Structure-guided directed evolution of alkenyl and arylmalonate decarboxylases.

The asymmetric decarboxylation of prochiral a-disubstituted malonic acids is an attractive route to produce homochiral asubstituted carboxylic acids. Various metal-catalysed and organocatalytic methods have been explored for related enantioselective decarboxylative protonation (EDP) reactions. However, the synthetic EDPmethods can only deliver chiral carboxylic acids or esters with moderate enantioselectivities (ee). On the other hand, enzymatic decarboxylation offers a cleaner, more sustainable and more efficient approach to deliver carboxylic acids of very high ee. Notably, the arylmalonate decarboxylase (AMDase) isolated from Bordetella bronchiseptica has been shown to be highly effective in the decarboxylation of a-arylmalonates to give enantiomerically pure a-arylcarboxylic acids (Scheme 1). The fact that the AMDase is highly robust and does not require co-factors further increases the potential of this enzyme for biocatalysis. Despite this, the pool of substrates accepted by the enzyme is limited to malonates that possess aaryl substituents. Indeed a number of other bacterial AMDases have been discovered from sequence similarity searches, or selective enrichment experiments. While all these enzymes could catalyse the decarboxylation of aarylmalonates, none have so far been demonstrated to decarboxylate malonates which do not possess a-aryl substituents. In order to rationalise this, and guide the development of new enzymes which could catalyse a wider range of reactions, we solved the X-ray crystal structure of the B. bronchiseptica AMDase. Our first high-resolution structure reveals a “dioxyanion hole” motif, which can donate six hydrogen bonds to potentially stabilize a putative high-energy enediolate intermediate, along with a small hydrophobic cavity likely favouring the formation of the neutral CO2. From this we were able to suggest a mechanism for the AMDase (Scheme 1). From comparison of the AMDase structure with structures of Asp/Glu racemases, we proposed that conserved dioxyanion hole motifs could have evolved to stabilise common enediolate intermediates in these mechanistically related enzymes. Indeed, up until our structure of the AMDase, there was no evidence to suggest how the AMDase or Asp/Glu racemases stabilised common putative high-energy enediolate intermediates. However, our proposal was based on a structure which only possessed phosphate as an active-site ligand. To lend further support to our proposal, we here present a second B. bronchiseptica AMDase structure with a mechanism-based inhibitor that more closely resembles the enediolate intermediate bound to the active site of the enzyme. This allowed us to rationalise the mechanism of this simple yet intriguing enzyme and design new and effective malonate substrates for the AMDase. Also, the new structure guided directed evolution of new AMDase mutants with significantly enhanced catalytic efficiency with a range of substrates. Scheme 1. Proposed mechanism of the AMDase. Preferred malonate substrates for the AMDase possess a larger substituent (L=aryl or alkenyl), which binds to a solvent-accessible cavity, and a smaller substituent (S=H, Me, OH, NH2). The neutral leaving group CO2 develops within the small hydrophobic pocket and the resulting enediolate is stabilised by the dioxyanion hole. In the case of the model substrate 2-methyl-2-phenylmalonate (L=Ph, S=Me) decarboxylation has been shown to occur through loss of the pro-R carboxylate and subsequent protonation of the si-face by Cys188, resulting in overall inversion of configuration.