Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii

• Published in: The Journal of biological chemistry Vol. 297; no. 5; p. 101280
• Main Authors: Watthaisong, Pratchaya, Binlaeh, Asweena, Jaruwat, Aritsara, Lawan, Narin, Tantipisit, Jirawat, Jaroensuk, Juthamas, Chuaboon, Litavadee, Phonbuppha, Jittima, Tinikul, Ruchanok, Chaiyen, Pimchai, Chitnumsub, Penchit, Maenpuen, Somchart
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.11.2021; American Society for Biochemistry and Molecular Biology
• Subjects: Acinetobacter baumannii - enzymology; Bacterial Proteins; Calcium - chemistry; Catalysis; Catalytic Domain; crystal structure; Crystallography, X-Ray; enzyme catalysis; Enzyme Stability; Fructose-Bisphosphate Aldolase - chemistry; metal-dependent enzyme; p-hydroxyphenylacetate degradation pathway; pyruvate-specific Class II metal aldolase; solvent-tolerant enzyme; stereoselectivity; stereospecificity; structure–function; Substrate Specificity; thermostable enzyme; Zinc - chemistry; (4S)-KDGlu; MPD; PPA; AbHpaI; LC-ESI-QTOF-MS; DTT; M2; HBA; PDB; metal-dependent enzyme; solvent-tolerant enzyme; BSA; LDH; NADH; MD; HOPA; Tm; stereospecificity; HKHD; ICP-OES; (NH4)2SO4; FPLC; SSA; (4R)-KDGal; thermostable enzyme; PYR; enzyme catalysis; MW; QM/MM; HNO3; DHAP; EDTA; p-hydroxyphenylacetate degradation pathway; OAA; EGTA; SEC; stereoselectivity; PEI; PMSF; HEPES; crystal structure; NaCl; EcHpaI; pyruvate-specific Class II metal aldolase; structure–function
• ISSN: 0021-9258; 1083-351X; 1083-351X
• DOI: 10.1016/j.jbc.2021.101280

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 (Tm 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.