Single mutations of residues outside the active center of the xylanase Xys1 Delta from Streptomyces halstedii JM8 affect its activity.
Mutagenesis of the xylanase Xys1 of Streptomyces halstedii JM8 has been done by error prone PCR. Mutants with modified hydrolytic activity were isolated, the recombinant variant proteins purified and the catalytic activities of each one determined and compared with the wild type enzyme. Two of the isolated single point mutants, m1 (G133D) and m8 (N148D), showed 22-25% increase in specific activity towards xylan compared to wild type xylanase. Two other mutants, m5a (D175A) and m7 (T160A), showed a significant reduction in specific activity of 40-50% with respect to the wild type enzyme. These residues are mainly located in the beta alpha-loops of the xylanase, the region showing the main structural divergences within family 10 of xylanases. This study shows the usefulness of random mutagenesis to point out some key residues not directly involved in the active center, but in which mutation produces subtle structural rearrangements affecting the enzymatic function.
Díaz
Margarita
M
Departamento de Microbiología y Genética, Instituto de Microbiología Bioquímica, Consejo Superior de Investigaciones Científicas (CSIC), Universidad de Salamanca, Edificio Departamental, Campus Miguel de Unamuno 37007, Salamanca, Spain.
Rodriguez
Sonia
S
Fernández-Abalos
José Manuel
JM
De Las Rivas
Javier
J
Ruiz-Arribas
Alberto
A
Shnyrov
Valery L
VL
Santamaría
Ramón I
RI
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D001665:Binding Sites; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D020125:Mutation, Missense; D017354:Point Mutation; D011487:Protein Conformation; D016415:Sequence Alignment; D013302:Streptomyces / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D014990:Xylans / Q000378:metabolism; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D014990:Xylans; D014995:Xylosidases; C107879:Xys1 protein, Streptomyces halstedii