Acinetobacter calcoaceticus encoded mutarotase: nucleotide sequence analysis of the gene and characterization of its secretion in Escherichia coli.
The nucleotide sequence of the mutarotase gene from Acinetobacter calcoaceticus has been determined. It reveals an open reading frame of 381 amino acids. The codon usage of A. calcoaceticus for this gene is similar to E. coli except for the amino acids Leu, Ala, Glu, and Arg where major differences exist. This did not interfere drastically with high level expression in E. coli. The regulatory sequences for the initiation of translation are similar to the ones described for E. coli. The N-terminal 20 amino acids, which are not found in the mature enzyme, show homology to signal sequences of exported proteins. In A. calcoaceticus and E. coli mutarotase is specifically secreted into the periplasmic space. Processing of the signal sequence occurs at identical sites in both organisms. The mature mutarotase consists of 361 amino acids and has a calculated molecular weight of 38457 Da. Expression of mutarotase at a high level in a recombinant E. coli destabilizes the outer membrane. This results in coordinated leakage of mutarotase and beta-lactamase into the culture broth.
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002238:Carbohydrate Epimerases / Q000235:genetics*; D002462:Cell Membrane / Q000201:enzymology; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D005786:Gene Expression Regulation; D008970:Molecular Weight; D021382:Protein Sorting Signals / Q000235:genetics
D021382:Protein Sorting Signals; D004262:DNA Restriction Enzymes; D002238:Carbohydrate Epimerases; C019646:aldose 1-epimerase