Manganese peroxidase (MnP) from the white rot fungus Phanerochaete chrysosporium contains a manganese-binding site that plays a critical role in its function. Previously, a Mn(II)-binding site was ...designed into cytochrome c peroxidase (CcP) based on sequence homology (Yeung et al. in Chem. Biol. 4:215-222, 1997; Gengenbach et al. in Biochemistry 38:11425-11432, 1999). Here, we report a redesign of this site based on X-ray structural comparison of MnP and CcP. The variant, CcP(D37E, V45E, H181E), displays 2.5-fold higher catalytic efficiency (k (cat)/K (M)) than the variant in the original design, mostly due to a stronger K (M) of 1.9 mM (vs. 4.1 mM). High-resolution X-ray crystal structures of a metal-free form and a form with Co(II) at the designed Mn(II) site were also obtained. The metal ion in the engineered metal-binding site overlays well with Mn(II) bound in MnP, suggesting that this variant is the closest structural model of the Mn(II)-binding site in MnP for which a crystal structure exists. A major difference arises in the distances of the ligands to the metal; the metal-ligand interactions in the CcP variant are much weaker than the corresponding interactions in MnP, probably owing to partial occupancy of metal ion at the designed site, difference in the identity of metal ions (Co(II) rather than Mn(II)) and other interactions in the second coordination sphere. These results indicate that the metal ion, the ligands, and the environment around the metal-binding site play important roles in tuning the structure and function of metalloenzymes.
G·A mismatched base pairs are frequently found in nucleic acids. Human centromere DNA sequences contain unusual repeating motifs, e.g., (GAATG) n ·(CATTC) n found in the human chromosome. The ...purine-rich strand of this repeating pentamer sequence forms duplex and hairpin structures with unusual stability. The high stability of these structures is contributed by the “sheared” G·A base pairs which present a novel recognition surface for ligands and proteins. We have solved the crystal structure, by the multiple-wavelength anomalous diffraction (MAD) method of d(CCGAATGAGG) in which the centromere core sequence motif GAATG is embedded. Three crystal forms were refined to near-atomic resolution. The structures reveal the detailed conformation of tandem G·A base pairs whose unique hydrogen-bonding surface has interesting interactions with bases, hydrated magnesium ions, cobalt(III)hexaammine, spermine, and water molecules. The results are relevant in understanding the structure associated with human centromere sequence in particular and G·A base pairs in nucleic acids (including RNA, like ribozyme) in general.
The questions of whether different tautomeric forms of nucleic acid bases exist to any significant extent in DNA, or what their possible roles in mutation may be, are under intense scrutiny. ...2‘-Deoxyisoguanosine (iG) has been suggested to have a propensity to adopt the enol form. Isoguanine (also called 2-hydroxyadenine) can be found in oxidatively damaged DNA generated from treating DNA with a Fenton-type reactive oxygen-generating system and is known to cause mutation. We have analyzed the three-dimensional structure of the DNA dodecamer d(CGCiGAATTTGCG) (denoted iG-DODE) by X-ray crystallography and NMR. The crystal structure of the iG-DODE complexed with the minor groove binder Hoechst 33342, refined to 1.4 Å resolution, showed that the two independent iG·T base pairs in the dodecamer duplex adopt different (one in Watson−Crick and the other in wobble) conformations. The high-resolution nature of the structure also affords unprecedented clear information about the conformation and interactions of the Hoechst drug. The Hoechst 33342 binds in the narrow minor groove at the iGAATT site, with the N-methylpiperazine ring near the iG4·T21 base pair. Three hydrogen bonds are found between the NH of the Hoechst ligand and T-O2 DNA atoms. In solution, the two iG·T base pairs in iG-DODE predominantly are in the wobble form at 2 °C. At higher temperatures, another duplex form (likely involving the enol form of iG) is in slow exchange with the keto form and becomes significantly populated, reaching ∼40% at 40 °C. Our data support the conclusion that iG pairs with T in a Watson−Crick configuration to a significant extent at physiological temperature (37 °C), which may explain the facile incorporation rate of T across from an iG during in vitro DNA replication.
The potent anticancer drug actinomycin D (ActD) acts by binding to DNA, thereby interfering with replication and transcription. ActD inhibits RNA polymerase far more specifically than DNA polymerase. ...Such discrimination is not easily understood by the conventional DNA binding mode of ActD. We have solved and refined at 1.7 Å resolution the crystal structure of ActD complexed to CGATCGATCG, which contains no canonical GpC binding sequence. The crystal data are space group P43212, a = b = 47.01 Å, and c = 160.37 Å. The structure was solved by the multiple wavelength anomalous diffraction method using a 5-bromo-U DNA. The asymmetric unit of the unit cell contains two independent dimers of a novel slipped duplex complex consisting of two decamer DNA strands bound with two ActD drug molecules. (The DNA in one dimer is numbered C1 to G10 in one strand and C11 to G20 in the complementary strand and in the second dimer, C101 to G110 and C111 to G120, respectively.) The structure reveals a highly unusual ActD binding mode in which the DNA adopts a slipped duplex with the A3-T4/A13-T14 dinucleotides looped out. ActD intercalates between G2-C11* (C11* being from a symmetry-related molecule) and C5-G20 base pairs. Two such slipped duplex−ActD complexes bound to each other by mutually intercalating their T4/T14 bases into the helix cavities (located between C5-G20 and G6-C19 base pairs) of neighboring complexes, forming a dimer of drug−DNA complexes. The binding site mimics the drug binding at the elongation point during transcription. Modeling studies show that the ActD−DNA complex fits snugly in the active site cavity in RNA polymerase but not in DNA polymerase. This may explain the strong preference of ActD inhibition toward transcription.
