Functional analysis of the methylmalonyl-CoA epimerase from Caenorhabditis elegans.
Methylmalonyl-CoA epimerase (MCE) is an enzyme involved in the propionyl-CoA metabolism that is responsible for the degradation of branched amino acids and odd-chain fatty acids. This pathway typically functions in the reversible conversion of propionyl-CoA to succinyl-CoA. The Caenorhabditis elegans genome contains a single gene encoding MCE (mce-1) corresponding to a 15 kDa protein. This was expressed in Escherichia coli and the enzymatic activity was determined. Analysis of the protein expression pattern at both the tissue and subcellular level by microinjection of green fluorescent protein constructs revealed expression in the pharynx, hypodermis and, most prominently in body wall muscles. The subcellular pattern agrees with predictions of mitochondrial localization. The sequence similarity to an MCE of known structure was high enough to permit a three-dimensional model to be built, suggesting conservation of ligand and metal binding sites. Comparison with corresponding sequences from a variety of organisms shows more than 1/6 of the sequence is completely conserved. Mutants allelic to mce-1 showed no obvious phenotypic alterations, demonstrating that the enzyme is not essential for normal worm development under laboratory conditions. However, survival of the knockout mutants was altered when exposed to stress conditions, with mutants surprisingly showing an increased resistance to oxidative stress.
Kühnl
Jochen
J
Department of Biochemistry, Bernhard-Nocht-Institute for Tropical Medicine, Hamburg, Germany.
Bobik
Thomas
T
Procter
James B
JB
Burmeister
Cora
C
Höppner
Jana
J
Wilde
Inga
I
Lüersen
Kai
K
Torda
Andrew E
AE
Walter
Rolf D
RD
Liebau
Eva
E
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology*; D029742:Caenorhabditis elegans Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016384:Consensus Sequence; D005091:Exons; D006801:Humans; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010802:Phylogeny; D017433:Protein Structure, Secondary; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D029742:Caenorhabditis elegans Proteins; D011994:Recombinant Proteins; D019998:Racemases and Epimerases; C021581:methylmalonyl-coenzyme A racemase