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, kcat and Kms for ATP, and both first (D-Ala1), and second (D-Ala2) 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-Ala1 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-Ala2 to attack the acyl phosphate form of D-Ala1. While all seven mutants that retain activities have a 300-2000 fold elevation in Km for D-Ala1 (1-2 microM in wild type), the S281A mutant has a 500-fold elevation in Km for D-Ala2, consistent with a proposed interaction with the COO- of D-Ala2. Similarly, the kinetics of inhibition by a slow-binding phosphinate inhibitor in the presence of ATP are most altered in the S281A mutant.
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Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Walsh
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D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D018360:Crystallography, X-Ray; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005798:Genes, Bacterial; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D011487:Protein Conformation
D017931:DNA Primers; D004269:DNA, Bacterial; D004151:Dipeptides; C002956:alanylalanine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C019638:D-alanylalanine synthetase