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Literature summary extracted from

  • Price, J.; Chen, L.; Whitaker, W.; Papoutsakis, E.; Chen, W.
    Scaffoldless engineered enzyme assembly for enhanced methanol utilization (2016), Proc. Natl. Acad. Sci. USA, 113, 12691-12696 .
    View publication on PubMedView publication on EuropePMC

Cloned(Commentary)

EC Number Cloned (Comment) Organism
4.1.2.43 expressed in Escherichia coli Bacillus methanolicus

Protein Variants

EC Number Protein Variants Comment Organism
1.1.1.244 additional information construction of multienzyme supramolecular complexes, which self-assemble into spatially defined architectures, to improve the efficiency of cascade reactions. Engineered supramolecular enzyme assemblies enhance hexose 6-phosphate and fructose 6-phosphate production and can be similarly created as a kinetic trap to enable fast and efficient methanol utilization, method, overview. Clustering Mdh3 with a bifunctional Hps-Phi fusion, further improves fructose 6-phosphate production, resulting in an overall 50fold improvement over the uncomplexed enzyme mixture. Compared with the unassembled enzyme system, a much lower level of formaldehyde is detected and only a small amount of hexose 6-phosphate is accumulated, indicating the effective channeling of formaldehyde toward fructose 6-phosphate by the supramolecular enzyme complex due to improved molecular proximity Bacillus methanolicus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
1.1.1.244 methanol + NAD+ Bacillus methanolicus in nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction formaldehyde + NADH + H+
-
 r
1.1.1.244 methanol + NAD+ Bacillus methanolicus MGA3 in nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction formaldehyde + NADH + H+
-
 r
1.1.1.244 additional information Bacillus methanolicus efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism ?
-
 ?
1.1.1.244 additional information Bacillus methanolicus MGA3 efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism ?
-
 ?
4.1.2.43 D-ribulose 5-phosphate + formaldehyde Bacillus methanolicus
-
 D-arabino-hex-3-ulose 6-phosphate
-
 ?
4.1.2.43 D-ribulose 5-phosphate + formaldehyde Bacillus methanolicus MGA3
-
 D-arabino-hex-3-ulose 6-phosphate
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
1.1.1.244 Bacillus methanolicus I3E949
-
-
1.1.1.244 Bacillus methanolicus MGA3 I3E949
-
-
4.1.2.43 Bacillus methanolicus
-
-
-
4.1.2.43 Bacillus methanolicus MGA3
-
-
-

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
1.1.1.244 methanol + NAD+
-
 Bacillus methanolicus formaldehyde + NADH + H+
-
 r
1.1.1.244 methanol + NAD+ in nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction Bacillus methanolicus formaldehyde + NADH + H+
-
 r
1.1.1.244 methanol + NAD+
-
 Bacillus methanolicus MGA3 formaldehyde + NADH + H+
-
 r
1.1.1.244 methanol + NAD+ in nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction Bacillus methanolicus MGA3 formaldehyde + NADH + H+
-
 r
1.1.1.244 additional information efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism Bacillus methanolicus ?
-
 ?
1.1.1.244 additional information efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism Bacillus methanolicus MGA3 ?
-
 ?
4.1.2.43 D-ribulose 5-phosphate + formaldehyde
-
 Bacillus methanolicus D-arabino-hex-3-ulose 6-phosphate
-
 ?
4.1.2.43 D-ribulose 5-phosphate + formaldehyde
-
 Bacillus methanolicus MGA3 D-arabino-hex-3-ulose 6-phosphate
-
 ?

Synonyms

EC Number Synonyms Comment Organism
1.1.1.244 MDH
-
 Bacillus methanolicus
1.1.1.244 Mdh3
-
 Bacillus methanolicus
1.1.1.244 NAD-dependent methanol dehydrogenase
-
 Bacillus methanolicus
4.1.2.43 HPS
-
 Bacillus methanolicus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
1.1.1.244 37
-
 assay at Bacillus methanolicus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
1.1.1.244 7.4
-
 assay at Bacillus methanolicus

Cofactor

EC Number Cofactor Comment Organism Structure
1.1.1.244 NAD+
-
 Bacillus methanolicus
1.1.1.244 NADH
-
 Bacillus methanolicus

General Information

EC Number General Information Comment Organism
1.1.1.244 additional information in nature, methanol dehydrogenase (Mdh), which converts methanol to formaldehyde, highly favors the reverse reaction, efficient coupling with the irreversible sequestration of formaldehyde by 3-hexulose-6-phosphate synthase (Hps) and 6-phospho-3-hexuloseisomerase (Phi) serves as the key driving force to pull the pathway equilibrium toward central metabolism. An emerging strategy to promote efficient substrate channeling is to spatially organize pathway enzymes in an engineered assembly to provide kinetic driving forces that promote carbon flux in a desirable direction. A scaffoldless, self-assembly strategy is applied to organize Mdh, Hps, and Phi into an engineered supramolecular enzyme complex using an SH3-ligand interaction pair, which enhances methanol conversion to fructose-6-phosphate. An NADH sink is created using Escherichia coli lactate dehydrogenase as an NADH scavenger, thereby preventing reversible formaldehyde reduction, to increase methanol consumption. Combination of the two strategies improves in vitro fructose 6-phosphate production by 97fold compared with unassembled enzymes. The beneficial effect of supramolecular enzyme assembly is also realized in vivo as the engineered enzyme assembly improves whole-cell methanol consumption rate by ninefold. This approach ultimately allows direct coupling of enhanced fructose 6-phosphate synthesis with other metabolic engineering strategies for the production of many desired metabolites from methanol Bacillus methanolicus