Der gezielte Entwurf funktioneller Enzyme mit hohem Turnover, insbesondere solcher mit komplexem aktivem Zentrum wie respiratorischer Oxidasen, ist eine Herausforderung. Durch Einführung zweier His‐ ...und eines Tyr‐Restes in Myoglobin wurden Enzyme erhalten, die O2 in mehr als 1000 Turnovers und mit minimaler Freisetzung reaktiver Sauerstoffspezies zu H2O reduzieren (rote Linie im Schema). Die Platzierung des Tyr‐Restes ist entscheidend für die Aktivität.
The concentration dependence of the surface tension of single and mixed electrolyte aqueous solutions is studied, based on the assumption that the surface layer can be treated as a separate phase ...located between vapor and bulk liquid phases. The mean spherical approximation modified by Lu et al. J.-F. Lu, Y.-X. Yu, Y.-G. Li, Fluid Phase Equilibria 85 (1993) 81–100 is used to calculate the activity coefficients of water in the surface and bulk liquid phases. The relation between the electrolyte concentration in the surface and bulk liquid phases is established and only one parameter needs to be determined. The surface tensions for 31 single electrolyte aqueous solutions are correlated and the overall average absolute deviation is 0.70%. The surface tensions at different temperatures are predicted with the parameters obtained at one fixed temperature. By introducing the proper mixing rules, the surface tensions for 14 mixed electrolyte aqueous solutions are predicted without any mixing parameters, and the total average absolute deviation is 0.63%. All the calculated results are compared with that of the surface tension model for aqueous electrolyte solutions proposed by Li et al. Z.-B. Li, Y.-G. Li, J.-F. Lu, Ind. Eng. Chem. Res. 38 (1999) 1133–1139.
Mononuclear cupredoxin proteins usually contain a coordinately saturated type 1 copper (T1Cu) center and function exclusively as electron carriers. Here we report a cupredoxin isolated from the ...nitrifying archaeon Nitrosopumilus maritimus SCM1, called Nmar1307, that contains a T1Cu center with an open binding site containing water. It displays a deep purple color due to strong absorptions around 413 nm (1880 M–1 cm–1) and 558 nm (2290 M–1 cm–1) in the UV–vis electronic spectrum. EPR studies suggest the protein contains two Cu(II) species of nearly equal population, one nearly axial, with hyperfine constant A∥ = 98 × 10–4 cm–1, and another more rhombic, with a smaller A∥ value of 69 × 10–4 cm–1. The X-ray crystal structure at 1.6 Å resolution confirms that it contains a Cu atom coordinated by two His and one Cys in a trigonal plane, with an axial H2O at 2.25 Å. Both UV–vis absorption and EPR spectroscopic studies suggest that the Nmar1307 can oxidize NO to nitrite, an activity that is attributable to the high reduction potential (354 mV vs SHE) of the copper site. These results suggest that mononuclear cupredoxins can have a wide range of structural features, including an open binding site containing water, making this class of proteins even more versatile.
Sso7d and Sac7d are two small chromatin proteins from the hyperthermophilic archaeabacterium
Sulfolobus solfataricus and
Sulfolobus acidocaldarius, respectively. The crystal structures of ...Sso7d-GTGATCGC, Sac7d-GTGATCGC and Sac7d-GTGATCAC have been determined and refined at 1.45 Å, 2.2 Å and 2.2 Å, respectively, to investigate the DNA binding property of Sso7d/Sac7d in the presence of a T-G mismatch base-pair. Detailed structural analysis revealed that the intercalation site includes the T-G mismatch base-pair and Sso7d/Sac7d bind to that mismatch base-pair in a manner similar to regular DNA. In the Sso7d-GTGATCGC complex, a new inter-strand hydrogen bond between T2O4 and C14N4 is formed and well-order bridging water molecules are found. The results suggest that the less stable DNA stacking site involving a T-G mismatch may be a preferred site for protein side-chain intercalation.
Formaldehyde (HCHO) cross-links the anticancer drug daunorubicin (DAU) to DNA efficiently. When DAU is mixed with DNA hexamers, d(CGCGCG) and d(CGTDCG), in the presence of HCHO, stable covalent ...adducts of DNA are formed, as shown by the HPLC analyses. The major adducts are identical with the materials in the respective crystals which can be readily obtained from the 1:1 mixture of DAU-d(CGCGCG) and DAU-d(CGTDCG) plus HCHO, but not from the solution without HCHO. The high-resolution (1.5 A) X-ray crystal structure of those adducts shows unambiguously that they contain a covalent methylene bridge between the N3' of daunosamine and the N2 of the guanine or 2-aminoadenine. The perfect juxtaposition of the two amino groups in the minor groove of the complex provides a template for an efficient addition of HCHO. The methylene bridge does not perturb the conformation of the drug-DNA complex, when compared to the structure of DAU-d(CGTACG). The results suggest new approaches for synthesizing a new type of potential anticancer drug by attaching a reactive (e.g., alkylating) functional group at the N3' amino position of daunorubicin/doxorubicin. The stable drug-DNA adduct may be useful as probes for other biological studies.
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