A heterologous overexpression system for mesophilic Pseudomonas aeruginosa holocytochrome c 551 (PA c 551 ) was established using Escherichia coli as a host organism. Amino acid residues were systematically substituted in three regions of PA c 551 with the corresponding residues from thermophilic Hydrogenobacter thermophilus cytochrome c 552 (HT c 552 ), which has similar main chain folding to PA c 551 , but is more stable to heat. Thermodynamic properties of PA c 551 with one of three single mutations (Phe-7 to Ala, Phe-34 to Tyr, or Val-78 to Ile) showed that these mutants had increased thermostability compared with that of the wild-type. Ala-7 and Ile-78 may contribute to the thermostability by tighter hydrophobic packing, which is indicated by the three dimensional structure comparison of PA c 551 with HT c 552 . In the Phe-34 to Tyr mutant, the hydroxyl group of the Tyr residue and the guanidyl base of Arg-47 formed a hydrogen bond, which did not exist between the corresponding residues in HT c 552 . We also found that stability of mutant proteins to denaturation by guanidine hydrochloride correlated with that against the thermal denaturation. These results and others described here suggest that significant stabilization of PA c 551 can be achieved through a few amino acid substitutions determined by molecular modeling with reference to the structure of HT c 552 . The higher stability of HT c 552 may in part be attributed to some of these substitutions.

Histone variants play important roles in the maintenance and regulation of the chromatin structure. In order to characterize the biochemical properties of the chromatin structure containing histone variants, we investigated the dynamic status of nucleosome core particles (NCPs) that were assembled with recombinant histones. We found that in the presence of nucleosome assembly protein I (NAP-I), a histone chaperone, H2A-Barr body deficient (H2A.Bbd) confers the most flexible nucleosome structure among the mammalian histone H2A variants known thus far. NAP-I mediated the efficient assembly and disassembly of the H2A.Bbd-H2B dimers from NCPs. This reaction was accomplished more efficiently when the NCPs contained H3.3, a histone H3 variant known to be localized in the active chromatin, than when the NCPs contained the canonical H3. These observations indicate that the histone variants H2A.Bbd and H3.3 are involved in the formation and maintenance of the active chromatin structure. We also observed that acidic histone binding proteins, TAF-I/SET and B23.1, demonstrated dimer assembly and disassembly activity, but the efficiency of their activity was considerably lower than that of NAP-I. Thus, both the acidic nature of NAP-I and its other functional structure(s) may be essential to mediate the assembly and disassembly of the dimers in NCPs.

In mammals, the resetting of DNA methylation patterns in early embryos and germ cells is crucial for development. De novo type DNA methyltransferases Dnmt3a and Dnmt3b are responsible for creating DNA methylation patterns during embryogenesis and in germ cells. Although their in vitro DNA methylation properties are similar, Dnmt3a and Dnmt3b methylate different genomic DNA regions in vivo. In the present study, we have examined the DNA methylation activity of Dnmt3a and Dnmt3b towards nucleosomes reconstituted from recombinant histones and DNAs, and compared it to that of the corresponding naked DNAs. Dnmt3a showed higher DNA methylation activity than Dnmt3b towards naked DNA and the naked part of nucleosomal DNA. On the other hand, Dnmt3a scarcely methylated the DNA within the nucleosome core region, while Dnmt3b significantly did, although the activity was low. We propose that the preferential DNA methylation activity of Dnmt3a towards the naked part of nucleosomal DNA and the significant methylation activity of Dnmt3b towards the nucleosome core region contribute to their distinct methylation of genomic DNA in vivo.

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. These reconstitute the water bridge. Based on these features, we suggest here a catalytic mechanism for hCAII: the tautomerization of His 64 can mediate the transfers of both protons and water molecules at a neutral pH with high efficiency, requiring no time-or energy-consuming processes.Carbonic anhydrase (CA) 2 (EC 4.2.1.1) is a ubiquitous enzyme that catalyzes the reversible hydration of carbon dioxide (1). Isozymes of carbonic anhydrase regulate or function in such diverse physiological processes as pH regulation, ion transport, water-electrolyte balance, bicarbonate secretion-absorption, bone resorption, maintenance of intraocular pressure, renal acidification, and brain development (2). Nonfunctioning CA is implicated in such diseases as osteopetrosis syndrome, glaucoma, respiratory acidosis, epilepsy, and Méni-ère syndrome. Diseases due to CA deficiency include those affecting bones, the brain, and the kidneys. Consequently determining the detailed structure/function relationships or mechanisms responsible for its catalytic properties is mandatory for developing inhibitors or replacement therapies.CA is present in at least three gene families (␣, ␤, and ␥), which has made it a popular model for the study of the evolution of gene families and protein folding, and for transgenic and gene target studies (2). Among the three families, the ␣ family is the best characterized, with 11 known isozymes identified in mammals. Earnhardt and co-workers have summarized maximal k cat and k cat /K m values for CO 2 hydration by isozyme I-VII (3). The human isozyme II (hCAII) has a remarkably high turnover rate or catalytic efficiency (k cat /K m ϭ 1.5 ϫ 10 8 M Ϫ1 s Ϫ1 ) that is very close to the frequency with which the enzyme and substrate molecules collide with each other in solution.It is widely accepted that the hydration of CO 2 catalyzed by hCAII proceeds through several chemical steps as shown in Scheme 1 (1, 4, 5): the direct nucleophilic attack of the zinc-bound hydroxide ion on the carbonyl carbon of substrate CO 2 (structures 1-2), the formation of a zinc-bound bicarbonate intermediate (structures 2-3), the isomerization of the bicarbonate ion (structures 3-4), the exchange of the product bicarbonate ion with a H 2 O (structures 4 -5), and the regeneration of the zinc-bound hydroxide ion by the transfer of a proton to bulk solvent (structures 1-5). The proton transfer step (structures 1-5) consists of two substeps: 1) an intra-molecular transfer of protons to another residue in the enzyme and 2) a release of protons to the outside of the enzyme with the aid of a base. The intra-molecular proton transfer is the rate-limiting step of the maximal turnover rate (10 6 s Ϫ1 ) at high concentrations of a base, whereas the proton release into the medium is rate-limiting at low buffer concentrations.