scholarly journals Functional Expression of Human Dihydroorotate Dehydrogenase (DHODH) in pyr4 Mutants of Ustilago maydis Allows Target Validation of DHODH Inhibitors In Vivo

2007 ◽  
Vol 73 (10) ◽  
pp. 3371-3379 ◽  
Author(s):  
Elke Zameitat ◽  
Gerald Freymark ◽  
Cornelia D. Dietz ◽  
Monika Löffler ◽  
Michael Bölker
Keyword(s):  
Biochemical Characterization ◽  
De Novo ◽  
Ustilago Maydis ◽  
Functional Expression ◽  
Cytostatic Drug ◽  
Dihydroorotate Dehydrogenase ◽  
Mitochondrial Targeting ◽  
Pyrimidine Nucleotides ◽  
Targeting Signal

ABSTRACT Dihydroorotate dehydrogenase (DHODH; EC 1.3.99.11) is a central enzyme of pyrimidine biosynthesis and catalyzes the oxidation of dihydroorotate to orotate. DHODH is an important target for antiparasitic and cytostatic drugs since rapid cell proliferation often depends on the de novo synthesis of pyrimidine nucleotides. We have cloned the pyr4 gene encoding mitochondrial DHODH from the basidiomycetous plant pathogen Ustilago maydis. We were able to show that pyr4 contains a functional mitochondrial targeting signal. The deletion of pyr4 resulted in uracil auxotrophy, enhanced sensitivity to UV irradiation, and a loss of pathogenicity on corn plants. The biochemical characterization of purified U. maydis DHODH overproduced in Escherichia coli revealed that the U. maydis enzyme uses quinone electron acceptor Q6 and is resistant to several commonly used DHODH inhibitors. Here we show that the expression of the human DHODH gene fused to the U. maydis mitochondrial targeting signal is able to complement the auxotrophic phenotype of pyr4 mutants. While U. maydis wild-type cells were resistant to the DHODH inhibitor brequinar, strains expressing the human DHODH gene became sensitive to this cytostatic drug. Such engineered U. maydis strains can be used in sensitive in vivo assays for the development of novel drugs specifically targeted at either human or fungal DHODH.

scholarly journals Complex Role of the Mitochondrial Targeting Signal in the Function of Steroidogenic Acute Regulatory Protein Revealed by Bacterial Artificial Chromosome Transgenesis in Vivo

2008 ◽  
Vol 22 (4) ◽  
pp. 951-964 ◽  
Author(s):  
Goro Sasaki ◽  
Tomohiro Ishii ◽  
Pancharatnam Jeyasuria ◽  
Youngah Jo ◽  
Assaf Bahat ◽  
...  
Keyword(s):  
Bacterial Artificial Chromosome ◽  
Adrenal Cortex ◽  
Regulatory Protein ◽  
Artificial Chromosome ◽  
Steroidogenic Acute Regulatory Protein ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Steroidogenic Cells ◽  
Steroidogenic Acute Regulatory

The steroidogenic acute regulatory protein (StAR) stimulates the regulated production of steroid hormones in the adrenal cortex and gonads by facilitating the delivery of cholesterol to the inner mitochondrial membrane. To explore key aspects of StAR function within bona fide steroidogenic cells, we used a transgenic mouse model to explore the function of StAR proteins in vivo. We first validated this transgenic bacterial artificial chromosome reconstitution system by targeting enhanced green fluorescent protein to steroidogenic cells of the adrenal cortex and gonads. Thereafter, we targeted expression of either wild-type StAR (WT-StAR) or a mutated StAR protein lacking the mitochondrial targeting signal (N47-StAR). In the context of mice homozygous for a StAR knockout allele (StAR−/−), all StAR activity derived from the StAR transgenes, allowing us to examine the function of the proteins that they encode. The WT-StAR transgene consistently restored viability and steroidogenic function to StAR−/− mice. Although the N47-StAR protein was reportedly active in transfected COS cells and mitochondrial reconstitution experiments, the N47-StAR transgene rescued viability in only 40% of StAR−/− mice. Analysis of lipid deposits in the primary steroidogenic tissues revealed a hierarchy of StAR function provided by N47-StAR: florid lipid deposits were seen in the adrenal cortex and ovarian theca region, with milder deposits in the Leydig cells. Our results confirm the ability of StAR lacking its mitochondrial targeting signal to perform some essential functions in vivo but also demonstrate important functional defects that differ from in vitro studies obtained in nonsteroidogenic cells.

