scholarly journals The α- and β-subunits of the Human UDP-N-acetylglucosamine:Lysosomal Enzyme Phosphotransferase Are Encoded by a Single cDNA

2005 ◽  
Vol 280 (43) ◽  
pp. 36141-36149 ◽  
Author(s):  
Mariko Kudo ◽  
Ming Bao ◽  
Anil D'Souza ◽  
Fu Ying ◽  
Huaqin Pan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Catalytic Domain ◽  
Amino Acid Sequences ◽  
Subunit Structure ◽  
Bac Clone ◽  
Significant Similarity ◽  
Α Subunit ◽  
Γ Subunit ◽  
Mannose 6 Phosphate ◽  
Α And Β Subunits

Lysosomal enzymes are targeted to the lysosome through binding to mannose 6-phosphate receptors because their glycans are modified with mannose 6-phosphate. This modification is catalyzed by UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase). Bovine GlcNAc-phosphotransferase was isolated using monoclonal antibody affinity chromatography, and an α2β2γ2-subunit structure was proposed. Although cDNA encoding the γ-subunit has been described, cDNAs for the α- and β-subunits have not. Using partial amino acid sequences from the bovine α- and β-subunits, we have isolated a human cDNA that encodes both the α- and β-subunits. Both subunits contain a single predicted membrane-spanning domain. The α- and β-subunits appear to be generated by a proteolytic cleavage at the Lys928-Asp929 bond. Transfection of 293T cells with the α/β-subunits-precursor cDNA with or without the γ-subunit cDNA results in a 3.6- or 17-fold increase in GlcNAc-phosphotransferase activity in cell lysates, suggesting that the precursor cDNA contains the catalytic domain. The sequence lacks significant similarity with any described vertebrate enzyme except for two Notch-like repeats in the α-subunit. However, a 112-amino acid sequence is highly similar to a group of bacterial capsular polymerases (46% identity). A BAC clone containing the gene that spanned 85.3 kb and was composed of 21 exons was sequenced and localized to chromosome 12q23. We now report the cloning of both the cDNA and genomic DNA of the precursor of Glc-NAc-phosphotransferase. The completion of cloning all three subunits of GlcNAc-phosphotransferase allows expression of recombinant enzyme and dissection of lysosomal targeting disorders.

scholarly journals Subtle Difference between Benzene and Toluene Dioxygenases of Pseudomonas putida

2005 ◽  
Vol 71 (3) ◽  
pp. 1570-1580 ◽  
Author(s):  
Claire Bagnéris ◽  
Richard Cammack ◽  
Jeremy R. Mason
Keyword(s):  
Amino Acids ◽  
Electron Transfer ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Pseudomonas Putida ◽  
Amino Acid Sequences ◽  
Subunit Structure ◽  
Subtle Difference ◽  
Α Subunit ◽  
Toluene Dioxygenase

ABSTRACT Benzene dioxygenase and toluene dioxygenase from Pseudomonas putida have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, α and β; the α subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the α subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase α subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the α subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.

Chemical Data on Pituitary Gonadotropins and Their Implication to Evolution

1982 ◽  
Vol 39 (1) ◽  
pp. 80-91 ◽  
Author(s):  
E. Burzawa-Gerard
Keyword(s):  
Amino Acid ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Quaternary Structure ◽  
Chelonia Mydas ◽  
Amino Acid Sequences ◽  
Chemical Data ◽  
Pituitary Hormone ◽  
Hybrid Molecules ◽  
Α And Β Subunits

Chemical data on gonadotropins from several vertebrate species are summarized and discussed from an evolutionary point of view. A high degree of homology has been observed between mammalian gonadotropins (LH and FSH) and thyrotropin (TSH). In non-mammalian species the existence of LH and FSH-like hormones has been demonstrated except for squamate and fish species. Especially in fish the number of GTHs is still controversial. One pituitary glycoprotein assumes various gonadotropic functions of the pituitary, and a second pituitary hormone (carbohydrate-poor) acts on fish ovarian growth. GTHs from bird, reptile, amphibian, and fish pituitaries have been purified and chemically characterized (amino acid composition, carbohydrate content). The existence of a quaternary structure has been demonstrated for several tetrapod LHs and fish GTHs. The amino acid composition of α and β subunits purified from turkey (Meleagris gallopavo), and turtle (Chelydra serpentira, Chelonia mydas) LHs and from common carp (Cyprinus carpio) and sturgeon (Acipenser stellatus) GTHs showed homology with the mammalian α and β subunits. The partial sequences of carp GTH subunits have shown that the carp GTH β was more closely related to mammalian LH β than to FSH β. Hybrid molecules could be obtained by association of heterologous subunits. The kinetics of subunit association has been studied in vitro. As compared to ovine LH, subunit association of carp GTH was more rapid and thermodependent. The subunit β seemed to determine the thermodependence. The various GTH subunits in living vertebrate probably derive from a common ancestral molecule.Key words: vertebrate gonadotropins, chemical characterizations, GTHs subunits, amino acid sequences, hybrid molecules, evolution.

