1.1.1.67: mannitol 2-dehydrogenase

This is an abbreviated version, for detailed information about mannitol 2-dehydrogenase, go to the full flat file.

Reaction

D-mannitol
+
NAD+
=
D-fructose
+
NADH
+
H+

Synonyms

D-mannitol dehydrogenase, DSF1, Hxt13, Hxt15, Hxt16, Hxt17, M2DH, Man1, MAN2, mannitol 2-dehydrogenase, mannitol dehydrogenase, mannitol dehydrogenase 1, mannitol dehydrogenase 2, mannitol transporter, mannitol-2-dehydrogenase, MDH, mt-dh, MtDH, MtlD, NADH-dependent mannitol dehydrogenase, pfMDH, polyol dehydrogenase, PsM2DH, TM0298, YNR073C

ECTree

     1 Oxidoreductases
         1.1 Acting on the CH-OH group of donors
             1.1.1 With NAD+ or NADP+ as acceptor
                1.1.1.67 mannitol 2-dehydrogenase

Engineering

Engineering on EC 1.1.1.67 - mannitol 2-dehydrogenase

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ENGINEERING
ORGANISM
UNIPROT
COMMENTARY hide
LITERATURE
D69A
-
site-directed mutagenesis, the mutant shows an altered cofactor specificity compared to the wild-type enzyme, which is switched to NADP(H), EC 1.1.1.138, NADP(H) is equally utilized as NAD(H); utilizes NAD(H) and NADP(H) with similar catalytic efficiencies. Uses NADP(H) almost as well as wild-type enzyme uses NAD(H)
E292A
-
mutation partially disrupts the catalytic cycle. Role for residue Glu292 as a gate in a water chain mechanism of proton translocation. Removal of gatekeeper control in the E292A mutant results in a selective, up to 120fold slowing down of microscopicsteps immediately preceding catalytic oxidation of mannitol, consistent with the notion that formation of the productive enzyme-NAD-mannitol complex is promoted by a corresponding position change of Glu292
E68K
-
site-directed mutagenesis, the mutant shows an altered cofactor specificity compared to the wild-type enzyme, which is switched to NADP(H), EC 1.1.1.138, NADP(H) is preferred by 10fold over NAD(H)
E68K/D69A
-
shows about a 10fold preference for NADP(H) over NAD(H), accompanied by a small decrease in catalytic efficiency for NAD(H)-dependent reactions as compared to wild-type enzyme
H303A
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 300fold below the wild-type value
K295M
-
2000000fold lower turnover number for D-mannitol oxidation at pH 10.0 than the wild-type enzyme
N191A
-
the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, between 540- and 2700fold. Partial disruption of the oxyanion hole in the single-site mutant causes an upshift, by about 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+-mannitol complex
N191A/N300A
-
the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, with additive effects in the double mutant
N191D
-
the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol is altered 10000- to 100000fold from being balanced in the wild-type enzyme to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3  0.1 and 6.3  0.2, respectively
N191D/N300D
-
mutant behaves as a slow fructose reductase at pH 5.2, lacking measurable activity for mannitol oxidation in the pH range 6.8-10
N191L
-
the rate constants for the overall hydride transfer to and from C-2 of mannitol are selectively slowed, between 540- and 2700fold. Partial disruption of the oxyanion hole in the single-site mutant causes an upshift, by about 1.2 pH units, in the kinetic pK of the catalytic acid-base Lys295 in the enzyme–NAD+-mannitol complex
N300A
-
mutant enzyme displays catalytic efficiency for NAD+-dependent oxidation of D-mannitol 1000fold below the wild-type value
N300D
-
the internal equilibrium of enzyme-NADH-fructose and enzyme-NAD+-mannitol is altered 10000- to 100000fold from being balanced in the wild-type enzyme to favoring enzyme-NAD+-mannitol in the single site mutants, N191D and N300D. N191D and N300D appear to lose fructose binding affinity due to deprotonation of the respective Asp above apparent pK values of 5.3  0.1 and 6.3  0.2, respectively
additional information
D-mannitol production by resting state whole cell biotransformation of D-fructose by heterologous mannitol dehydrogenase gene from Leuconostoc pseudomesenteroides and the formate dehydrogenase gene, gene fdh from Mycobacterium vaccae N10, expression in Bacillus megaterium, development of an in vivo system, overview