Yeast (Saccharomyces cerevisiae) fructose-1,6-bisphosphatase. Properties of phospho and dephospho forms and of two mutants in which serine 11 has been changed by site-directed mutagenesis.

Friedelin, a pentacyclic triterpene found in the leaves of the Celastraceae species, demonstrates numerous biological activities and is a precursor of quinonemethide triterpenes, which are promising antitumoral agents. Friedelin is biosynthesized from the cyclization of 2,3-oxidosqualene, involving a series of rearrangements to form a ketone by deprotonation of the hydroxylated intermediate, without the aid of an oxidoreductase enzyme. Mutagenesis studies among oxidosqualene cyclases (OSCs) have demonstrated the influence of amino acid residues on rearrangements during substrate cyclization: loss of catalytic activity, stabilization, rearrangement control or specificity changing. In the present study, friedelin synthase from Maytenus ilicifolia (Celastraceae) was expressed heterologously in Saccharomyces cerevisiae. Site-directed mutagenesis studies were performed by replacing phenylalanine with tryptophan at position 473 (Phe473Trp), methionine with serine at position 549 (Met549Ser) and leucine with phenylalanine at position 552 (Leu552Phe). Mutation Phe473Trp led to a total loss of function; mutants Met549Ser and Leu552Phe interfered with the enzyme specificity leading to enhanced friedelin production, in addition to α-amyrin and β-amyrin. Hence, these data showed that methionine 549 and leucine 552 are important residues for the function of this synthase.

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Finding the Binding Site of Peloruside A and its Secondary Effects in Saccharomyces Cerevisiae using a Chemical Genetics Approach

10.26686/wgtn.16969795.v1 ◽

2021 ◽

Author(s):

◽

Reem Hanna

Keyword(s):

Saccharomyces Cerevisiae ◽

Cell Cycle ◽

Amino Acids ◽

Flow Cytometry ◽

Binding Site ◽

Mammalian Cells ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Peloruside A ◽

Microtubule Function

<p>Peloruside A, a natural product isolated from the marine sponge Mycale hentscheli, is a microtubule-stabilising agent that has a similar mechanism of action to the anticancer drug paclitaxel and is cytotoxic to cultured mammalian cells. Peloruside appears to bind to a distinct site on mammalian tubulin that is different from that of the taxoid-site drugs. Because of the high sequence homology between yeast and mammalian tubulin, Saccharomyces cerevisiae (S. cerevisiae) was used as a model organism to characterise the peloruside-binding site with the aim of advancing our understanding about this site on mammalian tubulin. Wild type S. cerevisiae (BY4741) was sensitive to peloruside at uM concentrations; however, a strain that lacks the mad2 (Mitotic Arrest Deficient 2) gene showed increased sensitivity to the drug at much lower uM concentrations. This gene is a component of the spindle-assembly checkpoint complex that delays the onset of anaphase in cells with defects in mitotic spindle assembly. The main aims of this project were to define the binding site of peloruside A using yeast tubulin to see if microtubule function and/or morphology is altered in yeast by peloruside, and to identify any secondary drug targets "friends of the target" through chemical genetic interactions profiling (Homozygous deletion profiling microarray). Site-directed mutagenesis was used to mutate two conserved amino acids (A296T; R306H) known to confer resistance to peloruside in mammalian cells. Based on a published computer model of the peloruside binding site on mammalian tubulin, we also mutated three other amino acids, two that were predicted to affect peloruside binding (Q291M and N337L), and one that was predicted to affect laulimalide binding but have little affect on peloruside binding (V333W). We also included a negative control that was predicted to have no effect on peloruside binding (R282Q) and would affect epothilone binding. We found that of the six point mutations, only Q291M failed to confer resistance in yeast and instead it increased the inhibition to the drug. Using a bud index assay, confocal microscopy, and flow cytometry, 40-50 uM peloruside was shown to block cells in G2/M of the cell cycle, confirming a direct action of the drug on microtubule function. Homozygous profiling (HOP) microarray analysis of a deletion mutant set of yeast genes was also carried out to identify gene products that interact with peloruside in order to link the drug to specific networks or biochemical pathways in the cells. From site-directed mutagenesis, we concluded that peloruside binds to yeast B-tubulin in the region predicted by the published model of the binding site, and therefore mapping the site on yeast tubulin could provide useful information about the mammalian binding site for peloruside. The bud index, flow cytometry, and confocal microscopy experiments provided further evidence that peloruside interacts with yeast tubulin. From HOP we found that peloruside has roles in the cell cycle, as expected, and has effects on protein transport, secretion, cell wall synthesis, and steroid biosynthesis pathways.</p>

