ACTIVE-SITE MAPPING OF ESCHERICHIA-COLI D-ALA-D-ALA LIGASE BY STRUCTURE-BASED MUTAGENESIS

Eleven Escherichia coli D-Ala-D-Ala ligase (DdlB) mutants, at K144, K215, and E270 in the ATP binding site, at E15, S150, H63, and R255 in the first D-Ala subsite, and at Y216, S281, L282, and D257 in the second D-Ala subsite, were constructed, purified, and examined for steady-state kinetic parameters, k(cat) and K(m)s for ATP, and both first (D-Ala(1)), and second (D-Ala(2)) D-alanines. Of these, E270Q, K215A, R255A, and D257N retained very low or no detectable activity consistent with X-ray structure based predictions for roles in Mg2+ coordination to beta,gamma-P of ATP (E270), coordination to transferring gamma-PO3 of ATP (K215), and coordination/orientation of nucleophilic COO- of D-Ala(1) that attacks gamma-PO3 of ATP (R255, D257) and the side chain of R255, respectively. The substantial retention of activity in the Y216F mutant argues against the possibility that Y216 may be a catalytic base that deprotonates the alpha-NH3+ of D-Ala(2) to attack the acyl phosphate form of D-Ala(1). While all seven mutants that retain activities have a 300-2000 fold elevation in K-m for D-Ala(1) (1-2 mu M in wild type), the S281A mutant has a 500-fold elevation in K-m for D-Ala(2), consistent with a proposed interaction with the COO- of D-Ala(2). Similarly, the kinetics of inhibition by a slow-binding phosphinate inhibitor in the presence of ATP are most altered in the S281A mutant.