Summary auto-generated
This study describes the purification of two key metabolic enzymes encoded by the TOL plasmid pWW53 from Pseudomonas putida MT53: benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase. The researchers developed a multi-step chromatographic procedure involving DEAE-Sephacel, Matrex Gel Red A, Blue Sepharose, Phenyl Sepharose, and Matrex Gel Green A columns to isolate both enzymes in homogeneous form. The purified benzyl alcohol dehydrogenase has a subunit molecular weight of 43,000 Da and benzaldehyde dehydrogenase is 56,300 Da, with both likely existing as tetramers. Characterization revealed that both enzymes have pH optima around 9.3-9.4, show similar substrate specificities for benzyl alcohol and benzaldehyde and their methylated analogs, and prefer NAD+ over NADP+ as cofactor (though benzaldehyde dehydrogenase can use both). Remarkably, these plasmid-encoded enzymes show striking similarity to chromosomally encoded homologs from Acinetobacter calcoaceticus in their kinetic properties, substrate profiles, and structural features, suggesting evolutionary relationships between enzymes in different bacterial genera.
Key findings
- Benzyl alcohol dehydrogenase (MW 43,000 Da) and benzaldehyde dehydrogenase (MW 56,300 Da) from P. putida TOL plasmid were successfully purified to homogeneity using a novel multi-step chromatographic approach
- Both enzymes are likely tetrameric in native form, have pH optima near 9.3-9.4, and show similar substrate specificities for unsubstituted and methylated benzyl alcohols/aldehydes
- The plasmid-encoded benzaldehyde dehydrogenase uniquely retains significant activity with NADP+ (27% relative to NAD+) and requires monovalent cations for activity, resembling benzaldehyde dehydrogenase I from A. calcoaceticus
- Plasmid-encoded and chromosomal dehydrogenases from different bacterial genera show remarkable functional similarity in kinetics and heat stability, suggesting evolutionary links between these enzymes
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Abstract
Summary: A procedure was developed for purifying both the benzyl alcohol dehydrogenase and the benzaldehyde dehydrogenase encoded by the TOL plasmid pWW53 from a single batch of Pseudomonas putida MT53. The procedure involved disruption of the bacteria in the French pressure cell and preparation of a high-speed supernate, followed by chromatography on DEAE-Sephacel, Matrex Gel Red A and Blue Sepharose CL-6B which separated the two enzymes, Phenyl Sepharose CL-4B and Matrex Gel Green A. The final preparations gave single bands on electrophoresis under denaturing and non-denaturing conditions. The subunit Mr values of benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase are 43000 and 56300 respectively. Cross-linking studies with dimethylsuberimidate indicate that both enzymes are probably tetramers, although they run anomalously through gel-filtration columns. The benzyl alcohol dehydrogenase was fairly specific for NAD+ as cofactor but the benzaldehyde dehydrogenase had appreciable activity with NADP+ as well as with NAD+. The optimum pH values are 9.4 and 9.3 for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase respectively. Benzaldehyde dehydrogenase appears to require a monovalent cation for maximum activity. The apparent Km and maximum velocity values of the two plasmid-encoded dehydrogenases and of the chromosomally encoded benzyl alcohol dehydrogenase and benzaldehyde dehydrogenases I and II from Acinetobacter calcoaceticus were determined for NAD+ and for the unsubstituted substrates and for the monomethyl ring-substituted analogues. The corresponding apparent specificity constants were then calculated. All three benzaldehyde dehydrogenases had very similar substrate profiles, as did the two benzyl alcohol dehydrogenases. The plasmid-encoded benzaldehyde dehydrogenase resembles benzaldehyde dehydrogenase I from A. calcoaceticus (which also requires monovalent cations for activity) in being much more heat-stable than benzaldehyde dehydrogenase II. Overall, the plasmid-encoded benzyl alcohol dehydrogenase from P. putida appears to be remarkably similar to the chromosomally encoded benzyl alcohol dehydrogenase from A. calcoaceticus, and the plasmid-encoded benzaldehyde dehydrogenase is very similar to the two chromosomally encoded benzaldehyde dehydrogenases, particularly benzaldehyde dehydrogenase I.