Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Methylmalonic acidemia (MMA) can be caused by mutations in the gene coding for the methylmalonyl CoA mutase (MCM) apoenzyme or by mutations in genes required for provision of its adenosylcobalamin cofactor. We have characterized MCM activity, gene structure, and expression in a series of primary fibroblast cell lines derived from patients with MCM apoenzyme deficiency. Southern blot analysis reveals normal HindIII and TaqI polymorphisms but no gross insertions, deletions, rearrangements, or point mutations at restriction endonuclease recognition sequences. Northern blot analysis demonstrates that several cell lines have specifically decreased steady-state levels of MCM mRNA. At least six independent alleles can be delineated by a haplotype of HindIII and TaqI polymorphisms, the level of mRNA expression, and the biochemical phenotype of the cells. These studies confirm the wide phenotypic spectrum of MMA and provide molecular genetic evidence for a variety of independent alleles underlying this disorder.
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PMID:Heterogeneous alleles and expression of methylmalonyl CoA mutase in mut methylmalonic acidemia. 196 6

L-methylmalonyl-CoA mutase (MCM; E.C. 5,4,99,2) is the apoenzyme for catalyzing the isomerization of L-methylmalonyl-CoA to succinyl-CoA. Genetic deficiency of MCM leads to the accumulation of precursors and abnormal metabolites of L-methylmalonyl-CoA. This can be associated with fulminant metabolic acidosis, widespread secondary aberrations in systemic metabolic homeostasis, mental retardation, or even neonatal death. This disorder is termed methylmalonic acidemia (MMA). This report, describes the use of an authentic, full-length cloned human cDNA probe, MCM26, kindly provided by Dr. Fred Ledley, for Southern blot analysis of genomic DNA. The pattern of EcoRI, Sac I and Hind III restriction endonuclease sites is reported from 14 unrelated control individuals of Chinese background. A Southern blot by EcoRI to the MCM26b probe reveals invariant bands of 4.1, 3.8, and 2.2 kb respectively. By EcoRI to the MCM26c probe, 7.2 kb is invariant. By HindIII to the MCM26c probe, invariant bands are 4.8 and 2.7 kb respectively. By SacI to the MCMb probe, invariant bands are 17, 8.0, 6.0, 3.6 and 1.8 kb respectively, while the polymorphic band is at 5.6kb. When combined with more diverse samples and additional polymorphisms, this restriction fragment length polymorphism may be useful for genetic diagnostic and linkage studies of MCM in MMA.
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PMID:Restriction fragment length polymorphisms at the methylmalonyl CoA mutase locus in normal Chinese. 197 11

The stromal glycerol-3-phosphate acyltransferases (GPATs; EC 2.3.1.15) from spinach (Spinacia oleracea) and squash (Cucurbita moschata) were expressed in Escherichia coli and their activities with palmitoyl-CoA and oleoyl-CoA compared. The GPAT from squash, a chilling-sensitive plant, was found to have the greatest difference in activities between the two substrates, using palmitoyl-CoA over three times faster than oleoyl-CoA. In contrast, the enzyme from spinach, a chilling-tolerant plant, preferred oleoyl-CoA over palmitoyl-CoA. By using conserved restriction endonuclease sites each of the two genes was divided into three fragments of roughly equal size and recombined to create six different chimeras. All chimeras retained a large portion of their original activity but in most cases the specificity was greatly altered. The central third of the protein was found to contain the structural features which determine substrate specificity of the wild-type GPATs. Two of the chimeras, which have a spinach-derived central region and a squash-derived carboxyl region, were found to have greatly enhanced specificities for 18:1 acyl chains, potentially making them ideal for decreasing the level of saturation of plant membrane lipids through genetic engineering.
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PMID:Substrate specificity modification of the stromal glycerol-3-phosphate acyltransferase. 901 14

A shuttle mutagenesis system was developed for the dimorphic yeast Yarrowia lipolytica. This system combines transposon insertions generated in Escherichia coli with the transformation of yeast with the Tn-mutagenized DNA. The mini-transposon mTn-3xHA/GFP, used in Saccharomyces cerevisiae for producing stable insertions, was adapted for use in the yeast Y. lipolytica. The mTnYl1 transposon (for mini-Tn of Y. lipolytica) confers resistance to tetracycline in E. coli. It also contains the Y. lipolytica URA3 gene for selection of yeast transformants, and the coding sequence for the S65T mutant form of GFP. The rare cutter endonuclease, I-SceI, restriction site, which enables identification of the chromosomal localization of mutagenized genes, was also incorporated. mTnYl1 was first tested on the ACO1 gene, which encodes an Acyl CoA oxidase isozyme. The mutagenesis system was further validated on a Y. lipolytica genomic DNA library constructed in a pHSS6 derivative vector. Mutants with a particular morphology or defective for alkane, fatty acids and oil degradation were obtained.
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PMID:A shuttle mutagenesis system for tagging genes in the yeast Yarrowia lipolytica. 963 May 1

The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, are essential for viral transcription and replication. The PB2 subunit binds to the host RNA cap (7-methylguanosine triphosphate (m(7)GTP)) and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. However, the structure of PB2 is not fully understood, and the functional sites remain unknown. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is involved in interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m(7)GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m(7)GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of the valine and arginine residues or of all 3 residues of the VRG site to alanine significantly reduced the binding ability of PB2 to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication.
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PMID:A novel functional site in the PB2 subunit of influenza A virus essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. 2506 5

The PA, PB1, and PB2 subunits, components of the RNA-dependent RNA polymerase of influenza A virus, and the nucleoprotein (NP) interact with the genomic RNA of influenza viruses and form ribonucleoproteins. Especially, the PB2 subunit binds to the host RNA cap [7-methylguanosine triphosphate (m7GTP)] and supports the endonuclease activity of PA to "snatch" the cap from host pre-mRNAs. In this study, we describe a novel Val/Arg/Gly (VRG) site in the PB2 cap-binding domain, which is necessary for interaction with acetyl-CoA found in eukaryotic histone acetyltransferases (HATs). In vitro experiments revealed that the recombinant PB2 cap-binding domain that includes the VRG site interacts with acetyl-CoA; moreover, it was found that this interaction could be blocked by CoA and various HAT inhibitors. Interestingly, m7GTP also inhibited this interaction, suggesting that the same active pocket is capable of interacting with acetyl-CoA and m7GTP. To elucidate the importance of the VRG site on PB2 function and viral replication, we constructed a PB2 recombinant protein and recombinant viruses including several patterns of amino acid mutations in the VRG site. Substitutions of 2 or 3 amino acid residues of the VRG site to alanine significantly reduced PB2's binding ability to acetyl-CoA and its RNA polymerase activity. Recombinant viruses containing the same mutations could not be replicated in cultured cells. These results indicate that the PB2 VRG sequence is a functional site that is essential for acetyl-CoA interaction, RNA polymerase activity, and viral replication. I will also discuss some novel functions of NP in this review.
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PMID:Structural and Biochemical Analyses on the RNA-dependent RNA Polymerase of Influenza Virus for Development of Novel Anti-influenza Agents. 2815 33