<scp>VII</scp>. Yeast sequencing reports. Complete sequence of the <i>Saccharomyces cerevisiae LEU1</i> gene encoding isopropylmalate isomerase

Skála, J; Capieaux, Etienne; Balzi, Elisabetta; Chen, Wei-Ning; Goffeau, André

doi:10.1002/yea.320070310

Cited by 20 publications

(7 citation statements)

References 25 publications

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“…In order to test whether S. cerevisiae can functionally express other heterologous [4Fe-4S] ISCproteins from E. coli within its cytosol we decided to test if the S. cerevisiae strain JAY20, auxotrophic for leucine due to a deletion of the LEU1 gene, encoding the second step in the biosynthesis of leucine, catalyzed by the isopropylmalate isomerase, could be complemented by the E. coli heterodimeric isopropylmalate isomerase, encoded by leuC and leuD (Skala et al 1991). The leuC gene was inserted in the pESC-URA plasmid followed by leuD, hereby constructing plasmids pESC-leuC and pESC-leuC-D, respectively.…”

Section: Resultsmentioning

confidence: 99%

Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

Carlsen

Ajikumar

Formenti

et al. 2013

Appl Microbiol Biotechnol

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Transfer of a biosynthetic pathway between evolutionary distant organisms can create a metabolic shunt capable of bypassing the native regulation of the host organism, hereby improving the production of secondary metabolite precursor molecules for important natural products. Here, we report the engineering of Escherichia coli genes encoding the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway into the genome of Saccharomyces cerevisiae and the characterization of intermediate metabolites synthesized by the MEP pathway in yeast. Our UPLC-MS analysis of the MEP pathway metabolites from engineered yeast showed that the pathway is active until the synthesis of 2-C-methyl-D-erythritol-2,4-cyclodiphosphate, but appears to lack functionality of the last two steps of the MEP pathway, catalyzed by the [4Fe-4S] iron sulfur cluster proteins encoded by ispG and ispH. In order to functionalize the last two steps of the MEP pathway, we co-expressed the genes for the E. coli iron sulfur cluster (ISC) assembly machinery. By deleting ERG13, thereby incapacitating the mevalonate pathway, in conjunction with labeling experiments with U-¹³C₆ glucose and growth experiments, we found that the ISC assembly machinery was unable to functionalize ispG and ispH. However, we have found that leuC and leuD, encoding the heterodimeric iron-sulfur cluster protein, isopropylmalate isomerase, can complement the S. cerevisiae leu1 auxotrophy. To our knowledge, this is the first time a bacterial iron-sulfur cluster protein has been functionally expressed in the cytosol of S. cerevisiae under aerobic conditions and shows that S. cerevisiae has the capability to functionally express at least some bacterial iron-sulfur cluster proteins in its cytosol.

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Section: Resultsmentioning

confidence: 99%

Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

Carlsen

Ajikumar

Formenti

et al. 2013

Appl Microbiol Biotechnol

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“…PaAcnX and AcnX from P. aeruginosa PAO1; AtAcnX, A. tumefaciens C58; TrAcnX, AcnX from T. reesei QM6a; BxAcnX, Burkholderia xenovorans LB400; ApAcnX, Aeropyrum pernix K1; AcnB from E. coli 18; mAcn from bovine19; AcnD from Shewanella oneidensis MR-121; IRP from human20; IPMI from Saccharomyces cerevisiae 26; HACN1 and HACN2 from M. jannaschii 2342; HACN3 from Pyrococcus horikoshii OT343. “S” and “L” indicate small and large subunits, respectively.…”

Section: Figurementioning

confidence: 99%

“…The aconitase superfamily contains four functional hydro-lyase enzymes: aconitase (EC 4.2.1.3; Acn), 2-methylcitrate dehydratase (EC 4.2.1.79; AcnD), homoaconitase (EC 4.2.1.114; HACN), and isopropylmalate isomerase (EC 4.2.1.33; IPMI), which have been classified into eight phylogenetic subfamilies: (1) AcnA of bacteria17; (2) AcnB of bacteria18; (3) mitochondrial Acn (mAcn)19; (4) cytoplasmic Acn and iron regulatory protein (IRP) of mammalians20; (5) AcnD of bacteria21; (6) HACN of bacteria and archaea2223; (7) IPMI of bacteria, archaea, and fungi242526; (8) function unknown aconitase X (AcnX) (Fig. 2).…”

