Efficiency of selected heterogeneous paired reactions.

<p>Efficiency of selected heterogeneous paired reactions.</p

DHAP production <i>via</i> two step enzymatic cascade.

<p>DHAP production <i>via</i> two step enzymatic cascade.</p

Enzymatic cascades for the conversion of glycerol to DHAP via glycerol-3-phosphate.

<p>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 <i>L</i>- glycerol-3-phosphate oxidase enzyme (A) or a glycerol-3-phosphate dehydrogenase enzyme (B).</p

Details of the optimized cascade for the production of DHAP from glycerol.

<p>Phosphorylation of glycerol by ATP mediated by GlpK_<i>Tk</i> (EC 2.7.1.30) and Mg²⁺ <i>via</i> a phosphotransfer mechanism [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184183#pone.0184183.ref033" target="_blank">33</a>] was accompanied by regeneration of ATP from ADP by AceK_<i>Ms</i> (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[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184183#pone.0184183.ref034" target="_blank">34</a>]. Cytosolic glycerophosphate oxidase GlpO_<i>Mg</i>(EC 1.1.3.21) likely converts glycerol-3-phosphate to DHAP by a similar mechanism to the related GlpO from <i>Mycoplasma pneumoniae</i> (4X9M) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184183#pone.0184183.ref035" target="_blank">35</a>]), 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 <i>Micrococcus lysodeikticus</i>.</p

Production of rare chiral sugars by combining optimized multi-enzyme cascades for DHAP production with a DHAP-dependant fructose-1,6-biphosphate aldolase.

<p>Glycerol (10mM substrate) was converted to glycerol-3-phosphate by a glycerol kinase enzyme GlpK_<i>Tk</i> (28.6 pmoles) with concomitant regeneration of ATP by an acetate kinase enzyme AceK_<i>Ms</i> (40.2 pmoles). The glycerol-3-phopshate was then oxidized to DHAP by a novel <i>L</i>-glycerol-3-phosphate oxidase enzyme GlpO_<i>Mg</i> (154.2 pmoles), with mitigation of excess hydrogen peroxide by catalase (3U/mL) and an aldolase enzyme FruA_<i>Sc</i> (3.1 nmoles) converted this and acceptor aldehydes (provided at 10mM) into chiral sugars <i>D</i>-fructose-1,6-biphosphate (3<i>S</i>, 4<i>R</i>) and 3,4-dihydroxyhexulose phosphate (3<i>S</i>, 4<i>R</i>) as depicted.</p

Steady state kinetics for enzymes involved in conversion of glycerol to DHAP.

<p>Steady state kinetics for enzymes involved in conversion of glycerol to DHAP.</p