Academic literature on the topic 'His95'

Hydroxysteroid dehydrogenases (HSDHs) are from two superfamilies of short-chain dehydrogenase (SDR) and aldo–keto reductase (AKR). The HSDHs were summarized and classified according to their structural and functional differences. A typical pair of enzymes, 7α–hydroxysteroid dehydrogenase (7α–HSDH) and 7β–hydroxysteroid dehydrogenase (7β–HSDH), have been reported before. Molecular docking of 7-keto–lithocholic acid(7–KLA) to the binary of 7β–HSDH and nicotinamide adenine dinucleotide phosphate (NADP+) was realized via YASARA, and a possible binding model of 7β–HSDH and 7–KLA was obtained. The α side of 7–KLA towards NADP+ in 7β–HSDH, while the β side of 7–KLA towards nicotinamide adenine dinucleotide (NAD+) in 7α–HSDH, made the orientations of C7–OH different in products. The interaction between Ser193 and pyrophosphate of NAD(P)+ [Ser193–OG⋯3.11Å⋯O1N–PN] caused the upturning of PN–phosphate group, which formed a barrier with the side chain of His95 to make 7–KLA only able to bind to 7β–HSDH with α side towards nicotinamide of NADP+. A possible interaction of Tyr253 and C24 of 7–KLA may contribute to the formation of substrate binding orientation in 7β–HSDH. The results of sequence alignment showed the conservation of His95, Ser193, and Tyr253 in 7β–HSDHs, exhibiting a significant difference to 7α–HSDHs. The molecular docking of other two enzymes, 17β–HSDH from the SDR superfamily and 3(17)α–HSDH from the AKR superfamily, has furtherly verified that the stereospecificity of HSDHs was related to the substrate binding orientation.

PrP (prion-related protein) is a cell-surface Cu2+-binding glycoprotein which, when misfolded, is responsible for a number of transmissible spongiform encephalopathies. The co-ordination geometry, stoichiometry and affinity of Cu2+ for PrP are the subject of much debate. In the present paper, we review the recent progress we have made in these areas. As many as six Cu2+ ions bind to PrP with submicromolar affinity. Initially, two Cu2+ ions bind to full-length PrP in the amyloidogenic region, between the octarepeats and the structured domain, at His95 and His110. Only subsequent Cu2+ ions bind to single histidine residues within the octarepeat region. Competitive chelators have been used to determine the affinity of the first molar equivalent of Cu2+ bound to full-length PrP; this approach places the affinity in the nanomolar range. The affinity and number of Cu2+-binding sites support the suggestion that PrP could act as an antioxidant by binding potentially harmful Cu2+ ions and sacrificially quenching of free radicals generated as a result of copper redox cycling. Finally, the effect of Cu2+ on the prion structure and misassembly into oligomers and fibres is discussed.

ABSTRACT Bacillus subtilis contains three metalloregulatory proteins belonging to the ferric uptake repressor (Fur) family: Fur, Zur, and PerR. We have overproduced and purified Fur protein and analyzed its interaction with the operator region controlling the expression of the dihydroxybenzoate siderophore biosynthesis (dhb) operon. The purified protein binds with high affinity and selectivity to the dhb regulatory region. DNA binding does not require added iron, nor is binding reduced by dialysis of Fur against EDTA or treatment with Chelex. Fur selectively inhibits transcription from the dhb promoter by ςA RNA polymerase, even if Fur is added after RNA polymerase holoenzyme. Since neither DNA binding nor inhibition of transcription requires the addition of ferrous ion in vitro, the mechanism by which iron regulates Fur function in vivo is not obvious. Mutagenesis of the furgene reveals that in vivo repression of the dhb operon by iron requires His97, a residue thought to be involved in iron sensing in other Fur homologs. Moreover, we identify His96 as a second likely iron ligand, since a His96Ala mutant mediates repression at 50 μM but not at 5 μM iron. Our data lead us to suggest that Fur is able to bind DNA independently of bound iron and that the in vivo role of iron is to counteract the effect of an inhibitory factor, perhaps another metal ion, that antagonizes this DNA-binding activity.

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6–N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.

Human carbonic anhydrase II (CA-II) is a Zinc (Zn 2 + ) metalloenzyme responsible for maintenance of acid-base balance within the body through the reversible hydration of CO 2 to produce protons (H + ) and bicarbonate (BCT). Due to its importance, alterations to the amino acid sequence of the protein as a result of single nucleotide variations (nsSNVs) have detrimental effects on homeostasis. Six pathogenic CA-II nsSNVs, K18E, K18Q, H107Y, P236H, P236R and N252D were identified, and variant protein models calculated using homology modeling. The effect of each nsSNV was analyzed using motif analysis, molecular dynamics (MD) simulations, principal component (PCA) and dynamic residue network (DRN) analysis. Motif analysis identified 11 functionally important motifs in CA-II. RMSD data indicated subtle SNV effects, while PCA analysis revealed that the presence of BCT results in greater conformational sampling and free energy in proteins. DRN analysis showed variant allosteric effects, and the average betweenness centrality (BC) calculations identified Glu117 as the most important residue for communication in CA-II. The presence of BCT was associated with a reduction to Glu117 usage in all variants, suggesting implications for Zn 2 + dissociation from the CA-II active site. In addition, reductions to Glu117 usage are associated with increases in the usage of the primary and secondary Zn 2 + ligands; His94, His96, His119 and Asn243 highlighting potential compensatory mechanisms to maintain Zn 2 + within the active site. Compared to traditional MD simulation investigation, DRN analysis provided greater insights into SNV mechanism of action, indicating its importance for the study of missense mutation effects in proteins and, in broader terms, precision medicine related research.

