Characterization of the active site of Cystathionine y-Lyase from Saccharomyces Cerevisiae

SummaryPre-mRNA splicing is executed by the spliceosome. Structural characterization of the catalytically activated complex (B*) is pivotal for mechanistic understanding of catalysis of the branching reaction by the spliceosome. In this study, we assembled the B* complex on two different pre-mRNAs from Saccharomyces cerevisiae and determined the cryo-EM structures of four distinct B complexes at overall resolutions of 2.9-3.8 Å. The duplex between U2 snRNA and the branch point sequence (BPS) is located 13-20 Å away from the 5’-splice site (5’SS) in the B* complexes that are devoid of the step I splicing factors Yju2 and Cwc25. Recruitment of Yju2 into the active site brings the U2/BPS duplex into the vicinity of 5’SS, ready for branching. In the absence of Cwc25, the nucleophile from BPS is positioned about 4 Å away from, and remains to be activated by, the catalytic metal M2. This analysis reveals the functional mechanism of Yju2 and Cwc25 in branching. These four structures constitute compelling evidence for substrate-specific conformations of the spliceosome in a major functional state.

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Different specificities of two aldehyde dehydrogenases from Saccharomyces cerevisiae var. boulardii

Bioscience Reports ◽

10.1042/bsr20160529 ◽

2017 ◽

Vol 37 (2) ◽

Cited By ~ 5

Author(s):

Suprama Datta ◽

Uday S. Annapure ◽

David J. Timson

Keyword(s):

Saccharomyces Cerevisiae ◽

Steric Hindrance ◽

Active Site ◽

Limited Range ◽

Wild Type ◽

Yeast Saccharomyces Cerevisiae ◽

Aliphatic Aldehydes ◽

Aldehyde Dehydrogenases ◽

Equivalent Residue

Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous and endogenous aldehydes by catalysing their oxidation to carboxylic acid counterparts. The present study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes. Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met177, compared with the equivalent residue Leu196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates were partly driven by steric hindrance in the active site. A variant of wild-type Ald6p with the Met177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild-types. These data suggest that steric bulk in the active site of yeast aldehyde dehydrogenases is partially responsible for controlling specificity.

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Characterization of an Abnormal Antithrombin (Milano 2) with Defective Thrombin Binding

Thrombosis and Haemostasis ◽

10.1055/s-0038-1661681 ◽

1986 ◽

Vol 56 (03) ◽

pp. 349-352 ◽

Cited By ~ 2

Author(s):

A Tripodi ◽

A Krachmalnicoff ◽

P M Mannucci

Keyword(s):

Venous Thromboembolism ◽

Active Site ◽

Inhibitory Activity ◽

Antithrombin Iii ◽

Factor Xa ◽

Molecular Defect ◽

Affinity Adsorption ◽

Italian Family ◽

Crossed Immunoelectrophoresis

SummaryFour members of an Italian family (two with histories of venous thromboembolism) had a qualitative defect of antithrombin III reflected by normal antigen concentrations and halfnormal antithrombin activity with or without heparin. Anti-factor Xa activities were consistently borderline low (about 70% of normal). For the propositus’ plasma and serum the patterns of antithrombin III in crossed-immunoelectrophoresis with or without heparin were indistinguishable from those of normal plasma or serum. A normal affinity of antithrombin III for heparin was documented by heparin-sepharose chromatography. Affinity adsorption of the propositus’ plasma to human α-thrombin immobilized on sepharose beads revealed defective binding of the anti thrombin III to thrombin-sepharose. Hence the molecular defect of this variant appears to be at the active site responsible for binding and neutralizing thrombin, thus accounting for the low thrombin inhibitory activity.

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Taxonomic characterization and identification of Saccharomyces cerevisiae D8 for brandy production from pineapple

TAP CHI SINH HOC ◽

10.15625/0866-7160/v39n4.10864 ◽

2018 ◽

Vol 39 (4) ◽

pp. 474-482

Author(s):

Hoang Thi Le Thuong ◽

Nguyen Quang Hao ◽

Tran Thi Thuy

Keyword(s):

Saccharomyces Cerevisiae ◽

Low Ph ◽

Yeast Strains ◽

Biological Studies ◽

Taxonomic Characterization

Eight yeast strains (denoted as D1 to D8) were isolated from samples of natural fermented pineapple. Strain D8 showed highest alcoholic production at low pH and special aroma of pineapple has been chosen for further study. Taxonomic characterization of strain D8 using morphological, biochemical and molecular biological studies confirmed that strain D8 belong to Saccharomycetaceae family, Saccharomycetales order and Saccharomyces cerevisiae species. Therefore, we named this strain as Saccharomyces cerevisiae D8 for further study on Brandy production from pineapple. Citation: Hoang Thi Le Thuong, Nguyen Quang Hao, Tran Thi Thuy, 2017. Taxonomic characterization and identification of Saccharomyces cerevisiae D8 for brandy production from pineapple. Tap chi Sinh hoc, 39(4): 474- 482. DOI: 10.15625/0866-7160/v39n4.10864.*Corresponding author: [email protected] Received 5 December 2016, accepted 12 August 2017

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