Molecular characterization of a thermostable aldehyde dehydrogenase (ALDH) from the hyperthermophilic archaeon Sulfolobus tokodaii strain 7

Abstract

Aldehyde dehydrogenase (ALDH) is a widely distributed enzyme in nature. Although many ALDHs have been reported until now, the detailed enzymatic properties of ALDH from Archaea remain elusive. Herein, we describe the characterization of an ALDH from the hyperthermophilic archaeon Sulfolobus tokodaii. The enzyme (stALDH) could utilize various aldehydes as substrates, and maximal activity was found with acetaldehyde and the coenzyme NAD. The optimal temperature and pH were 80 °C and 8, respectively, and high thermostability was found with the half-life at 90 °C to be 4 h. The enzyme was considerably resistant to nitroglycerin (GTN) inhibition, which could be restored by reducing agent DTT or (±)-α-lipoic acid. Coenzyme NAD or NADP could regulate the enzymatic thermostability, as well as the esterase activity. Molecular modeling suggested that the enzyme harbored similar structural arrangement with its eukaryotic and bacterial counterparts. Sequence alignment showed the conserved catalytic residues E240 and C274 and cofactor interactive sites N142, K165, I168 and E370, the function of which were verified by site-directed mutagenesis analysis. This is the most thermostable ALDH reported until now and the unique property of this enzyme is potentially beneficial in the fields of biotechnology and biomedicine.

Functional characterization of a vanillin dehydrogenase in Corynebacterium glutamicum

“…Based on sequence alignment, E258 and C292 were identified as the candidate conserved catalytic residues whereas N157, K180 and E199 were identified as the candidate cofactor interactive sites in VDH ATCC13032 , and these residues were demonstrated to be essential for VDH from the hyperthermophilic archaeon Sulfolobus tokodaii 15 and Pseudomonas aeruginosa 16 . To investigate whether these five residues are also important for the VDH ATCC13032 catalytic activity, VDH ATCC13032 variants were constructed followed by in vitro catalytic assay using vanillin as the substrate in the presence of NAD(P) + .…”

“…Extensive works have been done to examine the active amino acid residues in the ALDH enzymes, and the examined residues have been well documented 15 16 23 . Sequence alignment revealed conserved sequences in VDH ATCC13032 with relatively high amino acid similarity with the active residues identified in ALDH and Pa BADH.…”

“…The optimal pH was determined in various pHs of 100mM buffer as follows: glycine-HCl buffer (pH 34), potassium phosphate buffer (pH48), Tris-HCl buffer (pH810) and glycine-HCl buffer (pH1012). One unit of enzyme activity was defined as the amount of enzyme producing 1mmol NAD(P)H min 1 as previously described 15 33 . Specific activities were given as unit mg 1 of protein 35 .…”

“…In detail, the following mutants were designed: N157A, K180A, E199A, E258A and C292A. Activities and kinetics parameters of all the mutants were determined with the experimental procedures of wild type enzyme described above 15 .…”

Archaeal Aldehyde Dehydrogenase ST0064 fromSulfolobus tokodaii, a Paralog of Non-Phosphorylating Glyceraldehyde-3-phosphate Dehydrogenase, Is a Succinate Semialdehyde Dehydrogenase

“…ST0064 is the closest paralog of GAPN from S. tokodaii (St-GAPN), at 34 and 54% amino acid sequence identity and similarity respectively, and was recently characterized as an aldehyde dehydrogenase with promiscuous substrate specificity using a number of aldehyde compounds. 8) The most effective aldehyde reported is acetaldehyde, with a K m of 6.38 mM, and a k cat of 1.14 s À1 at 80 C, but low maximum activity and an affinity for the aldehyde compounds tested suggest the presence of more physiological substrates of ST0064.…”