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-m of 87 degrees C) and solvent tolerance. All AbHpaI center dot M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGa1) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI center dot Zn2+ displaying the highest RIS stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI center dot M2+ only specifically forms (4R)-KDGal and not (45)-KDGlu and preferentially catalyzes condensation rather than cleavage by similar to 40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 angstrom 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 center dot Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.
