GLYCINE AND TAURINE CONJUGATION OF BILE-ACIDS BY A SINGLE ENZYME - MOLECULAR-CLONING AND EXPRESSION OF HUMAN LIVER BILE-ACID COA-AMINO ACIDN-ACYLTRANSFERASE

In order to establish whether a single enzyme in human liver was capab le of conjugating bile acids with both glycine and taurine, a cDNA enc oding human liver bile acid-CoA:amino acid N-acyltransferase (hBAT) ha s been isolated and characterized. A specific immunoaffinity-purified rabbit anti-hBAT polyclonal antibody was used to screen a lambda Zap X R human liver cDNA library resulting in the isolation of two unique cl ones. hBATS and hBAT9 (1669 and 1491 base pairs in length, respectivel y) were isolated following screening of 4 x 10(5) clones of the cDNA l ibrary. Restriction mapping and sequence analysis demonstrated that th e cDNAs were identical except hBAT8 contained an additional 178 bases of 5' sequence; hBAT8 was completely sequenced, characterized, and use d for all subsequent studies. hBAT8 consisted of a 184-nudeotide 5'-no ntranslated region, an open reading frame of 1,254 bases predicting a protein of 418 amino acids with a molecular mass of 46,296 Da, and a 3 '-nontranslated region of 209 nucleotides followed by a poly(A)(+) tai l. The identity of the cDNA was confirmed by the following findings: 1 ) the open reading frame began with an ATG codon and was followed by a nucleotide sequence which, when translated, corresponded exactly to t he first 17 NH2-terminal amino acids of purified human liver BAT; 2) c ytosol of Escherichia coli XL1-Blue cells transfected with hBAT8 subcl oned into an expression vector, pKK233-2, demonstrated significant enz ymatic activity for the conjugation of both taurine and glycine with c holic acid; 3) bacterial expression of hBAT8 generated a protein that comigrated with hBAT from human liver during SDS-polyacrylamide gel el ectrophoresis and cross-reacted with a specific polyclonal rabbit anti -hBAT antibody during immunoblot analysis; 4) kinetic characteristics of the expressed enzyme were very similar to those reported for purifi ed liver BAT. These data demonstrate that a single cDNA is present in human liver which codes for a protein capable of catalyzing the conjug ation of cholic acid with both glycine and taurine.