Demonstration of dimethylnonanoyl-CoA thioesterase activity in rat liver peroxisomes followed by purification and molecular cloning of the thioesterase involved.
Peroxisomes play an indispensable role in cellular fatty acid oxidation in higher eukaryotes by catalyzing the chain shortening of a distinct set of fatty acids and fatty acid derivatives including pristanic acid (2,6,10,14-tetramethylpentadecanoic acid). Earlier studies have shown that pristanic acid undergoes three cycles of beta-oxidation in peroxisomes to produce 4,8-dimethylnonanoyl-CoA (DMN-CoA) which is then transported to the mitochondria for full oxidation to CO(2) and H(2)O. In principle, this can be done via two different mechanisms in which DMN-CoA is either converted into the corresponding carnitine ester or hydrolyzed to 4,8-dimethylnonanoic acid plus CoASH. The latter pathway can only be operational if peroxisomes contain 4,8-dimethylnonanoyl-CoA thioesterase activity. In this paper we show that rat liver peroxisomes indeed contain 4,8-dimethylnonanoyl-CoA thioesterase activity. We have partially purified the enzyme involved from peroxisomes and identified the protein as the rat ortholog of a known human thioesterase using MALDI-TOF mass spectrometry in combination with the rat EST database. Heterologous expression studies in Escherichia coli established that the enzyme hydrolyzes not only DMN-CoA but also other branched-chain acyl-CoAs as well as straight-chain acyl-CoA-esters. Our data provide convincing evidence for the existence of the second pathway of acyl-CoA transport from peroxisomes to mitochondria by hydrolysis of the CoA-ester in peroxisomes followed by transport of the free acid to mitochondria, reactivation to its CoA-ester, and oxidation to CO(2) and H(2)O. (c)2002 Elsevier Science.
Ofman
R
R
Department of Clinical Chemistry, Emma Children's Hospital, Academic Medical Center, University of Amsterdam, Amsterdam, 1100 DE, The Netherlands.
el Mrabet
L
L
Dacremont
G
G
Spijer
D
D
Wanders
R J A
RJ
D018528:ATP-Binding Cassette Transporters; D000818:Animals; D002352:Carrier Proteins / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000737:chemistry / Q000235:genetics; D029968:Escherichia coli Proteins; D020224:Expressed Sequence Tags; D005227:Fatty Acids / Q000378:metabolism; D008099:Liver / Q000737:chemistry / Q000201:enzymology*; D008297:Male; D058619:Maltose-Binding Proteins; D008928:Mitochondria / Q000378:metabolism; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins; D020675:Peroxisomes / Q000737:chemistry / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013347:Subcellular Fractions / Q000737:chemistry / Q000201:enzymology; D013869:Thiolester Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D018528:ATP-Binding Cassette Transporters; D002352:Carrier Proteins; D029968:Escherichia coli Proteins; D005227:Fatty Acids; D058619:Maltose-Binding Proteins; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; C461599:maltose transport system, E coli; C002844:pristanic acid; C446295:4,8-dimethylnonanoyl-CoA thioesterase; D013869:Thiolester Hydrolases