STRUCTURE-FUNCTION RELATIONSHIP OF BACTERIAL PROLIPOPROTEIN DIACYLGLYCERYL TRANSFERASE - FUNCTIONALLY SIGNIFICANT CONSERVED REGIONS

The structure-function relationship of bacterial prolipoprotein diacyl glyceryl transferase (LGT) has been investigated by a comparison of th e primary structures of this enzyme in phylogenetically distant bacter ial species, analysis of the sequences of mutant enzymes, and specific chemical modification of the Escherichia coli enzyme, A clone contain ing the gene for LGT, Igt, of the gram-positive species Staphylococcus aureus was isolated by complementation of the temperature-sensitive I gt mutant bf E. coli (strain SK634) defective in LGT activity, In vivo and in vitro assays for prolipoprotein diacylglyceryl modification ac tivity indicated that the complementing clone restored the prolipoprot ein modification activity in the mutant strain, Sequence determination of the insert DNA revealed an open reading frame of 837 bp encoding a protein of 279 amino acids with a calculated molecular mass of 31.6 k Da. S. aureus LGT showed 24% identity and 47% similarity with E. coli, Salmonella typhimurium, and Haemophilus influenzae LGT, S. aureus LGT , while 12 amino acids shorter than the E. coli enzyme, had a hydropat hic profile and a predicted pi (10.4) similar to those of the E. coli enzyme, Multiple sequence alignment among E. coli, S. typhimurium, H. influenzae, and S. aureus LGT proteins revealed regions of highly cons erved amino acid sequences throughout the molecule, Three independent Igt mutant alleles from E. coli SK634, SK635, and SK636 and one Igt al lele from S. typhimurium SE5221, all defective in LGT activity at the nonpermissive temperature, were cloned by PCR and sequenced, The mutan t alleles were found to contain a single base alteration resulting in the substitution of a conserved amino acid, The longest set of identic al amino acids without any gap tvas H-103-GGLIG-108 in LGT from these four microorganisms. In E. coli Igt mutant SK634, Gly-104 in this regi on was mutated to Ser, and the mutant organism was temperature sensiti ve in growth and exhibited low LGT activity in vitro, Diethylpyrocarbo nate inactivated the E. coli LGT with a second-order rate constant of 18.6 M(-1) s(-1), and the inactivation of LGT activity was reversed by hydroxylamine at pH 7. The inactivation kinetics were consistent with the modification of a single residue, His or Tyr, essential for LGT a ctivity.