DNA拓扑异构酶Ⅱ与ⅠA的进化分析

DNA拓扑异构酶与断开DNA链和改变其拓扑结构有关。先前的研究认为,拓扑异构酶TypeⅡB是由TypeⅡA通过基因的复制、重组和缺失所造成。然而,本研究结果则显示,TypeⅠA与TypeⅡB的进化关系较近,而TypeⅡA与TypeⅡB的关系较远。因此,TypeⅡB可能是由TypeⅠA演化而来,或者说,TypeⅡB是TypeⅠA在古细菌中的特化形式;TypeⅡB可能是由TypeⅡA通过形成TypeⅠA后演化而成的,而不是由TypeⅡA直接通过基因的复制、重组和缺失所造成。在由TypeⅡA演化成为TypeⅠA,最后演化为TypeⅡB的过程中,DNA拓扑异构酶的催化机制也发生了变化从需要金属离子的协助,演化到了不需要镁离子的存在协助其催化。     

Syntheses, structures and properties of copper(II) complexes containing N-(2-hydroxybenzyl)-amino amide ligands

Four copper(II) complexes containing the reduced Schiff base ligands, namely, N-(2-hydroxybenzyl)-glycinamide (Hsglym) and N-(2-hydroxybenzyl)-l-alaninamide (Hsalam) have been synthesized and characterized. The crystal structures of [Cu₂(sglym)₂Cl₂] (1), [Cu₂(salam)₂(NO₃)₂] · H₂O (3), [Cu₂(salam)₂(NO₃)(H₂O)](NO₃) · 1.5H₂O (4), [Cu₂(salam)₂](ClO₄)₂ · 2H₂O (5) show that the Cu(II) atoms are bridged by two phenolato oxygen atoms in the dimers. The sglym ligand bonded to Cu(II) in facial manner while salam ligand prefers to bind to Cu(II) in meridonal geometry. Variable temperature magnetic studies of 3 showed it is antiferromagnetic. These Cu(II) complexes and [Cu₂(sglym)₂(NO₃)₂] (2), exhibit very small catecholase activity as compared to the corresponding complexes containing acid functional groups.     

Synthesis, crystal structure, spectral properties and catalytic activity of hexakis(imidazole)manganese(II) bisindole-2-carboxylato bisneocuproine bisdimethyl sulfoxide solvates {[Mn(Im)6] · 2(2-IC) · 2(NC) · 2(DMSO)}

Synthesis, X-ray crystal structure and IR spectrum of {[Mn^II(Im)₆] · 2(2-IC) · 2(NC) · 2(DMSO)} (Im = imidazole, 2-HIC = indole-2-carboxylic acid, NC = 2,9-dimethyl-1,10-phenanthroline, DMSO = dimethyl sulfoxide) are reported. The manganese(II) ion has octahedral geometry with a MnN₆ core. The crystal structure is completed by two NC, two 2-IC⁻ and two DMSO solvate molecules. The individual cations are linked into chains running parallel to the a axis by four intermolecular hydrogen bonding involving two 2-IC⁻solvate. Moreover, these chains are connected by π-π stacking interactions which occur between neocuproine molecules related through inversion center. In IR spectroscopy, the compound spectrum is roughly similar to the imidazole one: (i) above 1800 cm⁻¹, the bands are broad, but when focussing on some of them a doublet structure can be found; (ii) below 1800 cm⁻¹, the bands are sharp and it is then possible to point out the modification of S-O band when this later is involved in bifurcated hydrogen bonding to a second solvate 2-IC⁻. The compound catalyses the disproportionation of H₂O₂; moreover an additional quantity of imidazole increases the reaction rate.     

Characterization of the two catalytic domains in histone deacetylase 6

Histone deacetylase 6 (HDAC6) is the only known HDAC with two potentially functional catalytic domains, yet the role towards substrate played by these two domains remains ambiguous. Most studies report HDAC6 activities measured using either immune complexes or in vitro translated products. Here, we characterize the activity of highly purified recombinant HDAC6, mutants with active site histidine mutations in each domain (H216A and H611A), and individual catalytic domains. The deacetylase activities of these proteins, as well as their kinetic parameters, were measured using histone, alpha-tubulin, and fluorogenic acetylated lysine as substrates. Mutant H216A only slightly lowers the catalytic rate. However, mutant H611A decreases the catalytic rate more than 5000-fold. The first domain expressed alone is not catalytically active. In contrast, the second domain shows only a modest decrease in substrate binding and product formation rate. Our results indicate that the in vitro deacetylase activity of HDAC6 resides in the C-terminal second catalytic domain.     

Structural insights into the catalytic mechanism of sphingomyelinases D and evolutionary relationship to glycerophosphodiester phosphodiesterases

Spider venom sphingomyelinases D catalyze the hydrolysis of sphingomyelin via an Mg(2+) ion-dependent acid-base catalytic mechanism which involves two histidines. In the crystal structure of the sulfate free enzyme determined at 1.85A resolution, the metal ion is tetrahedrally coordinated instead of the trigonal-bipyramidal coordination observed in the sulfate bound form. The observed hyperpolarized state of His47 requires a revision of the previously suggested catalytic mechanism. Molecular modeling indicates that the fundamental structural features important for catalysis are fully conserved in both classes of SMases D and that the Class II SMases D contain an additional intra-chain disulphide bridge (Cys53-Cys201). Structural analysis suggests that the highly homologous enzyme from Loxosceles bonetti is unable to hydrolyze sphingomyelin due to the 95Gly-->Asn and 134Pro-->Glu mutations that modify the local charge and hydrophobicity of the interfacial face. Structural and sequence comparisons confirm the evolutionary relationship between sphingomyelinases D and the glicerophosphodiester phosphoesterases which utilize a similar catalytic mechanism.     