Characterization of the formate (for) locus, which encodes the cytosolic serine hydroxymethyltransferase of Neurospora crassa

1992 ◽  
Vol 12 (4) ◽  
pp. 1412-1421
Author(s):  
C R McClung ◽  
C R Davis ◽  
K M Page ◽  
S A Denome
Keyword(s):  
Amino Acid ◽  
Neurospora Crassa ◽  
Amino Acid Identity ◽  
Amino Acid Sequences ◽  
Serine Hydroxymethyltransferase ◽  
Central Position ◽  
Significant Similarity ◽  
C1 Metabolism ◽  
General Amino Acid Control ◽  
Growth Requirement

Serine hydroxymethyltransferase (SHMT) occupies a central position in one-carbon (C1) metabolism, catalyzing the reaction of serine and tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Methylenetetrahydrofolate serves as a donor of C1 units for the synthesis of numerous compounds, including purines, thymidylate, lipids, and methionine. We provide evidence that the formate (for) locus of Neurospora crassa encodes cytosolic SHMT. The for+ gene was localized to a 2.8-kb BglII fragment by complementation (restoration to formate-independent growth) of a strain carrying a recessive for allele, which confers a growth requirement for formate. The for+ gene encodes a polypeptide of 479 amino acids which shows significant similarity to amino acid sequences of SHMT from bacterial and mammalian sources (47 and 60% amino acid identity, respectively). The for+ mRNA has several different start and stop sites. The abundance of for+ mRNA increased in response to amino acid imbalance induced by glycine supplementation, suggesting regulation by the N. crassa cross-pathway control system, which is analogous to general amino acid control in Saccharomyces cerevisiae. This was confirmed by documenting that for+ expression increased in response to histidine limitation (induced by 3-amino-1,2,4-triazole) and that this response was dependent on the presence of a functional cross-pathway control-1 (cpc-1) gene, which encodes CPC1, a positively acting transcription factor. There are at least five potential CPC1 binding sites upstream of the for+ transcriptional start, as well as one that exactly matches the consensus CPC1 binding site in the first intron of the for+ gene.

Nucleotide and deduced amino acid sequences of the GABAA receptor α-subunit from human brain

1989 ◽  
Vol 15 (1) ◽  
pp. 33-38 ◽  
Author(s):  
M HIROUCHI ◽  
R KUWANO ◽  
K TAKASHI ◽  
T YASUO ◽  
K KURIYAMA
Keyword(s):  
Amino Acid ◽  
Human Brain ◽  
Gabaa Receptor ◽  
Amino Acid Sequences ◽  
Α Subunit

scholarly journals The Alkene Monooxygenase from Xanthobacter Strain Py2 Is Closely Related to Aromatic Monooxygenases and Catalyzes Aromatic Monohydroxylation of Benzene, Toluene, and Phenol

1999 ◽  
Vol 65 (4) ◽  
pp. 1589-1595 ◽  
Author(s):  
Ning-Yi Zhou ◽  
Alister Jenkins ◽  
Chan K. N. Chan Kwo Chion ◽  
David J. Leak
Keyword(s):  
Consensus Sequence ◽  
Detailed Comparison ◽  
Amino Acid Sequences ◽  
Effector Protein ◽  
Chromosomal Dna ◽  
Α Subunit ◽  
Γ Subunit ◽  
Genes Encoding ◽  
Benzene Toluene ◽  
Α Subunits

ABSTRACT The genes encoding the six polypeptide components of the alkene monooxygenase from Xanthobacter strain Py2 (Xamo) have been located on a 4.9-kb fragment of chromosomal DNA previously cloned in cosmid pNY2. Sequencing and analysis of the predicted amino acid sequences indicate that the components of Xamo are homologous to those of the aromatic monooxygenases, toluene 2-, 3-, and 4-monooxygenase and benzene monooxygenase, and that the gene order is identical. The genes and predicted polypeptides are aamA, encoding the 497-residue oxygenase α-subunit (XamoA); aamB, encoding the 88-residue oxygenase γ-subunit (XamoB); aamC, encoding the 122-residue ferredoxin (XamoC); aamD, encoding the 101-residue coupling or effector protein (XamoD); aamE, encoding the 341-residue oxygenase β-subunit (XamoE); andaamF, encoding the 327-residue reductase (XamoF). A sequence with >60% concurrence with the consensus sequence of ς54 (RpoN)-dependent promoters was identified upstream of the aamA gene. Detailed comparison of XamoA with the oxygenase α-subunits from aromatic monooxygenases, phenol hydroxylases, methane monooxygenase, and the alkene monooxygenase fromRhodococcus rhodochrous B276 showed that, despite the overall similarity to the aromatic monooxygenases, XamoA has some distinctive characteristics of the oxygenases which oxidize aliphatic, and particularly alkene, substrates. On the basis of the similarity between Xamo and the aromatic monooxygenases, Xanthobacterstrain Py2 was tested and shown to oxidize benzene, toluene, and phenol, while the alkene monooxygenase-negative mutants NZ1 and NZ2 did not. Benzene was oxidized to phenol, which accumulated transiently before being further oxidized. Toluene was oxidized to a mixture ofo-, m-, and p-cresols (39.8, 18, and 41.7%, respectively) and a small amount (0.5%) of benzyl alcohol, none of which were further oxidized. In growth studiesXanthobacter strain Py2 was found to grow on phenol and catechol but not on benzene or toluene; growth on phenol required a functional alkene monooxygenase. However, there is no evidence of genes encoding steps in the metabolism of catechol in the vicinity of theaam gene cluster. This suggests that the inducer specificity of the alkene monooxygenase may have evolved to benefit from the naturally broad substrate specificity of this class of monooxygenase and the ability of the host strain to grow on catechol.

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