Linked Life Data

1420191

Source:http://linkedlifedata.com/resource/pubmed/id/1420191

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
44
pubmed:dateCreated
1992-12-8
pubmed:databankReference
pubmed:abstractText
The gene encoding methylaspartase (EC 4.3.1.2) from Clostridium tetranomorphum has been cloned, sequenced, and expressed in Escherichia coli. The open reading frame (ORF) codes for a polypeptide of 413 amino acid residues (M(r) 45,539) of which seven are cysteine residues. The size of the ORF indicates that methylaspartase is a homodimer rather than an (AB)2 tetramer. The deduced primary structure of the protein shows no homology to enzymes that catalyze similar reactions or, indeed, any convincing homology with any other characterized protein. The recombinant protein is identical to the enzyme isolated directly from C. tetanomorphum as determined by several criteria. The enzyme is obtained in a highly active form (approximately 70% of the activity of the natural enzyme) and migrates as a single band (M(r) 49,000) in SDS-polyacrylamide gels. The kinetic parameters for the deamination of (2S,3S)-3-methylaspartic acid by the natural and recombinant proteins are very similar, and the proteins display identical potassium ion-dependent primary deuterium isotope effects for V and V/K when (2S,3S)-3-methylaspartic acid is employed as the substrate. In accord with the activity of the natural enzyme, the recombinant protein is able to catalyze the slow formation of (2S,3R)-3-methylaspartic acid, the L-erythro-epimer of the natural substrate, from mesaconic acid and ammonia. Earlier work in which the cysteine residues in the protein were labeled with N-ethylmaleimide had indicated that there were eight cysteine residues per protein monomer. One cysteine residue was protected by substrate. Here evidence is forwarded to suggest that the residue that was protected by the substrate is not a cysteine residue but the translation product of a serine codon. Kinetic data indicate that this serine residue may be modified in the active enzyme. The implications of these findings on the mechanism of catalysis are discussed within the context of a few emerging mode of action for methylaspartate ammonia-lyase.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Nov
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
10
pubmed:volume
31
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
10747-56
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed:year
1992
pubmed:articleTitle
Cloning, sequencing, and expression in Escherichia coli of the Clostridium tetanomorphum gene encoding beta-methylaspartase and characterization of the recombinant protein.
pubmed:affiliation
Division of Biotechnology, PHLS Centre for Applied Microbiology, Proton Down, Salisbury, Wiltshire, U.K.
pubmed:publicationType
Journal Article, Comparative Study, Research Support, Non-U.S. Gov't