Replacement of active-site residues of quinoline 2-oxidoreductase involved in substrate recognition and specificity.

Amino acid residues in the active site of quinoline 2-oxidoreductase (Qor) that are deemed important for substrate binding and turnover were replaced by site-directed mutagenesis. The apparent k(cat) values for quinoline were reduced 2.4-, 38-, 40-, and 199-fold in the protein variants QorA259G, QorW331G, QorV373A, and QorA546G, respectively. The substitution A259G did not significantly alter K(m app). Despite the presumed crucial role of W331 and V373 in substrate positioning, the replacements W331G (K(m app): 0.33 mM) and V373A (K(m app): 0.41 mM) only slightly affected affinity for quinoline (K(m app) of Qor: 0.12 mM). QorA546G showed an increased affinity for quinoline and quinoxaline, as suggested by its 4.3- and 7.5-fold decrease in K(m) (app (quinoline))and K(m app (quinoxaline)), respectively, compared with Qor. The relative activities of the protein variants towards substituted quinolines differed from those of Qor. QorW331G, for example, may be suitable for hydroxylation of quinoxaline and C4-substituted quinolines.