Linked Life Data

8241161

Source:http://linkedlifedata.com/resource/pubmed/id/8241161

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
48
pubmed:dateCreated
1994-1-6
pubmed:abstractText
The MutT protein, a 129-residue enzyme from Escherichia coli which prevents A.T-->C.G mutations, catalyzes the hydrolysis of nucleoside triphosphates (NTP) to nucleoside monophosphates (NMP) and pyrophosphate [Bhatnagar, S. K., Bullions, L. C., & Bessman, M. J. (1991) J. Biol. Chem. 266, 9050-9054], by a mechanism involving nucleophilic substitution at the rarely attacked beta-phosphorus of NTP [Weber, D. J., Bhatnagar, S. K., Bullions, L. C., Bessman, M. J., & Mildvan, A. S. (1992a) J. Biol. Chem. 267, 16939-16942]. The bacterial MutT gene was inserted into the plasmid pET-11b under control of the T7 promoter and overexpressed in minimal media, permitting labeling of MutT with 13C and/or 15N. The yield after purification of the soluble fraction was approximately 35 mg of homogeneous MutT/L with physical and enzymatic properties indistinguishable from those of the originally isolated enzyme. Essentially complete sequence-specific assignments of the backbone HN, N, C alpha, H alpha, and CO resonances of the free enzyme (1.5 mM) were made at pH 7.4 and 32 degrees C, by heteronuclear double- and triple-resonance experiments using a modified Bruker AM 600 NMR spectrometer. Specifically, 1H[15N]HSQC, 1H[15N]TOCSY-HMQC, and 1H[15N]NOESY-HMQC experiments were done with uniformly 15N-labeled enzyme. A 1H[15N] HSQC experiment was done with selective [alpha-15N]Lys-labeled enzyme. Also HNCA, HN(CO)CA, HNCO, constant time 1H[13C]HSQC, HCACO, and HCA(CO)N experiments were done with uniformly 13C- and 15N-labeled enzyme. Sequence-specific assignments were initiated from HN and 15N chemical shifts of Gly residues and of selectively labeled Lys residues in 1H[15N]HSQC experiments. They were confirmed by C alpha chemical shifts of Ala residues uniquely identified by residual coupling to C beta resonances in constant time 1H[13C]HSQC experiments. The sequence-specific assignments proceeded bidirectionally, terminating at Pro residues and at residues with undetectable NH signals, and the segments were linked to complete the backbone assignments. The backbone assignments reported here have permitted the interpretation of NOEs in the elucidation of the solution secondary structure of MutT, and the C alpha and H alpha chemical shifts have provided an independent approach to identifying secondary structural elements and to define their extent [Weber, D. J., Abeygunawardana, C., Bessman, M. J., & Mildvan, A. S. (1993) Biochemistry (following paper in this issue)].
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Dec
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
7
pubmed:volume
32
pubmed:geneSymbol
mutT
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
13071-80
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1993
pubmed:articleTitle
Sequence-specific assignments of the backbone 1H, 13C, and 15N resonances of the MutT enzyme by heteronuclear multidimensional NMR.
pubmed:affiliation
Department of Biological Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, P.H.S.