Abstract

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn²⁺ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M²⁺) cofactors, except Ca²⁺, for catalysis. We found that Zn²⁺ yielded the highest enzyme complex thermostability (T<inf>m</inf> of 87 ^°C) and solvent tolerance. All AbHpaI·M²⁺ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI·Zn²⁺ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M²⁺ cofactors). For the aldol condensation reaction, AbHpaI·M²⁺ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ~40-fold. Based on 11 X-ray structures of AbHpaI complexed with M²⁺ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M²⁺ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn²⁺-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca²⁺ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI·Zn²⁺ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.