PMID:2125053

Citation	Alpert, CA and Chassy, BM (1990) 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. J. Biol. Chem. 265:22561-8
Abstract	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.
Links	PubMed
Keywords	Base Sequence; Binding Sites; Cloning, Molecular/methods; Cysteine; DNA, Bacterial/genetics; Escherichia coli/genetics; Genes, Bacterial; Lactobacillus casei/enzymology; Lactobacillus casei/genetics; Lactococcus lactis/genetics; Molecular Sequence Data; Oligonucleotide Probes; Phosphoenolpyruvate Sugar Phosphotransferase System/genetics; Phosphoenolpyruvate Sugar Phosphotransferase System/metabolism; Phosphorylation; Protein Conformation; Restriction Mapping; Sequence Homology, Nucleic Acid; Staphylococcus aureus/enzymology
edit table

Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
LACCA:PTLCB		GO:0016310: phosphorylation	ECO:0000315:		P		Fig. 5 Lanes 1-4 indicate phosphorylation activity.	complete CACAO 3659
LACCA:PTLCB		GO:0046835: carbohydrate phosphorylation	ECO:0000315:		P		As seen in figure 7, lanes 1 through 6, when each cysteine residue was replaced in turn by serine, lactose phosphorylation was found in mutants in which cysteine residues 31, 137, 152, 235, 316, or 349 had been replaced. As seen in figure 7, lane 7, no phosphorylation of the substrate occurred when Cys483 was replaced with a serine. As seen in figure 7, lanes 7 through 9, to confirm that this effect was not due to an undetected secondary mutation, a total of five independently obtained Cys483+Ser483 mutants, from two different experiments, were assayed. It was found in all cases that these mutants did not phosphorylate lactose. As seen in figure 7, lanes 10 through 11, when Cys483 was replaced with histidine, an amino acid found to be involved in all other phosphoryltransfer reactions of the PTS, or tyrosine, no lactose phosphorylation was observed. The loss of phosphorylating activity appeared to be specifically due to the exchange of Cys483 and not due to disruption of the tertiary structure of the protein. Cys483 is the active center phosphorylated amino acid residue in EIIlacL.casei.	complete CACAO 4834
LACCA:PTLCB	involved_in	GO:0016310: phosphorylation	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete
LACCA:PTLCB	involved_in	GO:0046835: carbohydrate phosphorylation	ECO:0000315: mutant phenotype evidence used in manual assertion		P		Seeded From UniProt	complete

References

See Help:References for how to manage references in GONUTS.