A Conserved Tyrosyl−Glutamyl Catalytic Dyad in Evolutionarily Linked Enzymes: Carbapenam Synthetase and β-Lactam Synthetase

The compilation of genome sequence information has revealed genes encoding proteins of natural product biosynthetic pathways that differ in function from their primary metabolic ancestors. The clear evolution of these often well-studied enzymes to new, specialized roles is striking but frequently difficult to deduce from their primary sequence alone (1). Particularly in microorganisms, it is thought that survival under environmental stress will select mutant organisms whose overall fitness has improved (2, 3). The β-lactam-containing secondary metabolites, most widely known for their antibacterial properties, are an instructive example of this evolutionary process. Streptomyces clavuligerous β-lactam synthetase (β-LS) and Pectobacterium carotorova carbapenam synthetase (CPS)1 catalyze formation of the corresponding β-lactam rings in the clinically important β-lactamase inhibitor clavulanic acid (4) and (5R)-carbapenem-3-carboxylic acid (5), which is believed to be representative of the potent, broad spectrum carbapenem class of antibiotics. These pivotal transformations occur early in the respective biosyntheses. Based on both sequence and structure homology (4, 6), these β-lactam-forming enzymes show evolutionary roots in the broadly distributed asparagine synthetases, class B (AS-B), which convert the essential amino acid aspartate to asparagine. The sequence and structure similarity among AS-B, β-LS, and CPS are reflected in the overall chemistry leading to the synthesis of asparagine, deoxyguanidinoproclava-minic acid (DGPC), and (3S,5S)-carbapenam-3-carboxylate,2 respectively (Scheme 1). In their synthetase domains these three ATP/Mg2+-dependent reactions undergo two distinct chemical steps (or half-reactions): acyl-adenylation and nucleophilic acyl substitution (7–10). Recently, much has been learned about β-LS and CPS by detailed examination of their steady-state kinetics (11, 12). Both proteins exhibit an ordered Bi-Ter kinetic mechanism with ATP the first substrate to bind and PPi the last product to dissociate (7, 10). Additionally, a conformational change involving the opening of a catalytic loop associated with product release is at least partially rate-determining in both enzymes (11, 12). A large forward commitment exists to DGPC and carbapenam formation after acyl-adenylate activation of the respective substrates, implicating both substrate preorganization and the high chemical reactivity of the adenylated intermediate as important catalytic strategies for β-LS and CPS (7, 11). Since these β-lactam-producing enzymes share similar sequence and structure together with other common mechanistic features, it is proposed that this step of clavam and carbapenem biosynthesis is very closely related (13). Scheme 1 Chemical Reactions of β-LS, CPS, and AS-B: Acyl-Adenylate Activation and Nucleophilic Substitutiona The evolution to β-LS and CPS from their predecessor, AS-B, likely occurred independently through gene duplication and random mutations to enzymes of changed function (14). Crystal structures of β-LS provide hints to how this process occurred. While retaining the two subunits of AS-B (glutaminase and synthetase domains), an essential ammonia tunnel has collapsed and is blocked at the entrance to the synthetase domain of β-LS (15). Moreover, the pseudoglutaminase domain in β-LS lacks the catalytic cysteine essential for glutaminase activity in AS-B (16–18). Kinetic studies on β-LS and CPS reinforced the observation of an inactive glutaminase domain and demonstrated the complete loss of asparagine synthetase activity. Deprived of both a source and a path to transport “nascent” ammonia to an adenylated substrate in the synthetase domain, an intramolecular amine in N2-(2-carboxyethyl)-l-arginine (CEA) and (2S,5S)-carboxymethylproline (CMPr), both β-amino acids, is utilized in β-LS and CPS, respectively. In the case of β-LS, the active site is markedly elongated to accommodate the increased length of the substrate CEA (12 A) relative to aspartate (5 A) (19). A key substitution visualized in overlays of β-LS and CPS with AS-B was a tyrosine residue in the β-lactam-forming enzymes (β-LS, Y348; CPS, Y345), replacing an active site glutamate (E348) found in AS-B (Figure 1). E348 is universally conserved in AS-B enzymes and located at the end of the relatively hydrophobic ammonia tunnel (15). Site-directed mutagenesis of this glutamate to alanine in Escherichia coli AS-B resulted in undetectable asparagine formation and implicated it as the catalytic base responsible for ensuring deprotonation to a neutral ammonia nucleophile leading to Asn formation (unpublished data in ref 20). Figure 1 Sequence and structural comparison of homologues AS-B, β-LS, and CPS. Panel A gives a partial sequence alignment of AS-B, β-LS, and CPS with weak to identical matches in amino acid sequence shown as light blue to dark blue, respectively. ... Substitution of this strictly conserved glutamate in primary metabolism to a tyrosine in both β-LS and CPS could account for how AS-B enzymes altered their chemical mechanisms to produce β-lactam rings. While the first half-reaction of AS-B relative to β-LS and CPS is identical, the key difference in the second half-reaction is intermolecular nucleophilic attack by ammonia in AS-B versus intramolecular attack by a secondary amine in the case of β-LS and CPS. A more advanced mechanism of catalysis can be proposed from structural images of β-LS and CPS mediated by way of a Tyr-Glu catalytic dyad (Y348/E382 in β-LS; Y345/E380 in CPS). The dyad glutamate in β-LS and CPS corresponds to a similar amino acid in AS-B, D384. D384, however, is thought to be involved only in binding the α-amine of its aspartate substrate (19). The Tyr-Glu dyad in β-LS and CPS is proposed to deprotonate the secondary amine of their respective substrates, which is bound as its ammonium ion, to allow intramolecular nucleophilic substitution of the acyl-adenylate and ring cyclization. In the case of β-LS, N2-(carboxymethyl)-l-arginine (CMA), the lower homologue of CEA, cannot cyclize to the strained α-lactam. It could, however, undergo acyl-adenylation, which was visualized in high resolution and, thereby, captured an image of the high-energy intermediate generated in the first half-reaction. These pictures indicated that the dyad tyrosine Oη and glutamate Oδ are 2.5 A apart, and it is estimated that the corresponding hydrogen bound between these two residues is even closer (17). The potential functional importance suggested by these observations of a catalytic Tyr-Glu dyad observed in β-LS and CPS is demonstrated by site-directed mutagenesis and kinetic experiments that compare the wild-type enzymes to their respective mutant proteins using pH–rate profiles, 32P-incorporation experiments, solvent isotope effects (SIE), proton inventory, and viscosity variation.