to catalyze unwinding via translocation of a monomer on dsDNA, with a wedge domain facilitating strand separation. Conserved phenylalanines in the wedge are shown to be critical for DNA binding. When detached from the helicase domains, the wedge bound a Holliday junction with high affinity but failed to bind a replication fork structure. Further stabilizing contacts are identified in full-length RecG, which may explain fork binding. Detached from the wedge, the helicase region unwound junctions but had extremely low substrate affinity, arguing against the "classical inchworm" mode of translocation. We propose that the processivity of RecG on branched DNA substrates is dependent on the ability of the wedge to establish strong binding at the branch point. This keeps the helicase motor in contact with the substrate, enabling it to drive dsDNA translocation with high efficiency.