Linked Life Data

7873527

Source:http://linkedlifedata.com/resource/pubmed/id/7873527

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
8
pubmed:dateCreated
1995-4-4
pubmed:abstractText
Derivatization of 5,5'-dithiobis(2-nitrobenzoic acid)-treated trichodiene synthase with [methyl-14C]methyl methanethiosulfonate and analysis of the derived tryptic peptides suggested the presence of two cysteine residues at the active site. The corresponding C146A and C190A mutants were constructed by site-directed mutagenesis. The C190A mutant displayed partial but significantly reduced activity, with a reduction in kcat/Km of 3000 compared to the wild-type trichodiene synthase, while the C146A mutant was essentially inactive. A hybrid trichodiene synthase, constructed from amino acids 1-309 of the Fusarium sporotrichioides enzyme and amino acids 310-383 of the Gibberella pulicaris cyclase, had steady state kinetic parameters nearly identical to those of the wild-type F. sporotrichioides enzyme. From this parent hybrid, a series of mutants was constructed by site-directed mutagenesis in which the amino acids in the base-rich region, 302-306 (DRRYR), were systematically modified. Three of these mutants were overexpressed and purified to homogeneity. The importance of Arg304 for catalysis was established by the observation that the R304K mutant showed a more than 25-fold increase in Km, as well as a 200-fold reduction in kcat. In addition, analysis of the incubation products of the R304K mutant by gas chromatography-mass spectrometry (GC-MS) indicated that farnesyl diphosphate was converted not only to trichodiene but to at least two additional C15H24 hydrocarbons, mle 204. Replacement of the Tyr305 residue of trichodiene synthase with Phe had little effect on kcat, while increasing the Km by a factor of ca. 7-8.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:grant
pubmed:commentsCorrections
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Feb
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
28
pubmed:volume
34
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
2480-8
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed-meshheading:7873527-Amino Acid Sequence, pubmed-meshheading:7873527-Base Sequence, pubmed-meshheading:7873527-Binding Sites, pubmed-meshheading:7873527-Carbon-Carbon Lyases, pubmed-meshheading:7873527-Catalysis, pubmed-meshheading:7873527-Cloning, Molecular, pubmed-meshheading:7873527-Cysteine, pubmed-meshheading:7873527-DNA, Fungal, pubmed-meshheading:7873527-DNA Primers, pubmed-meshheading:7873527-Escherichia coli, pubmed-meshheading:7873527-Fusarium, pubmed-meshheading:7873527-Kinetics, pubmed-meshheading:7873527-Lyases, pubmed-meshheading:7873527-Methyl Methanesulfonate, pubmed-meshheading:7873527-Molecular Sequence Data, pubmed-meshheading:7873527-Molecular Structure, pubmed-meshheading:7873527-Mutagenesis, Site-Directed, pubmed-meshheading:7873527-Polyisoprenyl Phosphates, pubmed-meshheading:7873527-Sesquiterpenes, pubmed-meshheading:7873527-Substrate Specificity
pubmed:year
1995
pubmed:articleTitle
Trichodiene synthase. Identification of active site residues by site-directed mutagenesis.
pubmed:affiliation
Department of Chemistry, Brown University, Providence, Rhode Island 02912, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S., Research Support, Non-U.S. Gov't