Identification of the Catalytic Site of Phospholipase D2 (PLD2) Newly Described Guanine Nucleotide Exchange Factor Activity

We have demonstrated that phospholipase D2 (PLD2) is a guanine nucleotide exchange factor (GEF) for Rac2 and determined the PLD2 domains and amino acid site(s) responsible for its GEF activity. Experiments using GST fusion proteins or GST-free counterparts, purified proteins revealed that the PX domain is sufficient to exert GEF activity similar to full-length PLD2. The PLD2-GEF catalytic site is formed by a hydrophobic pocket of residues Phe-107, Phe-129, Leu-166, and Leu-173, all of which are in the PX domain. A nearby Arg-172 is also important in the overall activity. PX mutants altering any of those five amino acids fail to have GEF activity but still bind to Rac2, while their lipase activity was mostly unaffected. In addition to the PX domain, a region in the pleckstrin homology domain (Ile-306–Ala-310) aids in the PX-mediated GEF activity by providing a docking site to hold Rac2 in place during catalysis. We conclude that PLD2 is a unique GEF, with the PX being the major catalytic domain for its GEF activity, whereas the pleckstrin homology domain assists in the PX-mediated activity. The physiological relevance of this novel GEF in cell biology is demonstrated here in chemotaxis and phagocytosis of leukocytes, as the specific PX and PH mutants abolished cell function. Thus, this study reveals for the first time the catalytic site that forms the basis for the mechanism behind the GEF activity of PLD2.     

8R-Lipoxygenase-catalyzed synthesis of a prominent cis-epoxyalcohol from dihomo-γ-linolenic acid: a distinctive transformation compared with S-lipoxygenases

Conversion of fatty acid hydroperoxides to epoxyalcohols is a well known secondary reaction of lipoxygenases, described for S-specific lipoxygenases forming epoxyalcohols with a trans-epoxide configuration. Here we report on R-specific lipoxygenase synthesis of a cis-epoxyalcohol. Although arachidonic and dihomo-γ-linolenic acids are metabolized by extracts of the Caribbean coral Plexaura homomalla via 8R-lipoxygenase and allene oxide synthase activities, 20:3ω6 forms an additional prominent product, identified using UV, GC-MS, and NMR in comparison to synthetic standards as 8R,9S-cis-epoxy-10S-erythro-hydroxy-eicosa-11Z,14Z-dienoic acid. Both oxygens of (18)O-labeled 8R-hydroperoxide are retained in the product, indicating a hydroperoxide isomerase activity. Recombinant allene oxide synthase formed only allene epoxide from 8R-hydroperoxy-20:3ω6, whereas two different 8R-lipoxygenases selectively produced the epoxyalcohol.A biosynthetic scheme is proposed in which a partial rotation of the reacting intermediate is required to give the observed erythro epoxyalcohol product. This characteristic and the synthesis of cis-epoxy epoxyalcohol may be a feature of R-specific lipoxygenases.     

Catalytic mechanism of rhomboid protease GlpG probed by 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate

Rhomboid proteases have many important biological functions. Unlike soluble serine proteases such as chymotrypsin, the active site of rhomboid protease, which contains a Ser-His catalytic dyad, is submerged in the membrane and surrounded by membrane-spanning helices. Previous crystallographic analyses of GlpG, a bacterial rhomboid protease, and its complex with isocoumarin have provided insights into the mechanism of the membrane protease. Here, we studied the interaction of GlpG with 3,4-dichloroisocoumarin and diisopropyl fluorophosphonate, both mechanism-based inhibitors for the serine protease, and describe the crystal structure of the covalent adduct between GlpG and diisopropyl fluorophosphonate, which mimics the oxyanion-containing tetrahedral intermediate of the hydrolytic reaction. The crystal structure confirms that the oxyanion is stabilized by the main chain amide of Ser-201 and by the side chains of His-150 and Asn-154. The phosphorylation of the catalytic Ser-201 weakens its interaction with His-254, causing the catalytic histidine to rotate away from the serine. The rotation of His-254 is accompanied by further rearrangement of the side chains of Tyr-205 and Trp-236 within the substrate-binding groove. The formation of the tetrahedral adduct is also accompanied by opening of the L5 cap and movement of transmembrane helix S5 toward S6 in a direction different from that predicted by the lateral gating model. Combining the new structural data with those on the isocoumarin complex sheds further light on the plasticity of the active site of rhomboid membrane protease.     

Molecular imprint of enzyme active site by camel nanobodies: rapid and efficient approach to produce abzymes with alliinase activity

Screening of inhibitory Ab1 antibodies is a critical step for producing catalytic antibodies in the anti-idiotypic approach. However, the incompatible surface of the active site of the enzyme and the antigen-binding site of heterotetrameric conventional antibodies become the limiting step. Because camelid-derived nanobodies possess the potential to preferentially bind to the active site of enzymes due to their small size and long CDR3, we have developed a novel approach to produce antibodies with alliinase activities by exploiting the molecular mimicry of camel nanobodies. By screening the camelid-derived variable region of the heavy chain cDNA phage display library with alliinase, we obtained an inhibitory nanobody VHHA4 that recognizes the active site. Further screening with VHHA4 from the same variable domain of the heavy chain of a heavy-chain antibody library led to a higher incidence of anti-idiotypic Ab2 abzymes with alliinase activities. One of the abzymes, VHHC10, showed the highest activity that can be inhibited by Ab1 VHHA4 and alliinase competitive inhibitor penicillamine and significantly suppressed the B16 tumor cell growth in the presence of alliin in vitro. The results highlight the feasibility of producing abzymes via anti-idiotypic nanobody approach.