scholarly journals Detection and location of the enzymes of de novo pyrimidine biosynthesis in mammalian spermatozoa

Reproduction ◽  
2002 ◽  
pp. 757-768 ◽  
Author(s):  
EA Carrey ◽  
C Dietz ◽  
DM Glubb ◽  
M Loffler ◽  
JM Lucocq ◽  
...  
Keyword(s):  
De Novo ◽  
Pyrimidine Biosynthesis ◽  
Dihydroorotate Dehydrogenase ◽  
Aspartate Transcarbamylase ◽  
Carbamyl Phosphate ◽  
Pyrimidine Nucleotides ◽  
Carbamyl Phosphate Synthetase ◽  
De Novo Biosynthesis ◽  
Ump Synthase ◽  
Mammalian Spermatozoa

Enzymes of the pathway for de novo biosynthesis of pyrimidine nucleotides have been reported in spermatozoa from fruitfly and mammals. The aim of the present study was to test the hypothesis that the enzymes for biosynthesis of uridine monophosphate (UMP) are concentrated near the mitochondria, which are segregated in the mid-piece of spermatozoa. Baby hamster kidney fibroblasts were compared with spermatozoa from rams, boars, bulls and men. Antibodies raised against synthetic peptides from sequences of the multienzyme polypeptides containing glutamine-dependent carbamyl phosphate synthetase, aspartate transcarbamylase and dihydroorotase (CAD) and UMP synthase, which catalyse reactions 1-3 and 5-6, respectively, were used, together with an affinity-purified antibody raised against dihydroorotate dehydrogenase (DHODH), the mitochondrial enzyme for step 4. Western blot analysis, immunofluorescent microscopy and immunoelectron microscopy confirmed that CAD and UMP synthase are found in the cytoplasm around and outside the mitochondria; DHODH is found exclusively inside the mitochondria. CAD was also located in the nucleus, where it has been reported in the nuclear matrix, and in the cytoplasm, apparently associated with the cytoskeleton. It is possible that CAD in the cytoplasm has a role unconnected with pyrimidine biosynthesis.

scholarly journals Recombinant production, crystallization and crystal structure determination of dihydroorotate dehydrogenase fromLeishmania (Viannia) braziliensis

2015 ◽  
Vol 71 (5) ◽  
pp. 547-552 ◽  
Author(s):  
Renata Almeida Garcia Reis ◽  
Eder Lorenzato ◽  
Valeria Cristina Silva ◽  
Maria Cristina Nonato
Keyword(s):  
Crystal Structure ◽  
De Novo ◽  
Cell Metabolism ◽  
Dihydroorotate Dehydrogenase ◽  
Recombinant Production ◽  
High Degree ◽  
Respective Host

The enzyme dihydroorotate dehydrogenase (DHODH) is a flavoenzyme that catalyses the oxidation of dihydroorotate to orotate in thede novopyrimidine-biosynthesis pathway. In this study, a reproducible protocol for the heterologous expression of active dihydroorotate dehydrogenase fromLeishmania (Viannia) braziliensis(LbDHODH) was developed and its crystal structure was determined at 2.12 Å resolution.L. (V.) braziliensisis the species responsible for the mucosal form of leishmaniasis, a neglected disease for which no cure or effective therapy is available. Analyses of sequence, structural and kinetic features classifyLbDHODH as a member of the class 1A DHODHs and reveal a very high degree of structural conservation with the previously reported structures of orthologous trypanosomatid enzymes. The relevance of nucleotide-biosynthetic pathways for cell metabolism together with structural and functional differences from the respective host enzyme suggests that inhibition ofLbDHODH could be exploited for antileishmanicidal drug development. The present work provides the framework for further integratedin vitro,in silicoandin vivostudies as a new tool to evaluate DHODH as a drug target against trypanosomatid-related diseases.

scholarly journals Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases.

