Heterologous expression and characterization of wild-type and mutant forms of a 26 kDa endochitinase from barley (Hordeum vulgare L.).
To investigate structure-function relationships in plant chitinases, we have developed a heterologous expression system for the 26 kDa endochitinase from Hordeum vulgare L. (barley). Escherichia coli cells harbouring the gene in a T7 RNA polymerase-based expression vector synthesized completely insoluble recombinant protein under standard induction conditions at 37 degrees C. However, a concentration of soluble recombinant protein of approx. 15 mg/l was achieved by inducing bacteria at low temperature (15 degrees C). Recombinant endochitinase was purified to homogeneity and shown to be structurally and functionally identical to the seed protein. An average of three disulphide bonds are present in the recombinant enzyme, consistent with the number found in the natural form. The seed and recombinant proteins showed the same specific activity towards a high-molecular-mass substrate and exhibited similar anti-fungal activity towards Tricoderma reesei. Site-directed mutagenesis was used to replace residues that are likely to be involved in the catalytic event, based on structural similarities with lysozyme and on sequence alignments with related chitinases. The Glu67-->Gln mutation resulted in a protein with undetectable activity, while the Glu89-->Gln mutation yielded an enzyme with 0. 25% of wild-type specific activity. This suggests that two acidic residues are essential for catalytic activity, similar to the situation with many other glycosyl hydrolases. Examination of conserved residues stretching into the proposed substrate binding cleft suggests that Asn124 also plays an important functional role.
Andersen
M D
MD
Department of Protein Chemistry, Institute of Molecular Biology, University of Copenhagen, Oster Farimagsgade 2A, DK-1353 Copenhagen K, Denmark.
Jensen
A
A
Robertus
J D
JD
Leah
R
R
Skriver
K
K
D002688:Chitinases / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D018506:Gene Expression Regulation, Plant; D001467:Hordeum / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
D018076:DNA, Complementary; D011994:Recombinant Proteins; D002688:Chitinases