The anticancer complexes cisplatin and carboplatin are known to bind to both the Nδand the N∊atoms of His15 of hen egg-white lysozyme (HEWL) in the presence of dimethyl sulfoxide (DMSO). However, neither binds in aqueous media after 4 d of crystallization and crystal growth, suggesting that DMSO facilitates cisplatin/carboplatin binding to the N atoms of His15 by an unknown mechanism. Crystals of HEWL cocrystallized with cisplatin in both aqueous and DMSO media, of HEWL cocrystallized with carboplatin in DMSO medium and of HEWL cocrystallized with cisplatin andN-acetylglucosamine (NAG) in DMSO medium were stored for between seven and 15 months. X-ray diffraction studies of these crystals were carried out on a Bruker APEX II home-source diffractometer at room temperature. Room-temperature X-ray diffraction data collection removed the need for cryoprotectants to be used, ruling out any effect that the cryoprotectants might have had on binding to the protein. Both cisplatin and carboplatin still bind to both the Nδand N∊atoms of His15 in DMSO media as expected, but more detail for the cyclobutanedicarboxylate (CBDC) moiety of carboplatin was observed at the N∊binding site. However, two molecules of cisplatin were now observed to be bound to His15 in aqueous conditions. The platinum peak positions were identified using anomalous difference electron-density maps as a cross-check withFo−FcOMIT electron-density maps. The occupancies of each binding site were calculated usingSHELXTL. These results show that over time cisplatin binds to both N atoms of His15 of HEWL in aqueous media, whereas this binding is speeded up in the presence of DMSO. The implication of cisplatin binding to proteins after a prolonged period of time is an important consideration for the length of treatment in patients who are given cisplatin.

SummaryThe cleavage of bovine κ-casein at the Phe105–Met106* bond by chymosin or pepsin is the first stage in casein micelle coagulation and casein digestion. The nature of the interaction of the peptide His98–Pro–His–Pro–His–Leu–Ser–Phe105–Met–Ala–Ile–Pro-Pro-Lys111 with chymosin and porcine pepsin was investigated using molecular modelling and energy minimization techniques. This study verified and extended a proposed model that electrostatic binding (involving His98, His100, His102 and Lys111 or Lys112) at either end of the active site cleft of chymosin is important for the positioning of residues 103–108 in the cleft. The peptide conformation remained unchanged in going from solution to binding into the active site cleft, with the exception that optimum binding of substrate to chymosin required the isomerization of the His98–Pro99 peptide bond from the trans to the cis conformation. The study also identified an acidic region in porcine pepsin that is in a position to form strong electrostatic interactions with the histidines at the N-terminus of the peptide.

Xerostomia, also known as dry mouth, is caused by a reduction in salivary secretion and by changes in the composition of saliva associated with the malfunction of salivary glands. Xerostomia decreases quality of life. In the present study, we investigated the effects of peptides derived from β-lactoglobulin C on age-dependent atrophy, gene expression profiles, and the dysfunction of salivary glands. Long-term oral administration of Leu57-Leu58-His59-Lys60 (LLHK), Leu58-His59-Lys60 (LHK) and His59-Lys60 (HK) peptides induced salivary secretion and prevented and/or reversed the age-dependent atrophy of salivary glands in older rats. The transcripts of 78 genes were upregulated and those of 81 genes were downregulated by more than 2.0-fold (p ≤ 0.05) after LHK treatment. LHK upregulated major salivary protein genes such as proline-rich proteins (Prpmp5, Prb3, Prp2, Prb1, Prp15), cystatins (Cst5, Cyss, Vegp2), amylases (Amy1a, Amy2a3), and lysozyme (Lyzl1), suggesting that LLHK, LHK, and HK restored normal salivary function. The AP-2 transcription factor gene (Tcfap2b) was also induced significantly by LHK treatment. These results suggest that LLHK, LHK, and HK-administration may prevent and/or reverse the age-dependent atrophy and functional decline of salivary glands by affecting gene expression.

