Human NEIL3 is mainly a monofunctional DNA glycosylase removing spiroimindiohydantoin and guanidinohydantoin.

Base excision repair is the major pathway for removal of oxidative DNA base damage. This pathway is initiated by DNA glycosylases, which recognize and excise damaged bases from DNA. In this work, we have purified the glycosylase domain (GD) of human DNA glycosylase NEIL3. The substrate specificity has been characterized and we have elucidated the catalytic mechanisms. GD NEIL3 excised the hydantoin lesions spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) in single-stranded (ss) and double-stranded (ds) DNA efficiently. NEIL3 also removed 5-hydroxy-2'-deoxycytidine (5OHC) and 5-hydroxy-2'-deoxyuridine (5OHU) in ssDNA, but less efficiently than hydantoins. Unlike NEIL1 and NEIL2, which possess a ß,δ-elimination activity, NEIL3 mainly incised damaged DNA by ß-elimination. Further, the base excision and strand incision activities of NEIL3 exhibited a non-concerted action, indicating that NEIL3 mainly operate as a monofunctional DNA glycosylase. The site-specific NEIL3 mutant V2P, however, showed a concerted action, suggesting that the N-terminal amino group in Val2 is critical for the monofunctional modus. Finally, we demonstrated that residue Lys81 is essential for catalysis.

Schizosaccharomyces pombe Ofd2 is a nuclear 2-oxoglutarate and iron dependent dioxygenase interacting with histones.

2-Oxoglutarate (2OG) dependent dioxygenases are ubiquitous iron containing enzymes that couple substrate oxidation to the conversion of 2OG to succinate and carbon dioxide. They participate in a wide range of biological processes including collagen biosynthesis, fatty acid metabolism, hypoxic sensing and demethylation of nucleic acids and histones. Although substantial progress has been made in elucidating their function, the role of many 2OG dioxygenases remains enigmatic. Here we have studied the 2OG and iron (Fe(II)) dependent dioxygenase Ofd2 in Schizosaccharomyces pombe, a member of the AlkB subfamily of dioxygenases. We show that decarboxylation of 2OG by recombinant Ofd2 is dependent on Fe(II) and a histidine residue predicted to be involved in Fe(II) coordination. The decarboxylase activity of Ofd2 is stimulated by histones, and H2A has the strongest effect. Ofd2 interacts with all four core histones, however, only very weakly with H4. Our results define a new subclass of AlkB proteins interacting with histones, which also might comprise some of the human AlkB homologs with unknown function.

Expression and characterization of endochitinase C from Serratia marcescens BJL200 and its purification by a one-step general chitinase purification method.

In this study we cloned, expressed, purified, and charaterized chitinase C1 from Serratia marcescens strain BJL200. As expected, the BJL200-ChiC1 amino acid sequence of this strain was highly similar to sequences of ChiC1 identified in two other strains of S. marcescens. BJL200-ChiC1 was overproduced in E. coli by the T7 expression system, and purified by a one-step hydrophobic interaction chromatography (HIC) with phenyl-sepharose. BJL200-ChiA and BJL200-ChiB had an approximately 30-fold higher k(cat) and 15 fold-lower K(m) than BJL200-ChiC1 for the oligomeric substrate 4-methylumbelliferyl-beta-D-N-N'-N''-triacetylchitotrioside, while BJL200-ChiC1 was 10-15 times faster than BJL200-ChiB and BJL200-ChiA in degrading the polymeric substrate CM-chitin-RBV. BJL200-ChiC1 degradation of beta-chitin resulted in a range of different chito-oligosaccharides (GlcNAc)(2) (main product), GlcNAc, (GlcNAc)(3), (GlcNAc)(4), and (GlcNAc)(5), indicating endo activity. The purification method used for BJL200-ChiC1 in this study is generally applicable to family 18 chitinases and their mutants, including inactive mutants, some of which tend to bind almost irreversibly to chitin columns. The high specificity of the interaction with the (non-chitinous) column material is mediated by aromatic residues that occur in the substrate-binding clefts and surfaces of the enzymes.

Thermodynamic analysis of L-arginine and N omega-hydroxy-L-arginine binding to nitric oxide synthase.

Isothermal titration calorimetry has been used to determine thermodynamic parameters of substrate binding to the oxygenase domain of neuronal nitric oxide synthase (nNOS(oxy)) in the presence of the cofactor tetrahydrobiopterin. The intermediate N(omega)-hydroxy-L-arginine (NHA) has a larger affinity than L-Arginine (L-Arg) for nNOS(oxy), with K(d)=0.4+/-0.1 microM and 1.7+/-0.3 microM at 25 degrees C, respectively. nNOS(oxy) binds NHA and L-Arg with DeltaH -4.1+/-0.2 and -1.0+/-0.1 kcal/mol and DeltaS=15 and 23 cal/Kmol respectively. NHA binding is more exothermic probably due to formation of an extra hydrogen bond in the active site compared to L-Arg. The changes in heat capacity (DeltaC(p)) are relatively small for binding of both NHA and L-Arg (-53+/-18 and -95+/-23 cal/L mol, respectively), which indicates that hydrophobic interactions contribute little to binding.

Thermodynamic analysis of allosamidin binding to a family 18 chitinase.

Inhibition of family 18 chitinases is emerging as a target for pest and fungal control as well as asthma and inflammatory therapy. One of the best known inhibitors for these enzymes is allosamidin, a natural product. While interactions of this compound with family 18 chitinases have been studied in much detail by X-ray crystallography and standard enzymology, details of the driving forces behind its tight binding remain unknown. We have studied the thermodynamics of allosamidin binding to chitinase B (ChiB), a family 18 chitinase from Serratia marcescens, using isothermal titration calorimetry. At pH 6.0, Kd is 0.16 +/- 0.04 microM, and the binding reaction is entropically driven (DeltaSr = 44 cal/K mol) with an enthalpic penalty (DeltaHr = 3.8 +/- 0.2 kcal/mol). Dissection of the entropic term shows that a favorable conformational change in the allosamidin-ChiB complex (DeltaSconf = 37 cal/K mol) is the main contributor to the reaction. At pH 8.5, Kd decreases to 0.03 muM and the binding reaction is less entropically favorable (DeltaSr = 30 cal/K mol). While the solvation entropy change (DeltaSsolv) increases from 15 cal/K mol at pH 6.0 to 46 cal/K mol at pH 8.5, DeltaSconf becomes small and negative (-8 cal/K mol) because of an enthalpy-entropy compensation. Analyses of proton transfer showed that at pH 6.0 binding of allosamidin requires deprotonation of the Asp142-Glu144 catalytic diad. At pH 8.5, the 142-144 diad is ionized in the native enzyme, relieving the deprotonation penalty of binding and explaining why binding becomes enthalpically favorable (DeltaHr = -1.2 +/- 0.2 kcal/mol).