Thermotoga maritima TM0298 is a highly thermostable mannitol dehydrogenase.
Thermotoga maritima TM0298 is annotated as an alcohol dehydrogenase, yet it shows high identity and similarity to mesophilic mannitol dehydrogenases. To investigate this enzyme further, its gene was cloned and expressed in Escherichia coli. The purified recombinant enzyme was most active on fructose and mannitol, making it the first known hyperthermophilic mannitol dehydrogenase. T. maritima mannitol dehydrogenase (TmMtDH) is optimally active between 90 and 100 degrees C and retains 63% of its activity at 120 degrees C but shows no detectable activity at room temperature. Its kinetic inactivation follows a first-order mechanism, with half-lives of 57 min at 80 degrees C and 6 min at 95 degrees C. Although TmMtDH has a higher V (max) with NADPH than with NADH, its catalytic efficiency is 2.2 times higher with NADH than with NADPH and 33 times higher with NAD(+) than with NADP(+). This cofactor specificity can be explained by the high density of negatively charged residues (Glu193, Asp195, and Glu196) downstream of the NAD(P) interaction site, the glycine motif. We demonstrate that TmMtDH contains a single catalytic zinc per subunit. Finally, we provide the first proof of concept that mannitol can be produced directly from glucose in a two-step enzymatic process, using a Thermotoga neapolitana xylose isomerase mutant and TmMtDH at 60 degrees C.
Song
Seung Hoon
SH
Department of Biochemistry and Molecular Biology, Michigan State University, 110 Biochemistry Building, East Lansing, MI 48824, USA.
Ahluwalia
Nitasha
N
Leduc
Yvonne
Y
Delbaere
Louis T J
LT
Vieille
Claire
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D005947:Glucose / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D008354:Mannitol Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D020124:Thermotoga maritima / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D008353:Mannitol; D008354:Mannitol Dehydrogenases; D005947:Glucose