碩士
國立交通大學
生物資訊研究所
97
Myoglobin (Mb) is a heme-protein, functioning as oxygen carrier in vertebrates. In previous study, MbH64D/V68L/I107M had been engineered into an enzyme with peroxidase activity. We further investigated the L29X/H64D/V68L/I107M and site-saturated H93X mutational effects on peroxidase activity. A well established peroxidase activity with one-electron oxidation reacted with 2’-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) was observed by the UV/Vis spectrophotometer and demonstrated its capability in electron transfer reaction. When ABTS was used as substrate, the results showed that the activity of quadruple mutants was less than triple mutant, indicating the important role of Leu-29 in maintaining peroxidase function, especially the catalysis of ABTS. The catalyses of H2¬¬O¬¬2 of MbL29I/H64D/V68L/I107M, MbL29F/H64D/V68L/I107M, and MbL29M/H64D/V68L/I107M were as good as that of triple mutants. The H93X mutants exhibited no activity, indicating that the peroxidase activity was related with the environment.

One of the emerging technologies is the use of microbial agents for the control of postharvest diseases of fruits and vegetables. A number of antagonistic microorganisms have been discovered which have the potential to effectively control postharvest diseases. Some of this technology has been patented and commercial products such as AspireTM (Ecogen Corporatin, Langhorne, PA, USA), Biosave 10TM and Biosave 11TM (Ecoscience Inc., Worchester, MA, USA) have been registered for commercial use. The principal investigator of this project was involved in developing the yeast-based biofungicide-AspireTM and testing its efficacy under commercial conditions. This research project was initiated to fill the gap between the knowledge available on development and commercial implementation of yeast biocontrol agents and basic understanding of various aspects related to introducing yeast antagonists to fruit surfaces, along with verification of population genetics. The main objectives of this study were: Study ecology, population dynamics and genetic diversity of the yeast antagonists Candida guilliermondii, C. oleophila, and Debaryomyces hansenii, and study the effect of preharvest application of the yeast antagonist C. oleophila naturally occurring epiphytic microbial population and on the development of postharvest diseases of citrus fruit during storage. Our findings, which were detailed in several publications, have shown that an epiphytic yeast population of grapefruit able to grow under high osmotic conditions and a wide range of temperatures was isolated and characterized for its biocontrol activity against green mold decay caused by Penicillium digitatum. Techniques based on random amplified polymorphic DNA (RAPD) and arbitrary primed polymerase chain reaction (ap-PCR), as well as homologies between sequences of the rDNA internal transcribed spacers (ITS) and 5.8S gene, were used to characterize the composition of the yeast population and to determine the genetic relationship among predominant yeast species. Epiphytic yeasts exhibiting the highest biocontrol activity against P. digitatum on grapefruit were identified as Candida guilliermondii, C. oleophila, C. sake, and Debaryomyces hansenii, while C. guilliermondii was the most predominant species. RAPD and ap-PCR analysis of the osmotolerant yeast population showed two different, major groups. The sequences of the ITS regions and the 5.8S gene of the yeast isolates, previously identified as belonging to different species, were found to be identical. Following the need to develop a genetically marked strain of the yeast C. oleophila, to be used in population dynamics studies, a transformation system for the yeast was developed. Histidine auxotrophy of C. oloephila produced using ethyl methanesulfonate were transformed with plasmids containing HIS3, HIS4 and HIS5 genes from Saccharomyces cerevisiae. In one mutant histidin auxotrophy was complemented by the HIS5 gene of S. cerevisiae is functionally homologous to the HIS5 gene in V. oleophila. Southern blot analysis showed that the plasmid containing the S. cerevisiae HIS5 gene was integrated at a different location every C. oleophila HIS+ transformant. There were no detectable physiological differences between C. oleophila strain I-182 and the transformants. The biological control ability of C. oleophila was not affected by the transformation. A genetically marked (with b-glucuronidase gene) transformant of C. oleophila colonized wounds on orange fruits and its population increased under field conditions. Effect of preharvest application of the yeast C. oleophila on population dynamics of epiphytic microbial population on wounded and unwounded grapefruit surface in the orchard and after harvest was also studied. In addition, the effect of preharvest application of the yeast C. oleophila on the development of postharvest decay was evaluated. Population studies conducted in the orchard showed that in control, non-treated fruit, colonization of wounded and unwounded grapefruit surface by naturally occurring filamentous fungi did not vary throughout the incubation period on the tree. On the other hand, colonization of intact and wounded fruit surface by naturally occurring yeasts was different. Yeasts colonized wounded surface rapidly and increased in numbers to about two orders of magnitude as compared to unwounded surface. On fruit treated with the yeast and kept on the tree, a different picture of fungal and yeast population had emerged. The detected fungal population on the yeast-treated intact surface was dramatically reduced and in treated wounds no fungi was detected. Yeast population on intact surface was relatively high immediately after the application of AspireTM and decreased to than 70% of that detected initially. In wounds, yeast population increased from 2.5 x 104 to about 4x106 after 72 hours of incubation at 20oC. Results of tests conducted to evaluate the effect of preharvest application of AspireTM on the development of postharvest decay indicated the validity of the approach.