Linked Life Data

15096031

Source:http://linkedlifedata.com/resource/pubmed/id/15096031

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
16
pubmed:dateCreated
2004-4-20
pubmed:databankReference
pubmed:abstractText
The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported [Raynaud, C., et al. (2003) Proc. Natl. Acad. Sci. U.S.A. 100, 5010-5015]. In this work, we have further characterized this system by biochemical and crystallographic methods. Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions. In this form, both the GD and GD-AE were inactive. A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE. Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions. We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms. The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif. Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented. This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group. Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme. A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Apr
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
27
pubmed:volume
43
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
4635-45
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:15096031-Acetyltransferases, pubmed-meshheading:15096031-Amino Acid Sequence, pubmed-meshheading:15096031-Bacterial Proteins, pubmed-meshheading:15096031-Binding Sites, pubmed-meshheading:15096031-Clostridium, pubmed-meshheading:15096031-Crystallization, pubmed-meshheading:15096031-Crystallography, X-Ray, pubmed-meshheading:15096031-Culture Media, pubmed-meshheading:15096031-Enzyme Activation, pubmed-meshheading:15096031-Enzyme Reactivators, pubmed-meshheading:15096031-Glycerol, pubmed-meshheading:15096031-Hydro-Lyases, pubmed-meshheading:15096031-Molecular Sequence Data, pubmed-meshheading:15096031-Propanediol Dehydratase, pubmed-meshheading:15096031-Propylene Glycol, pubmed-meshheading:15096031-Structure-Activity Relationship, pubmed-meshheading:15096031-Substrate Specificity, pubmed-meshheading:15096031-Vitamin B 12
pubmed:year
2004
pubmed:articleTitle
Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602, USA.
pubmed:publicationType
Journal Article