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Identification of pre-mRNA polyadenylation sites in Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.4215-4229.1992 ◽

1992 ◽

Vol 12 (9) ◽

pp. 4215-4229

Author(s):

S Heidmann ◽

B Obermaier ◽

K Vogel ◽

H Domdey

Keyword(s):

Saccharomyces Cerevisiae ◽

Signal Sequence ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

Yeast Saccharomyces Cerevisiae ◽

Adh1 Gene ◽

Polyadenylation Sites ◽

Yeast Genes

In contrast to higher eukaryotes, little is known about the nature of the sequences which direct 3'-end formation of pre-mRNAs in the yeast Saccharomyces cerevisiae. The hexanucleotide AAUAAA, which is highly conserved and crucial in mammals, does not seem to have any functional importance for 3'-end formation in yeast cells. Instead, other elements have been proposed to serve as signal sequences. We performed a detailed investigation of the yeast ACT1, ADH1, CYC1, and YPT1 cDNAs, which showed that the polyadenylation sites used in vivo can be scattered over a region spanning up to 200 nucleotides. It therefore seems very unlikely that a single signal sequence is responsible for the selection of all these polyadenylation sites. Our study also showed that in the large majority of mRNAs, polyadenylation starts directly before or after an adenosine residue and that 3'-end formation of ADH1 transcripts occurs preferentially at the sequence PyAAA. Site-directed mutagenesis of these sites in the ADH1 gene suggested that this PyAAA sequence is essential for polyadenylation site selection both in vitro and in vivo. Furthermore, the 3'-terminal regions of the yeast genes investigated here are characterized by their capacity to act as signals for 3'-end formation in vivo in either orientation.

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Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7836-7849.1993 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7836-7849

Author(s):

P Russo ◽

W Z Li ◽

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

In Vitro Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Element ◽

A Site ◽

Derivatives Of ◽

Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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Gene Knockouts, in vivo Site-Directed Mutagenesis and Other Modifications Using the Delitto Perfetto System in Saccharomyces cerevisiae

Methods in Enzymology - Laboratory Methods in Enzymology: Cell, Lipid and Carbohydrate ◽

10.1016/b978-0-12-420067-8.00008-8 ◽

2013 ◽

pp. 103-131 ◽

Cited By ~ 24

Author(s):

Samantha Stuckey ◽

Francesca Storici

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Knockouts

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Site-directed Mutagenesis of Residues at Subunit Interfaces of Porcine Fructose-1,6-bisphosphatase

Journal of Biological Chemistry ◽

10.1074/jbc.271.6.3005 ◽

1996 ◽

Vol 271 (6) ◽

pp. 3005-3010 ◽

Cited By ~ 13

Author(s):

Lie-Fen Shyur ◽

Alexander E. Aleshin ◽

Richard B. Honzatko ◽

Herbert J. Fromm

Keyword(s):

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Fructose 1,6 Bisphosphatase

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Site-directed mutagenesis of the active site of diacylglycerol kinase α: calcium and phosphatidylserine stimulate enzyme activity via distinct mechanisms

Biochemical Journal ◽

10.1042/bj20031052 ◽

2003 ◽

Vol 375 (3) ◽

pp. 673-680 ◽

Cited By ~ 26

Author(s):

Takahiro ABE ◽

Xiaolan LU ◽

Ying JIANG ◽

Clark E. BOCCONE ◽

Shaomin QIAN ◽

...

