Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii
| Autor: | Pimchai Chaiyen, Jirawat Tantipisit, Asweena Binlaeh, Litavadee Chuaboon, Juthamas Jaroensuk, Somchart Maenpuen, Penchit Chitnumsub, Ruchanok Tinikul, Aritsara Jaruwat, Jittima Phonbuppha, Pratchaya Watthaisong, Narin Lawan |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
structure–function
p-hydroxyphenylacetate degradation pathway Acinetobacter baumannii Enzyme complex PMSF phenyl methane sulfonyl fluoride QM/MM quantum mechanics/molecular mechanics DHAP dihydroxyacetone phosphate SSA succinic semialdehyde Crystallography X-Ray Biochemistry HKHD 4-hydroxy-2-ketoheptane-1 7-dioate MW molecular weight Substrate Specificity FPLC fast protein liquid chromatography LC-ESI-QTOF-MS liquid chromatography-electrospray ionization-quadrupole-time-of-flight mass spectrometer NaCl sodium chloride Aldol reaction stereospecificity Catalytic Domain Fructose-Bisphosphate Aldolase Enzyme Stability Tm melting temperature PPA propionaldehyde biology solvent-tolerant enzyme LDH lactate dehydrogenase Chemistry MD molecular dynamics Enzyme structure HOPA 4-hydroxy-2-oxopentanoate Zinc (NH4)2SO4 ammonium sulfate PYR pyruvate metal-dependent enzyme Research Article crystal structure HNO3 nitric acid SEC size-exclusion chromatography ICP-OES inductively coupled plasma-optical emission spectrometry Stereochemistry (4R)-KDGal (4R)-2-keto-3-deoxy-D-galactonate stereoselectivity enzyme catalysis Catalysis Enzyme catalysis Stereospecificity (4S)-KDGlu (4S)-2-keto-3-deoxy-D-gluconate Bacterial Proteins PDB Protein Data Bank NADH the reduced β-nicotinamide adenine dinucleotide thermostable enzyme Molecular Biology M2+ divalent meatl ion PEI polyethyleneimine MPD 2-methyl-2 4-pentanediol Aldolase A EGTA ethylene glycol-bis(2-aminoethylether)-N N N′ N′-tetraacetic acid HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Cell Biology AbHpaI 4-hydroxy-2-ketoheptane-1 7-dioate aldolase from Acinetobacter baumannii HBA 4-hydroxybenzaldehyde EcHpaI 4-hydroxy-2-ketoheptane-1 7-dioate aldolase from Escherichia coli Biocatalysis DTT dithiothreitol biology.protein Aldol condensation BSA bovine serum albumin EDTA ethylenediaminetetraacetatic acid Calcium OAA oxaloacetate pyruvate-specific Class II metal aldolase |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X 0021-9258 |
| Popis: | 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 A 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. |
| Databáze: | OpenAIRE |
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