Extradiol dioxygenases play a key role in determining the specificities of the microbial aromatic catabolic pathways in which they occur. To identify the structural determinants of specificity in this class of enzymes, variants of 2,3-dihydroxybiphenyl (DHB) 1,2-dioxygenase (DHBD) were investigated. Structural data of the DHBD/DHB complex informed the design of seven variants at four positions: V148W, V148L. M175W, A200I, A200W, P280W, and V148L/A2001. All variants had reduced specificity for DHB. In addition, the V148W. V148L, A200I, and V148L/A2001 variants had increased specificity for catechol. Indeed, the V148W variant had a higher apparent specificity for 3-Me catechol than for DHB, although the substitution reduced the k(cat) for all tested substrates as well as the rate constant of suicide inactivation of the enzyme. These results are consistent with available structural data which suggest that the larger residue at position 148 may partially occlude O-2 binding. The results further indicate that in addition to defining substrate specificity, the binding pocket orientates the bound catechol to minimize oxidative inactivation of the enzyme during catalysis. (c) 2005 Published by Elsevier Inc.