8 b S Supporting Information 28 ' INTRODUCTION 29 Penicillin-binding proteins (PBPs) are DD-peptidases involved 30 in the late stages of peptidoglycan synthesis. 1,2 Their natural 31 substrate is the C-terminal D-Ala-D-Ala dipeptide of the stem 32 pentapeptide necessary to cross-link the glycan chains of the 33 peptidoglycan. 3 A series of compounds, mainly containing variations 34 on the theme of the β-lactam ring, have proven their efficacy as PBP 35 inhibitors. For over 60 years,

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... cephalosporins, monobac-36 tams, and carbapenems have been used to combat all types of 37 bacterial infections and continuously developed in response to the 38 progressive bacterial resistance to β-lactam antibiotics. 39 Recent research has intensified the investigation of non-β-lactam 40 inhibitors of PBPs, e.g., lactivicin, a bicyclic molecule with a γ-lactam/ 41 γ-lactone ring structure, 4,5 and shown that boronic acids repre-42 sent potential inhibitors of PBPs. 6À8 It has been known for a long 43 time that serine amidohydrolases are inhibited by boronic acids, 44 and structures of complexes between boronic acids and trypsin, 45 chymotrypsin, subtilisin, elastase, R-lytic protease, serine β-lactamases, 46 prolyl tripeptidyl aminopeptidase, and hormone-processing pro-47 65 γ-chymotrypsin and D-p-chlorophenyl-or D-1-naphthylboronic 66 acids. 15 Both compounds linked to Ser195Oγ and His57Nε2, 67 two residues of the catalytic triad of γ-chymotrypsin. Likewise, 68 dicovalent complexes with borate and benzylboronate linked to ABSTRACT: Boronic acids bearing appropriate side chains are good inhibitors of serine amidohy-10 drolases. The boron usually adopts a tetrahedral conformation, bound to the nucleophilic serine of the 11 active site and mimicking the transition state of the enzymatic reaction. We have solved the structures 12 of complexes of a penicillin-binding protein, the DD-peptidase from Actinomadura sp. R39, with four 13 amidomethylboronic acids (2,6-dimethoxybenzamidomethylboronic acid, phenylacetamidomethyl-14 boronic acid, 2-chlorobenzamidomethylboronic acid, and 2-nitrobenzamidomethylboronic acid) and 15 the pinacol ester derived from phenylacetamidomethylboronic acid. We found that, in each case, the 16 boron forms a tricovalent adduct with Oγ of Ser49, Ser298, and the terminal amine group of Lys410, 17 three key residues involved in the catalytic mechanism of penicillin-binding proteins. This represents 18 the first tricovalent enzymeÀinhibitor adducts observed by crystallography. In two of the five R39-19 boronate structures, the boronic acid is found as a tricovalent adduct in two monomers of the asymmetric unit and as a 20 monocovalent adduct with the active serine in the two remaining monomers of the asymmetric unit. Formation of the tricovalent 21 complex from a classical monocovalent complex may involve rotation around the Ser49 CRÀCβ bond to place the boron in a 22 position to interact with Ser298 and Lys410, and a twisting of the side-chain amide such that its carbonyl oxygen is able to hydrogen 23 bond to the oxyanion hole NH of Thr413. Biphasic kinetics were observed in three of the five cases, and details of the reaction 24 between R39 and 2,6-dimethoxybenzamidomethylboronic acid were studied. Observation of biphasic kinetics was not, however, 25 thought to be correlated to formation of tricovalent complexes, assuming that the latter do form in solution. On the basis of the 26 crystallographic and kinetic results, a reaction scheme for this unexpected inhibition by boronic acids is proposed.