Molecular cloning and DNA sequence of lacE, the gene encoding the lactose-specific enzyme II of the phosphotransferase system of Lactobacillus casei. Evidence that a cysteine residue is essential for sugar phosphorylation.
The gene coding for the lactose-specific Enzyme II of the Lactobacillus casei phosphoenolpyruvate-dependent phosphotransferase system, lacE, has been isolated by molecular cloning and expressed in Escherichia coli. The DNA sequence of the lacE gene and the deduced amino acid sequence are presented. The putative translation product comprises a hydrophobic protein of 577 amino acids with a calculated molecular mass of 62,350 Da. The deduced polypeptide has a high degree of sequence similarity with the corresponding lactose-specific enzymes II of Staphylococcus aureus and Lactococcus lactis. The sequence surrounding cysteine 483 was strongly conserved in the three proteins. The identity of the lacE product as the Enzyme IIlacL.casei was demonstrated by in vitro lactose phosphorylation assays using the protein expressed in E. coli. Single replacement of each of the histidine and cysteine residues by site-directed mutagenesis pointed to cysteine 483 as an amino acid residue essential for the phosphoryl group transfer reaction.
Alpert
C A
CA
Laboratory for Microbial Ecology, National Institute for Dental Research, National Institutes of Health, Bethesda, Maryland 20892.
Chassy
B M
BM
D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine; D004269:DNA, Bacterial / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics*; D013294:Lactococcus lactis / Q000235:genetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D013211:Staphylococcus aureus / Q000201:enzymology
D004269:DNA, Bacterial; D015345:Oligonucleotide Probes; D010731:Phosphoenolpyruvate Sugar Phosphotransferase System; C029022:phosphoenolpyruvate-lactose dependent phosphotransferase system; D003545:Cysteine