1989 ◽  
Vol 9 (4) ◽  
pp. 1611-1620 ◽  
Author(s):  
S R Ellis ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Protein Import ◽  
Amino Terminus ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Trna Methyltransferase ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Passenger Protein

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.

scholarly journals Mitochondrial ribosomal proteins developed unconventional mitochondrial targeting signals due to structural constraints

2021 ◽  
Author(s):  
Yury Bykov ◽  
Tamara Flohr ◽  
Felix Boos ◽  
Johannes M. Herrmann ◽  
Maya Schuldiner
Keyword(s):  
Ribosomal Proteins ◽  
Protein Targeting ◽  
Mitochondrial Protein ◽  
Nuclear Genome ◽  
Structural Data ◽  
Structural Constraints ◽  
Mitochondrial Targeting ◽  
Targeting Signal ◽  
Targeting Signals

Mitochondrial ribosomes are complex molecular machines indispensable for respiration. Their assembly involves the import of several dozens of mitochondrial ribosomal proteins (MRPs), encoded in the nuclear genome, into the mitochondrial matrix. Available proteomic and structural data as well as computational predictions indicate that up to 25% of MRPs do not have a conventional N-terminal mitochondrial targeting signal (MTS). We characterized a set of 15 yeast MRPs in vivo and showed that 30% of them use internal mitochondrial targeting signals. We isolated a novel internal targeting signal from the conserved MRP Mrp17 (bS6). The Mrp17 targeting signal shares some properties as well as import components with conventional MTS-containing proteins but is not reliably predicted indicating that mitochondrial protein targeting is more versatile than expected. We hypothesize that internal targeting signals arose in MRPs when the N-terminus extension was constrained by ribosome assembly interfaces.

Amino-terminal extension generated from an upstream AUG codon increases the efficiency of mitochondrial import of yeast N2,N2-dimethylguanosine-specific tRNA methyltransferases

1989 ◽  
Vol 9 (4) ◽  
pp. 1611-1620
Author(s):  
S R Ellis ◽  
A K Hopper ◽  
N C Martin
Keyword(s):  
Protein Import ◽  
Amino Terminus ◽  
Mitochondrial Targeting ◽  
Amino Terminal ◽  
Trna Methyltransferase ◽  
Targeting Signal ◽  
Targeting Signals ◽  
Passenger Protein

Fusions between the TRM1 gene of Saccharomyces cerevisiae and COXIV or DHFR were made to examine the mitochondrial targeting signals of N2,N2-dimethylguanosine-specific tRNA methyltransferase [tRNA (m2(2)G)dimethyltransferase]. This enzyme is responsible for the modification of both mitochondrial and cytoplasmic tRNAs. We have previously shown that two forms of the enzyme are translated from two in-frame ATGs in this gene, that they differ by a 16-amino-acid amino-terminal extension, and that both the long and short forms are imported into mitochondria. Results of studies to test the ability of various TRM1 sequences to serve as surrogate mitochondrial targeting signals for passenger protein import in vitro and in vivo showed that the most efficient signal derived from tRNA (m2(2)G)dimethyltransferase included a combination of sequences from both the amino-terminal extension and the amino terminus of the shorter form of the enzyme. The amino-terminal extension itself did not serve as an independent mitochondrial targeting signal, whereas the amino terminus of the shorter form of tRNA (m2(2)G)dimethyltransferase did function in this regard, albeit inefficiently. We analyzed the first 48 amino acids of tRNA (m2(2)G)dimethyltransferase for elements of primary and secondary structure shared with other known mitochondrial targeting signals. The results lead us to propose that the most efficient signal spans the area around the second ATG of TRM1 and is consistent with the idea that there is a mitochondrial targeting signal present at the amino terminus of the shorter form of the enzyme and that the amino-terminal extension augments this signal by extending it to form a larger, more efficient mitochondrial targeting signal.

