275, 15605-15608). We now show that this enzyme also activates chenodeoxycholate, the secondary bile acids deoxycholate and lithocholate, and 3␣,7␣,12␣-trihydroxy-5␤-cholestanoic acid. In contrast, VLCS activated 3␣,7␣,12␣-trihydroxy-5␤-cholestanoate, but did not utilize any of the C 24 bile acids as substrates. We hypothesize that the primary function of homolog 2 is in the reactivation and recycling of C 24 bile acids, whereas VLCS participates in the de novo synthesis pathway. Results of in situ hybridization, topographic orientation, and inhibition studies are consistent with the proposed roles of these enzymes in bile acid metabolism.

No abstract

Unconjugated bile acids must be activated to their CoA thioesters before conjugation to taurine or glycine can occur. A human homolog of very long-chain acylCoA synthetase, hVLCS-H2, has two requisite properties of a bile acid:CoA ligase, liver specificity and an endoplasmic reticulum subcellular localization. We investigated the ability of this enzyme to activate the primary bile acid, cholic acid, to its CoA derivative. When expressed in COS-1 cells, hVLCS-H2 exhibited cholate:CoA ligase (choloyl-CoA synthetase) activity with both nonisotopic and radioactive assays. Other long-and very long-chain acyl-CoA synthetases were incapable of activating cholate. Endogenous choloyl-CoA synthetase activity was also detected in liver-derived HepG2 cells but not in kidney-derived COS-1 cells. Our results are consistent with a role for hVLCS-H2 in the re-activation and re-conjugation of bile acids entering liver from the enterohepatic circulation rather than in de novo bile acid synthesis.

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, we cloned the associated human gene and expressed its protein product. The cDNA was cloned with the use of a reverse genetics approach based on the amino acid sequence obtained from purified L-pipecolic acid oxidase from monkey. The complete cDNA, obtained by conventional library screening and 5' rapid amplification of cDNA ends, encompassed an open reading frame of 1170 bases, translating to a 390-residue protein. The translated protein terminated with the sequence AHL, a peroxisomal targeting signal 1. Indirect immunofluorescence studies showed that the protein product was expressed in human fibroblasts in a punctate pattern that co-localized with the peroxisomal enzyme catalase. A BLAST search with the amino acid sequence showed 31% identity and 53% similarity with Bacillus sp. NS-129 monomeric sarcosine oxidase, as well as similarity to all sarcosine oxidases and dehydrogenases. No similarity was found to the peroxisomal D-amino acid oxidases. The recombinant enzyme oxidized both L-pipecolic acid and sarcosine. However, PBD patients who lack the enzyme activity accumulate only L-pipecolic acid, suggesting that in humans in vivo, this enzyme is involved mainly in the degradation of L-pipecolic acid.