Roles of histidine-103 and tyrosine-235 in the function of the prolipoprotein diacylglyceryl transferase of Escherichia coli.
Phosphatidylglycerol:prolipoprotein diacylglyceryl transferase (Lgt) is the first enzyme in the posttranslational sequence of reactions resulting in the lipid modification of lipoproteins in bacteria. A previous comparison of the primary sequences of the Lgt enzymes from phylogenetically distant bacterial species revealed several highly conserved amino acid sequences throughout the molecule; the most extensive of these 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. coli Lgt with diethylpyrocarbonate were consistent with the modification of a single essential histidine or tyrosine residue. The current study was conducted in an attempt to identify this essential amino acid residue in order to further define structure-function relationships in Lgt. Accordingly, all of the histidine residues and seven of the tyrosine residues of E. coli Lgt were altered by site-directed mutagenesis, and the in vitro activities of the altered enzymes, as well the abilities of the respective mutant lgt alleles to complement the temperature-sensitive phenotype of E. coli 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 that Tyr-235 plays an important role in the function of this enzyme. Although other histidine and tyrosine residues were not found to be essential for Lgt activity, alterations of His-196 resulted in a significant reduction of in vitro activity.
Sankaran
K
K
Department of Microbiology and Immunology, The Uniformed Services University of the Health Sciences, Bethesda, Maryland 20814-4799, USA.
Gan
K
K
Rash
B
B
Qi
H Y
HY
Wu
H C
HC
Rick
P D
PD
D000595:Amino Acid Sequence; D017124:Conserved Sequence; D004047:Diethyl Pyrocarbonate / Q000494:pharmacology; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005816:Genetic Complementation Test; D006639:Histidine; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D010802:Phylogeny; D017354:Point Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014166:Transferases / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine
D010446:Peptide Fragments; D011994:Recombinant Proteins; D014443:Tyrosine; D006639:Histidine; D014166:Transferases; C042308:phosphatidylglycerol glyceryl transferase; D004047:Diethyl Pyrocarbonate