Fig 1.
Aldolase enzymes are involved in both gluconeogenesis and glycolysis (a) and are classified into two classes by two distinct mechanisms of action for nucleophilic activation. Class I aldolases exhibit a strictly conserved lysine residue which forms a covalent Schiff base with the donor molecule to generate an enamine nucleophile (b) whilst class II aldolases use a divalent metal cation cofactor to promote enolization of the donor molecule via bidentate Lewis acid complexation (c).
Fig 2.
Enzymatic cascades for the conversion of glycerol to DHAP via glycerol-3-phosphate.
Glycerol is converted to glycerol-3-phosphate by a glycerol kinase enzyme with concomitant regeneration of ATP by an acetate or pyruvate kinase enzyme. The glycerol-3-phopshate is then oxidized to DHAP by either an L- glycerol-3-phosphate oxidase enzyme (A) or a glycerol-3-phosphate dehydrogenase enzyme (B).
Table 1.
Steady state kinetics for enzymes involved in conversion of glycerol to DHAP.
Table 2.
Efficiency of selected heterogeneous paired reactions.
Table 3.
DHAP production via two step enzymatic cascade.
Fig 3.
Details of the optimized cascade for the production of DHAP from glycerol.
Phosphorylation of glycerol by ATP mediated by GlpKTk (EC 2.7.1.30) and Mg2+ via a phosphotransfer mechanism [33] was accompanied by regeneration of ATP from ADP by AceKMs (EC 2.7.2.1), which catalyzes reversibly the phosphorylation of acetate in the presence of a divalent cation and ATP with the formation of acetylphosphate and ADP[34]. Cytosolic glycerophosphate oxidase GlpOMg(EC 1.1.3.21) likely converts glycerol-3-phosphate to DHAP by a similar mechanism to the related GlpO from Mycoplasma pneumoniae (4X9M) [35]), Similarly to other flavoprotein oxidases, glycerophosphate oxidase GlpO enzymes follow a hydride transfer mechanism to stabilize a positive charge on the flavin N(5)-sulfite adduct (C). The hydrogen peroxide generated from the oxidation of enzymatic FADH2 was converted to water by the addition of catalase from Micrococcus lysodeikticus.
Fig 4.
Production of rare chiral sugars by combining optimized multi-enzyme cascades for DHAP production with a DHAP-dependant fructose-1,6-biphosphate aldolase.
Glycerol (10mM substrate) was converted to glycerol-3-phosphate by a glycerol kinase enzyme GlpKTk (28.6 pmoles) with concomitant regeneration of ATP by an acetate kinase enzyme AceKMs (40.2 pmoles). The glycerol-3-phopshate was then oxidized to DHAP by a novel L-glycerol-3-phosphate oxidase enzyme GlpOMg (154.2 pmoles), with mitigation of excess hydrogen peroxide by catalase (3U/mL) and an aldolase enzyme FruASc (3.1 nmoles) converted this and acceptor aldehydes (provided at 10mM) into chiral sugars D-fructose-1,6-biphosphate (3S, 4R) and 3,4-dihydroxyhexulose phosphate (3S, 4R) as depicted.