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The molecular structure of the D-alanine:D-alanine ligase of the ddlB gene of Escherichia coli, co-crystallized with an S,R-methylphosphinate and adenosine triphosphate, was determined by x-ray diffraction to a resolution of 2.3 angstroms. A catalytic mechanism for the ligation of two D-alanine substrates is proposed in which a helix dipole and a hydrogen-bonded triad of tyrosine, serine, and glutamic acid assist binding and deprotonation steps. From sequence comparison, it is proposed that a different triad exists in a recently discovered D-alanine:D-lactate ligase (VanA) present in vancomycin-resistant enterococci. A molecular mechanism for the altered specificity of VanA is suggested.

The structure of the class C ampC 3lactam-ase (cephalosporinase) from Enterobacter cloacae strain P99 has been established by x-ray crystallography to 2-resolution and compared to a class A ,B-lactamase (penicillinase) struc- comparison of overall tertary folding shows that the cephalosporinase, more than the penicillinase, is broadly similar to the ancestral 3lactam-inhibited enzymes of bacterial cell wall synthesis. On this basis, it is proposed that the cephalosporinase is the older of the two 3lactamases, and, therefore, that a local refolding in the active site, rather than a simple point mutation, was required for the primordial class C &3-lactamase to evolve to the dass A -lactamase having an improved ability to catalyze the deacylation step of 3lactam hydrolysis.

The crystal structure of a complex formed on reaction of the Enterobacter cloacae P99 cephalosporinase (beta-lactamase) with a phosphonate monoester inhibitor, m-carboxyphenyl [[N-[(p-iodophenyl)acetyl]amino]methyl]phosphonate, has been obtained at 2.3-A resolution. The structure shows that the inhibitor has phosphonylated the active site serine (Ser64) with loss of the m-carboxyphenol leaving group. The inhibitor is positioned in the active site in a way that can be interpreted in terms of a transition-state analog. The arylacetamido side chain is placed as anticipated from analogous beta-lactamoyl complexes of penicillin-recognizing enzymes, with the amino group hydrogen-bonded to the backbone carbonyl of Ser318 (of the B3 beta-strand) and to the amides of Gln120 and Asn152. There is support in the asymmetry of the hydrogen bonding of this side chain to the protein and in the 2-fold disorder of the benzyl group for the considerable breadth in substrate specificity exhibited by class C beta-lactamases. One phosphonyl oxygen atom is in the oxyanion hole, hydrogen-bonded to main-chain NH groups of Ser318 and Ser64, while the other oxygen is solvated, not within hydrogen-bonding distance of any amino acid side chain. The closest active site functional group to the solvated oxygen atom is the Tyr150 hydroxyl group (3.4A); Lys67 and Lys315 are quite distant (4.3 and 5.7 A, respectively). Rather, Tyr150 and Lys67 are more closely associated with Ser64O gamma (2.9 and 3.3 A). This arrangement is interpreted in terms of the transition state for breakdown of the tetrahedral intermediate in the deacylation step of catalysis, where the Tyr150 phenol seems the most likely general acid. Thus, Tyr150, as the phenoxide anion, would be the general base catalyst in acylation, as proposed by Oefner et al. [Nature (1990) 343, 284-288]. The structure is compared with that of a similar phosphonate derivative of a class A beta-lactamase [Chen et al. (1993) J. Mol. Biol. 234, 165-178], and mechanistic comparisons are made. The sensitivity of serine beta-lactamases, as opposed to serine proteinases, toward inhibition by phosphonate monoanions is supported by electrostatic calculations showing a net positive potential only in the catalytic sites of the beta-lactamases.

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