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231.
The B domain of CGTase has been generally accepted as a domain involved in thermostability. However, limited work has been performed in which entire B domain is substituted with the thermostable counterpart. Using overlap extension PCR, we replaced the B domain of a variant of CGTase Bacillus sp. G1 by six other B domains from thermostable CGTases. Likely due to distortion in the substrate-binding cleft adjacent to the active site, variants with the domain replacements from Thermoanaerobacter, Thermococcus, Thermococcus kodakarensis, Anaerobranca gottschalkii and Pyrococcus furiosus completely lost their catalytic function. A mutant designated Cgt_ET1 with a domain replacement from a Bacillus stearopthermophilus ET1 CGTase was the only variant that retained activity after domain exchange. Both the parental enzyme and the mutant Cgt_ET1 had an identical optimum temperature at 60 °C. The activity half-life was 22 min for the parental CGTase, whereas a marked increase to 57 min was observed for the mutant. Further mutagenesis on Cgt_ET1 was performed at residue 188 by replacing a Phe residue with Tyr. The mutant Cgt_ET1_F188Y displayed a decreased activity half-life of 28 min. Both mutants exhibited a better cyclodextrin-forming ability and a faster turnover rate (kcat) than the parental CGTase. 相似文献
232.
David F. Conga Mark Bowler Manuel Tantalean Daniel Montes Nicolau Maués Serra‐Freire Pedro Mayor 《Journal of medical primatology》2014,43(2):130-133
Parasites are important in the management of the health of primate populations. We examined 36 fecal samples from Peruvian red uakari monkeys (Cacajao calvus ucayalii) collected from wild animals in the northeastern Peruvian Amazon. Samples were positive for helminth infection. Nematodes egg: Strongyloididae, Trypanoxyuris sp., Spirurid, and a cestode egg were identified. 相似文献
233.
Identification of an intermediate of delta-aminolevulinate biosynthesis in Chlamydomonas by high-performance liquid chromatography 总被引:4,自引:0,他引:4
The first committed intermediate of the chlorophyll biosynthetic pathway is delta-aminolevulinic acid (ALA). In plant cells, ALA is formed from glutamate by a pathway not yet clearly defined. One of the proposed pathways involves the reduction of glutamate to glutamate-1-semialdehyde (GSA) via a glutamyl-tRNA intermediate. GSA is then converted to ALA by an aminotransferase. We are studying this pathway using partially purified components from Chlamydomonas reinhardtii in in vitro reactions with [3H]L-glutamate as the substrate and analysis of the radioactive reaction products via HPLC. In reactions either lacking GSA-aminotransferase or containing gabaculine (an inhibitor of aminotransferase), a radioactive intermediate is formed which cochromatographs with synthetic GSA. As observed previously for ALA synthesis, the synthesis of this intermediate has an absolute requirement for RNA, ATP, and active enzymes, while the requirement for NADPH is less stringent. Both the accumulated intermediate and the synthetic GSA can be converted to ALA by the aminotransferase without any additional substrates or cofactors. These results support previous observations that GSA or a very similar compound is an intermediate of ALA synthesis. 相似文献
234.
235.
236.
Amit K Singh Nisha Pandey Mau Sinha Punit Kaur Sujata Sharma Tej P Singh 《International Journal of Biochemistry and Molecular Biology》2011,2(4):328-339
Lactoperoxidase (LPO) is a member of the family of mammalian heme peroxidases. It catalyzes the oxidation of halides and pseudohalides in presence of hydrogen peroxide. LPO has been co-crystallized with inorganic substrates, SCN-, I-, Br- and Cl-. The structure determination of the complex of LPO with above four substrates showed that all of them occupied distinct positions in the substrate binding site on the distal heme side. The bound substrate ions were separated from each other by one or more water molecules. The heme iron is coordinated to His-351 Nϵ2 on the proximal side while it is coordinated to conserved water molecule W-1 on the distal heme side. W-1 is hydrogen bonded to Br- ion which is followed by Cl- ion with a hydrogen bonded water molecule W-5′ between them. Next to Cl- ion is a hydrogen bonded water molecule W-7′ which in turn is hydrogen bonded to W-8′ and N atom of SCN-. W-80 is hydrogen bonded to W-9′ which is hydrogen bonded to I-. SCN- ion also interacts directly with Asn-230 and through water molecules with Ser-235 and Phe-254. Therefore, according to the locations of four substrate anions, the order of preference for binding to lactoperoxidase is observed as Br- > Cl- > SCN- > I-. The positions of anions are further defined in terms of subsites where Br- is located in subsite 1, Cl- in subsite 2, SCN- in subsite 3 and I- in subsite 4. 相似文献
237.
Amit K. Singh Nagendra Singh Ashutosh Tiwari Mau Sinha Gajraj S. Kushwaha Punit Kaur A. Srinivasan Sujata Sharma T. P. Singh 《Journal of biological inorganic chemistry》2010,15(7):1099-1107
The mode of binding of aromatic ligands in the substrate binding site on the distal heme side in heme peroxidases is well
understood. However, the mode of diffusion through the extended hydrophobic channel and the regulatory role of the channel
are not yet clear. To provide answers to these questions, the crystal structure of the complex of lactoperoxidase and 3-amino-1,2,4-triazole
(amitrole) has been determined, which revealed the presence of two ligand molecules, one in the substrate binding site and
the second in the hydrophobic channel. The binding of ligand in the channel induced a remarkable conformational change in
the side chain of Phe254, which flips from its original distant position to interact with the trapped ligand in the hydrophobic
channel. As a result, the channel is completely blocked so that no ligand can diffuse through it to the substrate binding
site. Another amitrole molecule is bound to lactoperoxidase in the substrate binding site by replacing three water molecules,
including the crucial iron-bound water molecule, W1. In this arrangement, the amino nitrogen atom of amitrole occupies the
position of W1 and interacts directly with ferric iron. As a consequence, it prevents the binding of H2O2 to heme iron. Thus, the interactions of amitrole with lactoperoxidase obstruct both the passage of ligands through the hydrophobic
channel as well as the binding of H2O2. This explains the amitrole toxicity. From binding studies, the dissociation constant (K
d) for amitrole with lactoperoxidase was found to be approximately 5.5 × 10−7 M, indicating high affinity. 相似文献
238.
The “expensive tissue hypothesis” states that large brains are active at high metabolic rates, which have to be financed by a significant trade-off with other organs such as the alimentary tract. Recent morphological findings on primate brains and guts support this idea also considering the importance of high-energy diets as a possible driving power of this process. However, the trade-off correlation between brain and alimentary tract, the essence of the “expensive tissue hypothesis”, has not yet been tested using molecular data to complement morpho-functional findings. We therefore hypothesize that the activity of marker proteins expressed both in brain and alimentary tract should parallel functional morphology in organs at the molecular level. Thus, in animals feeding on hard to digest diet, we would expect a high concentration per unit mass of that marker protein in the digestive tract and reversely a lower concentration in the brain. In contrast, in animals feeding on easily-digested, high-energy food we would expect the reverse pattern. Recent preliminary studies suggest that carbonic anhydrase II (CA-II) could act as a marker. The enzyme concentration was found to increase in the brain with higher cerebral activity from cattle to humans and to reversely decrease in salivary secretions. The reverse concentration of CA-II in saliva and brains of cattle and primates might be the first molecular evidence of the validity of the “expensive tissue hypothesis”. 相似文献