Engineering and directed evolution of a Ca2+ binding site A-deficient AprE mutant reveal an essential contribution of the loop Leu75-Leu82 to enzyme activity.
An aprE mutant from B. subtilis 168 lacking the connecting loop Leu(75)-Leu(82) which is predicted to encode a Ca(2+) binding site was constructed. Expression of the mutant gene (aprEDeltaLeu(75)-Leu(82)) produced B. subtilis colonies lacking protease activity. Intrinsic fluorescence analysis revealed spectral differences between wild-type AprE and AprEDeltaL(75)-L(82). An AprEDeltaL(75)-L(82) variant with reestablished enzyme activity was selected by directed evolution. The novel mutations Thr(66)Met/Gly(102)Asp located in positions which are predicted to be important for catalytic activity were identified in this variant. Although these mutations restored hydrolysis, they had no effect with respect to thermal inactivation of AprEDeltaL(75)-L(82) T(66)M G(102)D. These results support the proposal that in addition to function as a calcium binding site, the loop that connects beta-sheet e3 with alpha-helix c plays a structural role on enzyme activity of AprE from B. subtilis 168.
Romero-García
Eliel R
ER
Departamento de Biología, Universidad de Guanajuato, Colonia Noria Alta S/N, Guanajuato, 36050 Guanajuato, Mexico.
Téllez-Valencia
Alfredo
A
Trujillo
María F
MF
Sampedro
José G
JG
Nájera
Hugo
H
Rojo-Domínguez
Arturo
A
García-Soto
Jesús
J
Pedraza-Reyes
Mario
M
D001412:Bacillus subtilis / Q000201:enzymology / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D019020:Directed Molecular Evolution / Q000379:methods*; D004795:Enzyme Stability; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed / Q000379:methods*; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship
C088074:AprE protein, Bacteria; D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D002118:Calcium