Study of ALDH from Thermus thermophilus-Expression, Purification and Characterisation of the Non-Substrate Specific, Thermophilic Enzyme Displaying Both Dehydrogenase and Esterase Activity.

Kim Shortall,Tewfik Soulimane,Edel Durack,Edmond Magner

doi:10.3390/cells10123535

Abstract

Aldehyde dehydrogenases (ALDH), found in all kingdoms of life, form a superfamily of enzymes that primarily catalyse the oxidation of aldehydes to form carboxylic acid products, while utilising the cofactor NAD(P)+. Some superfamily members can also act as esterases using p-nitrophenyl esters as substrates. The ALDHTt from Thermus thermophilus was recombinantly expressed in E. coli and purified to obtain high yields (approximately 15–20 mg/L) and purity utilising an efficient heat treatment step coupled with IMAC and gel filtration chromatography. The use of the heat treatment step proved critical, in its absence decreased yield of 40% was observed. Characterisation of the thermophilic ALDHTt led to optimum enzymatic working conditions of 50 °C, and a pH of 8. ALDHTt possesses dual enzymatic activity, with the ability to act as a dehydrogenase and an esterase. ALDHTt possesses broad substrate specificity, displaying activity for a range of aldehydes, most notably hexanal and the synthetic dialdehyde, terephthalaldehyde. Interestingly, para-substituted benzaldehydes could be processed efficiently, but ortho-substitution resulted in no catalytic activity. Similarly, ALDHTt displayed activity for two different esterase substrates, p-nitrophenyl acetate and p-nitrophenyl butyrate, but with activities of 22.9% and 8.9%, respectively, compared to the activity towards hexanal.

Highlights

Found in all kingdoms of life, aldehyde dehydrogenases (ALDH) (EC;1.2.1.3) constitute a large family of NAD(P)+ -dependent enzymes with a molecular mass of ca.50–60 kDa, and are composed of 450–500 amino acids
The 59 kDa his-tagged ALDHTt was overexpressed in E. coli BL21(DE3), and purified to apparent homogeneity using immobilized metal affinity chromatography (IMAC) and gel filtration chromatography
12 aldehyde substrates were tested for efficient conversion by the ALDHTt, with eight aliphatic, aromatic, and dialdehydes demonstrating catalytic activity

Summary

Introduction

Found in all kingdoms of life, aldehyde dehydrogenases (ALDH) (EC;1.2.1.3) constitute a large family of NAD(P)+ -dependent enzymes with a molecular mass of ca.50–60 kDa, and are composed of 450–500 amino acids. ALDHs exist as dimers [1], tetramers [2,3,4] and hexamers [5]; the latter are less prevalent, with only two resolved structures available to date [6,7,8]. Their structures consist of three conserved domains, the catalytic domain, the cofactor binding domain, and the oligomerisation domain [1], which work together to catalyse the conversion of an aldehyde substrate using. The dehydrogenase activity of ALDH occurs in five distinct steps [13]

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Conclusion