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confidence: 99%

Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

Watanabe

Tajima

Fujii

et al. 2016

Sci Rep

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In the aconitase superfamily, which includes the archetypical aconitase, homoaconitase, and isopropylmalate isomerase, only aconitase X is not functionally annotated. The corresponding gene (LhpI) was often located within the bacterial gene cluster involved in L-hydroxyproline metabolism. Screening of a library of (hydroxy)proline analogues revealed that this protein catalyzes the dehydration of cis-3-hydroxy-L-proline to Δ1-pyrroline-2-carboxylate. Furthermore, electron paramagnetic resonance and site-directed mutagenic analyses suggests the presence of a mononuclear Fe(III) center, which may be coordinated with one glutamate and two cysteine residues. These properties were significantly different from those of other aconitase members, which catalyze the isomerization of α- to β-hydroxy acids, and have a [4Fe-4S] cluster-binding site composed of three cysteine residues. Bacteria with the LhpI gene could degrade cis-3-hydroxy-L-proline as the sole carbon source, and LhpI transcription was up-regulated not only by cis-3-hydroxy-L-proline, but also by several isomeric 3- and 4-hydroxyprolines.

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“…The sources of the sequences used in the comparisons are as follows: porcine heart (Zheng et al, 1990) and S. cerevisiae (Gangloff et al, 1990) mitochondrial ACN; B. suhtilis ACN, partial sequence (Dingman and Sonenshein, 1987;revised according to Wilde, 1988); human IRE-BP (Rouault et al, 1990, revised as in Rouault et al, 1991 and extended by 20 N-terminal residues according to Hentze and Argos, 1991); IPMI of Mucor circinelloides (Roncero et al, 1989, revised as shown in Fig. 4), Phycomyces blakesleeams (Iturriaga et al, 1990), S. cerevisiae (Skala et al, 1991) and S. typhimurium, LuC (Rosenthal and Calvo, 1990) and leuD (Friedberg et al, 1985); CatR, the Pseudomonas putida transcriptional regulator for benzoate degradation (Rothmel et al, 1990); and two E. coli fumarases, FUMA (Miles and Guest, 1984b) and FUMB (Bell et al, 1989).…”

Section: Alignment Of Amino Acid Sequencesmentioning

confidence: 99%

The aconitase of Escherichia coli

Prodromou¹,

Artymiuk²,

Guest³

1992

European Journal of Biochemistry

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The nucleotide sequence of the aconitase gene (acn) of Escherichia coli was determined and used to deduce the primary structure of the enzyme. The coding region comprises 2670 bp (890 codons excluding the start and stop codons) which define a product having a relative molecular mass of 97513 and an N-terminal amino acid sequence consistent with those determined previously for the purified enzyme. The acn gene is flanked by the cysB gene and a putative riboflavin biosynthesis gene resembling the rihA gene of Bacillus subtilis. The 1004-bp cysB-acn intergenic region contains several potential promoter and regulatory sequences.The amino acid sequence of the E. coli aconitase is similar to the mitochondrial aconitases (27 -29% identity) and the isopropylmalate isomerases (20-21% identity) but it is most similar to the human iron-responsive-element-binding protein (53% identity). The three cysteine residues involved in ligand binding to the [4Fe-4S] centre are conserved in all of these proteins. Of the remaining 17 active-site residues assigned for porcine aconitase, 16 are conserved in both the bacterial aconitase and the iron-responsive-element-binding protein and 14 in the isopropylmalate isomerases. It is concluded that the bacterial and mitochondrial aconitases, the isopropylmalate isomerases and the iron-responsive-element-binding protein form a family of structurally related proteins, which does not include the Fe-S-containing fumarases. These relationships raise the possibility that the ironresponsive-element-binding protein may be a cytoplasmic aconitase and that the E. coli aconitase may have an iron-responsive regulatory function.Aconitase catalyses the reversible isomerization of citrate and isocitrate via the dehydration product cis-aconitate. The mitochondrial aconitases are monomeric proteins of M , 79000 -83 000, which contain single [4Fe-4S] clusters. They have been purified from porcine heart (Villafranca and Mildvan, 1971), bovine heart (Ryden et al., 1984) and Sac-ckaromyces cerevisiae (Scholze, 1983). Primary structures for the porcine and yeast enzymes have been deduced from the nucleotide sequences of the corresponding cDNA or gene (Zheng et al., 1990;Gangloff et al., 1990). The three-dimensional structure of porcine aconitase has been determined (Robbins and Stout, 1989a and1989b); it contains three Correspondence to J. R.

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VII. Yeast sequencing reports. Complete sequence of the Saccharomyces cerevisiae LEU1 gene encoding isopropylmalate isomerase

Cited by 20 publications

References 25 publications

Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

Heterologous expression and characterization of bacterial 2-C-methyl-d-erythritol-4-phosphate pathway in Saccharomyces cerevisiae

Functional characterization of aconitase X as a cis-3-hydroxy-L-proline dehydratase

The aconitase of Escherichia coli

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