scholarly journals Biochemical Characterization and Physiological Properties of Escherichia coli UDP-N-Acetylmuramate:l-Alanyl-γ-d-Glutamyl-meso- Diaminopimelate Ligase

2007 ◽  
Vol 189 (11) ◽  
pp. 3987-3995 ◽  
Author(s):  
Mireille Hervé ◽  
Audrey Boniface ◽  
Stanislav Gobec ◽  
Didier Blanot ◽  
Dominique Mengin-Lecreulx
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
De Novo ◽  
Diaminopimelic Acid ◽  
Kinetic Properties ◽  
Gene Encoding ◽  
Peptidoglycan Biosynthesis ◽  
Physiological Properties

ABSTRACT The UDP-N-acetylmuramate:l-alanyl-γ-d-glutamyl-meso-diaminopimelate ligase (murein peptide ligase [Mpl]) is known to be a recycling enzyme allowing reincorporation into peptidoglycan (murein) of the tripeptide l-alanyl-γ-d-glutamyl-meso-diaminopimelate released during the maturation and constant remodeling of this bacterial cell wall polymer that occur during cell growth and division. Mpl adds this peptide to UDP-N-acetylmuramic acid, thereby providing an economical additional source of UDP-MurNAc-tripeptide available for de novo peptidoglycan biosynthesis. The Mpl enzyme from Escherichia coli was purified to homogeneity as a His-tagged form, and its kinetic properties and parameters were determined. Mpl was found to accept tri-, tetra-, and pentapeptides as substrates in vitro with similar efficiencies, but it accepted the dipeptide l-Ala-d-Glu and l-Ala very poorly. Replacement of meso-diaminopimelic acid by l-Lys resulted in a significant decrease in the catalytic efficacy. The effects of disruption of the E. coli mpl gene and/or the ldcA gene encoding the ld-carboxypeptidase on peptidoglycan metabolism were investigated. The differences in the pools of UDP-MurNAc peptides and of free peptides between the wild-type and mutant strains demonstrated that the recycling activity of Mpl is not restricted to the tripeptide and that tetra- and pentapeptides are also directly reused by this process in vivo. The relatively broad substrate specificity of the Mpl ligase indicates that it is an interesting potential target for antibacterial compounds.

The in vivo distribution and dynamics of DNA topoisomerase II in Drosophila embryonic nuclei and chromosomes

1993 ◽  
Vol 51 ◽  
pp. 74-75
Author(s):  
Jason R. Swedlow ◽  
Neil Osheroff ◽  
Tim Karr ◽  
John W. Sedat ◽  
David A. Agard
Keyword(s):  
Topoisomerase Ii ◽  
Biochemical Characterization ◽  
Developmental Cycle ◽  
Dna Topoisomerase Ii ◽  
Chromosomal Distribution ◽  
Dna Topoisomerase ◽  
Ideal System ◽  
Dnase Treatment

DNA topoisomerase II is an ATP-dependent double-stranded DNA strand-passing enzyme that is necessary for full condensation of chromosomes and for complete segregation of sister chromatids at mitosis in vivo and in vitro. Biochemical characterization of chromosomes or nuclei after extraction with high-salt or detergents and DNAse treatment showed that topoisomerase II was a major component of this remnant, termed the chromosome scaffold. The scaffold has been hypothesized to be the structural backbone of the chromosome, so the localization of topoisomerase II to die scaffold suggested that the enzyme might play a structural role in the chromosome. However, topoisomerase II has not been studied in nuclei or chromosomes in vivo. We have monitored the chromosomal distribution of topoisomerase II in vivo during mitosis in the Drosophila embryo. This embryo forms a multi-nucleated syncytial blastoderm early in its developmental cycle. During this time, the embryonic nuclei synchronously progress through 13 mitotic cycles, so this is an ideal system to follow nuclear and chromosomal dynamics.

Sign in / Sign up

Export Citation Format

Share Document