Readthrough of the Bacillus subtilis stop codon produces an extended enzyme displaying a higher polymerase activity.
It has been generally accepted that the structural sacB gene of Bacillus subtilis levansucrase encodes a 50,000 Da extracellular protein. However, examination of the DNA sequence of the sacB flanking regions shows a putative open reading frame coding for a 20 amino acid peptide downstream immediately following the terminal TAA stop codon. By site-directed mutagenesis we have changed this stop codon to a glutamine codon. This stop codon readthrough leads to the synthesis and secretion by B. subtilis of a levansucrase possessing an extended polypeptide chain. The extended levansucrase has a molecular weight of 53,000 with a new carboxyl-terminus, rich in basic and hydrophobic amino acids and possessing one cysteine residue. This enzyme synthesizes fructosyl polymer levan of higher molecular weight than the shorter levansucrase. The increase in molecular weight was achieved by increasing the number of branches. These results suggest that the C-terminal part of the enzyme plays a specific role in the degree of branching of the synthesized polymer. Moreover, the extended enzyme is able to form an active dimer from two polypeptide chains linked by an S-S bridge.
Chambert
R
R
Institut Jacques Monod, Université Paris VII, France.
Rain-Guion
M C
MC
Petit-Glatron
M F
MF
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics*; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D003062:Codon; D004591:Electrophoresis, Polyacrylamide Gel; D005630:Fructans / Q000737:chemistry / Q000378:metabolism; D006602:Hexosyltransferases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D013728:Terminator Regions, Genetic
D003062:Codon; D005630:Fructans; D006602:Hexosyltransferases; C021513:levansucrase