ROLES OF HISTIDINE-103 AND TYROSINE-235 IN THE FUNCTION OF THE PROLIPOPROTEIN DIACYLGLYCERYL TRANSFERASE OF ESCHERICHIA-COLI

Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) i s the first enzyme in the posttranslational sequence of reactions resu lting in the lipid modification of lipoproteins in bacteria. A previou s comparison of the primary sequences of the Lgt enzymes from phylogen etically distant bacterial species revealed several highly conserved a mino acid sequences throughout the molecule; the most extensive of the se was the region 103HGGLIG108 in the Escherichia coli Lgt (H.-Y. Qi, K. Sankaran, K. Gan, and H. C. Wu, J. Bacteriol. 177:6820-6824, 1995). These studies also revealed that the kinetics of inactivation of E. c oli Lgt with diethylpyrocarbonate were consistent with the modificatio n of a single essential histidine or tyrosine residue. The current stu dy was conducted in an attempt to identify this essential amino acid r esidue in order to further define structure-function relationships in Lgt, Accordingly, all of the histidine residues and seven of the tyros ine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abili ties of the respective mutant Ist alleles to complement the temperatur e-sensitive phenotype of E. call SK634 defective in Lgt activity, were determined, The data obtained from these studies, in conjunction with additional chemical inactivation studies, support the conclusion that His-103 is essential for Lgt activity. These studies also indicated t hat Tyr-235 plays an important role in the function of this enzyme. Al though other histidine and tyrosine residues were not found to be esse ntial for Lgt activity, alterations of His-196 resulted in a significa nt reduction of in vitro activity.