Keyword(s):

Catalytic Domain ◽

Regulatory Region ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Kinase C ◽

Acidic Phospholipids ◽

Ef Hands ◽

Mutant Forms

Diacylglycerol kinases (DAGKs) catalyse ATP-dependent phosphorylation of sn-1,2-diacylglycerol that arises during stimulated phosphatidylinositol turnover. DAGKα is activated in vitro by Ca2+ and by acidic phospholipids. The regulatory region of DAGKα includes an N-terminal RVH motif and EF hands that mediate Ca2+-dependent activation. DAGKα also contains tandem C1 protein kinase C homology domains. We utilized yeast, Saccharomyces cerevisiae, which lacks an endogenous DAGK, to express DAGKα and to determine the enzymic activities of different mutant forms of pig DAGKα in vitro. Six aspartate residues conserved in all DAGKs were individually examined by site-directed mutagenesis. Five of these aspartate residues reside in conserved blocks that correspond to sequences in the catalytic site of phosphofructokinases. Mutation of D434 (Asp434) or D650 abolished all DAGKα activity, whereas substitution of one among D465, D497, D529 and D697 decreased the activity to 6% or less of that for wild-type DAGKα. Roles of homologous residues in phosphofructokinases suggested that the N-terminal half of the DAGK catalytic domain binds Mg-ATP and the C-terminal half binds diacylglycerol. A DAGKα mutant with its entire regulatory region deleted showed a much decreased activity that was not activated by Ca2+, but still exhibited PS (phosphatidylserine)-dependent activation. Moreover, mutations of aspartate residues at the catalytic domain had differential effects on activation by Ca2+ and PS. These results indicate that Ca2+ and PS stimulate DAGKα via distinct mechanisms.

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Signals that produce 3' termini in CYC1 mRNA of the yeast Saccharomyces cerevisiae.

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7836 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7836-7849 ◽

Cited By ~ 32

Author(s):

P Russo ◽

W Z Li ◽

Z Guo ◽

F Sherman

Keyword(s):

Saccharomyces Cerevisiae ◽

Site Directed Mutagenesis ◽

Yeast Cells ◽

In Vitro Mutagenesis ◽

Yeast Saccharomyces Cerevisiae ◽

Upstream Element ◽

A Site ◽

Derivatives Of ◽

Acting In Concert

The cyc1-512 mutant was previously shown to contain a 38-bp deletion, 8 nucleotides upstream from the major wild-type poly(A) site, in the CYC1 gene, which encodes iso-1-cytochrome c of the yeast Saccharomyces cerevisiae. This 38-bp deletion caused a 90% reduction in the CYC1 transcripts, which were heterogeneous in size, aberrantly long, and presumably labile (K. S. Zaret and F. Sherman, Cell 28:563-573, 1982). Site-directed mutagenesis in and adjacent to the 38-bp region was used to identify signals involved in the formation and positioning of CYC1 mRNA 3' ends. In addition, combinations of various putative 3' end-forming signals were introduced by in vitro mutagenesis into the 3' region of the cyc1-512 mutant. The combined results from both studies suggest that 3' end formation in yeast cells involves signals having the following three distinct but integrated elements acting in concert: (i) the upstream element, including sequences TATATA, TAG ... TATGTA, and TTTTTATA, which function by enhancing the efficiency of downstream elements; (ii) downstream elements, such as TTAAGAAC and AAGAA, which position the poly(A) site; and (iii) the actual site of polyadenylation, which often occurs after cytidine residues that are 3' to the so-called downstream element. While the upstream element is required for efficient 3' end formation, alterations of the downstream element and poly(A) sites generally do not affect the efficiency of 3' end formation but appear to alter the positions of poly(A) sites. In addition, we have better defined the upstream elements by examining various derivatives of TATATA and TAG ... TATGTA, and we have examined the spatial requirements of the three elements by systematically introducing or deleting upstream and downstream elements and cytidine poly(A) sites.

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