Purification, characterization and cloning of aldehyde dehydrogenase from Rhodococcus erythropolis UPV-1.
The enzyme responsible for formaldehyde removal in industrial wastewaters by cells of Rhodococcus erythropolis UPV-1 was identified as a broad-specific aldehyde dehydrogenase (EC 1.2.1.3). The enzyme was purified to electrophoretic homogeneity from ethanol-grown cells with a specific activity of 19.5 U mg-1 protein and an activity recovery of 56%. The enzyme showed an isoelectric point (pI) of 5.3 and was a trimer of 162 kDa consisting of three identical 54-kDa subunits. It was specific for NAD+ and showed hyperbolic kinetics for this coenzyme (Km=90 microM), but sigmoidal kinetics for the aliphatic aldehydes used as substrates. The enzyme affinity for aldehydes increased with their hydrocarbon chain length, ranging from 333 microM for formaldehyde to 85 nM for n-octanal. The corresponding calculated Hill coefficients were in the 1.55-2.77 range. With n-propanal as substrate, the optimum pH and temperature for activity were 9.5-10.0 and 47.5 degrees C, respectively, with an Ea for catalysis of 28.6 kJ mol-1. NAD+ protected the enzyme against thermal inactivation, but aldehydes were ineffective. The activity was severely inhibited by p-hydroxymercuribenzoate, indicating that a thiol was essential for catalysis. The 1,524-bp aldhR gene encoding a 507-amino-acid protein was expressed in cells of Escherichia coli M15 as a hexahistidine-tagged protein.
Jaureguibeitia
Arrate
A
Enzyme and Cell Technology Group, Department of Biochemistry and Molecular Biology, Faculty of Science and Technology, University of the Basque Country, P.O. Box 644, 48080, Bilbao, Spain.
Saá
Laura
L
Llama
María J
MJ
Serra
Juan L
JL
D000444:Aldehyde Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism; D003001:Cloning, Molecular; D003067:Coenzymes / Q000494:pharmacology; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D006902:Hydroxymercuribenzoates / Q000494:pharmacology; D007526:Isoelectric Point; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000494:pharmacology; D021122:Protein Subunits; D012240:Rhodococcus / Q000201:enzymology* / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D000447:Aldehydes; D003067:Coenzymes; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D006902:Hydroxymercuribenzoates; D021122:Protein Subunits; D009243:NAD; C030601:4-hydroxymercuribenzoate; D000444:Aldehyde Dehydrogenase