Abstract
NAD(P)+-dependent alcohol dehydrogenases (ADH) are widely distributed in all phyla. These proteins can be assigned to three nonhomologous groups of isozymes, with group III being highly diverse with regards to catalytic activity and primary structure. Members of group III ADHs share a conserved stretch of amino acid residues important for cofactor binding and metal ion coordination, while sequence identities for complete proteins are highly diverse (<20 to >90 %). A putative group III ADH PaYqhD has been identified in BLAST analysis from the plant pathogenic enterobacterium Pectobacterium atrosepticum. The PaYqhD gene was expressed in the heterologous host Escherichia coli, and the recombinant protein was purified in a two-step purification procedure to homogeneity indicating an obligate dimerization of monomers. Four conserved amino acid residues involved in metal ion coordination were substituted with alanine, and their importance for catalytic activity was confirmed by circular dichroism spectrum determination, in vitro, and growth experiments. PaYqhD exhibits optimal activity at 40 °C with short carbon chain aldehyde compounds and NADPH as cofactor indicating the enzyme to be an aldehyde reductase. No oxidative activities towards alcoholic compounds were detectable. EDTA completely inhibited catalytic activity and was fully restored by the addition of Co2+. Activity measurements together with sequence alignments and structure analysis confirmed that PaYqhD belongs to the butanol dehydrogenase-like enzymes within group III of ADHs.
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Acknowledgments
The authors would like to express their gratitude to Henning Piascheck for his help with the fermentations. Stéphane Boivin and Marvin Boothby at the “Sample Preparation and Characterization” facility of the EMBL at Hamburg are thanked for their help with the CD measurements. This work was funded by the Excellence Cluster in the Excellence Initiative by the State of Hamburg “Fundamentals of Synthetic Biological Systems (SynBio).”
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Elleuche, S., Fodor, K., von der Heyde, A. et al. Group III alcohol dehydrogenase from Pectobacterium atrosepticum: insights into enzymatic activity and organization of the metal ion-containing region. Appl Microbiol Biotechnol 98, 4041–4051 (2014). https://doi.org/10.1007/s00253-013-5374-z
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DOI: https://doi.org/10.1007/s00253-013-5374-z