Interspecific luciferase beta subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi.
Bacterial luciferase (EC 1.14.14.3) is a heterodimer composed of alpha- and beta-chains encoded by luxA and luxB, respectively. Although some interspecific combinations of these subunits lead to active enzyme, others do not. The beta subunits of Vibrio fischeri and Photobacterium leiognathi form active enzyme with the alpha subunits of V.fischeri, P.leiognathi and Vibrio harveyi, while the beta subunit from V.harveyi only complements the alpha subunit of V.harveyi. Inactivity is caused by a lack of dimerization of the beta subunit of V.harveyi with the alpha subunits of V.fischeri and P.leiognathi. These observations served as the basis for a search to discover which segment of the beta polypeptide confers the ability to dimerize with the alpha subunits of V.fischeri and P.leiognathi. Intragenic beta subunit hybrids were made between V.harveyi, V.fischeri and P.leiognathi. Unique restriction sites were introduced into the respective luxB genes to divide them into four roughly equal segments. In all, 78 hybrids were constructed by in vitro techniques. The N-terminal segment of the peptide contains the signals that differentiate between the beta subunits of V.fischeri and P. leiognathi and the beta subunit of V. harveyi, and allow the former to dimerize with their alpha subunits. The second segment has no major effect on enzyme activity but does exhibit some context effects. Important interactions were found between the third and fourth segments of the polypeptide with respect to enzymatic activity.
Faculty of Biology, Technion, Israel Institute of Technology, Haifa, Israel.
S
S
Almashanu
I
I
Gendler
R
R
Hadar
J
J
Kuhn
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D004262:DNA Restriction Enzymes; D004269:DNA, Bacterial / Q000235:genetics; D019281:Dimerization; D006824:Hybridization, Genetic; D008156:Luciferases / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D010776:Photobacterium / Q000201:enzymology / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014733:Vibrio / Q000201:enzymology / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D008156:Luciferases; D004262:DNA Restriction Enzymes