Analysis of the amino terminus of maize branching enzyme II by polymerase chain reaction random mutagenesis.
Maize endosperm branching enzyme II (mBEII) plays a pivotal role in determining the quality of starch by catalyzing the synthesis of the alpha-1,6-branch points. While the central (alpha/beta)8-barrel and the C-terminal domains of mBEII have been analyzed previously, the possible role of its amino terminus in catalysis is still poorly understood. Because the amino terminus of mBEII shares very little sequence homology with other amylolytic enzymes, the Met1-Gly276 region of mBEII was randomly mutagenized under error-prone PCR conditions. Subsequent screening by a heterologous complementation system, utilizing an Escherichia coli strain devoid of the endogenous glycogen branching enzyme (glgB-), led to the recovery of mBEII mutants with altered iodine-staining patterns and reduced branching enzyme activities. The NR-625 mutant enzyme, which lacks the N-terminal 39 residues of mBEII due to a frameshift mutation introduced during the random mutagenesis, retained more than 70% of the wild-type activity. The chain transfer pattern and substrate preference of the truncated enzyme were almost identical to those of the wild-type mBEII. It appears that the N-terminal 39 residues of mBEII are neither required for catalysis nor involved in chain transfer. On the other hand, the Gln-to-Arg substitution at position 270 of mBEII resulted in the loss of more than 90% of branching activity. The Gln270 of mBEII, located at the beginning of the (alpha/beta)8-barrel domain, may be required for maximum enzyme activity.
Hong
S
S
Department of Biochemistry, Michigan State University, East Lansing 48824, USA.
Mikkelsen
R
R
Preiss
J
J
D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics; D015153:Blotting, Western; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000378:metabolism; D016368:Frameshift Mutation; D016296:Mutagenesis; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D016133:Polymerase Chain Reaction / Q000379:methods*; D003313:Zea mays / Q000201:enzymology*
D015061:1,4-alpha-Glucan Branching Enzyme; C112590:starch-branching enzyme II