Synthesis and n.m.r. characterization of intermediates in the l-type pentose phosphate cycle

Most of the "repressor, open reading frame, kinase" (ROK) proteins already characterized so far, and exhibiting a kinase activity, take restrictedly D-glucose as substrate. By exploring the sequenced bacterial diversity, 61 ATP-dependent kinases belonging to the ROK family have been identified and experimentally assayed for the phosphorylation of hexoses. These kinases were mainly found to be thermotolerant and highly active toward D-mannose and D-fructose with notable activities toward D-tagatose. Among them, the ATP-dependent kinase from the mesophile Streptococcus mitis (named ScrK) was biochemically characterized and its substrate spectrum further studied. This enzyme possessed impressive catalytic efficiencies toward D-mannose and D-fructose of 1.5 10 s M and 2.7 10 s M, respectively, but also significant ones toward D-tagatose (3.5 10 s M) and the unnatural monosaccharides D-altrose (1.1 10 s M) and D-talose (3.4 10 s M). Specific activities measured for all hexoses showed a high stereopreference for D- over L-series. As proof of concept, 8 hexoses were phosphorylated in moderate to good yields, some of them described for the first time like L-sorbose-5-phosphate unusually phosphorylated in position 5. Its thermotolerance, its wide pH tolerance (from 7 to 10), and temperature range (> 85% activity between 40 and 70 °C) open the way to applications in the enzymatic synthesis of monophosphorylated hexoses.

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“…Isolated yield: 57% (25 mg of white solid). NMR C was identical to the one found in the literature (Franke et al 1985). HRMS ESI , m/z calcd.…”

Section: Purification By Ion Exchange Chromatographysupporting

confidence: 72%

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Vergne-Vaxelaire¹,

Mariage²,

Petit³

et al. 2018

Appl Microbiol Biotechnol

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“…β- d -Allose 6-Phosphate (55). Phosphorylation of 22 according to the general procedure gave 0.516 g (85%) of 55 monosodium salt: mp 125−126 °C; 1 H NMR (400 MHz) δ 4.82 (d, J = 8.3 Hz, 1H, H-1), 4.12 (t, J = 2.8 Hz, 1H), 4.04 (m, 1H), 3.93 (m, 1H), 3.82 (dd, J = 3.7, 10.4 Hz, 1H), 3.65 (dd, J = 2.7, 10.0 Hz, 1H), 3.37 (dd, J = 3.0, 8.3 Hz, 1H); 31 P NMR δ 3.55; HRMS calcd for C 6 H 12 Na 2 O 9 P (monosodium salt + Na + ) 305.0014, found 305.0025.…”

Section: Methodsmentioning

confidence: 99%

Synthetic Utility of Yeast Hexokinase. Substrate Specificity, Cofactor Regeneration, and Product Isolation

Hornberger

1997

J. Org. Chem.

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Yeast hexokinase (EC 2.7.1.1) catalyzes the phosphorylation of pyranose and furanose analogs of glucose at 0.01-125% of the rate of glucose. The enzyme is highly tolerant of structural changes at C-2 and C-3 of glucopyranose and less tolerant of changes at C-1 and C-4. Preparative phosphorylations were performed on compounds having 0.01-100% of the activity of glucose, using phosphoenolpyruvate and pyruvate kinase to regenerate ATP. The effects of inhibition of hexokinase by phosphoenolpyruvate and acetyl phosphate on cofactor regeneration are discussed.

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“…A simulation scheme is defined by the reaction niecha- c, a-Fru(l,6)P2f, PI (x-furanose [15], 23% of total derived from "P spectra); f, g, b-and u-Glc6Pp (60%, 40% of total, respectively [16]); h, Sed7P; i, RuSPIXuSP (unresolved); j, m, b-Sed(l,7)PJ; 74% oftotal [14]; 1, /l-Fru(l,6)P2f, P1,77% of total [15]; n, GrnP (ketone) [15], 55% of total); o, Fru6P/~-Fru(l,6)P~(unresolved); p, RibSP; q, u-Fru(l,6)PZf, P6, [15]; r, AMP; U1 -U3 refer to unassigned resonances; the asterixed peaks arc spectrometer artefacts…”

Section: Numericul Methodsmentioning

confidence: 99%

“…Saturation transfer experiments [18] were also used to confirm the assignments of some peaks from rapidly exchanging sugar phosphates, such as some of the conformers of Sed(l,7)Pz [14]. Titrations were performed on ultrafiltered lysates to ascertain the pH dependence of the chemical shift of each resonance (not shown).…”

Section: Assignment Of the Sugur-phosphate Regionmentioning

confidence: 99%

Comparison of computer simulations of the F‐type and L‐type non‐oxidative hexose monophosphate shunts with ³¹P‐NMR experimental data from human erythrocytes

Mcintyre

Thorburn

Bubb

et al. 1989

European Journal of Biochemistry

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Mathematical modelling was used to predict the behaviour of the two most favoured schemes for the operation of the non-oxidative hexose monophosphate shunt (HMS), the F-type and the L-type pathways. The models simulate the time courses of sugar-phosphate concentrations when various substrates are metabolized via each pathway. A 31P-NMR technique, with which to observe time courses of concentrations of sugar phosphates in a human red cell lysate, was developed. The accuracy of each hypothesised scheme was then evaluated by comparing predicted with observed data.The results were more consistent with time courses of sugar-phosphate levels predicted by the F-type (classical) pathway than those predicted by the L-type model. However, the accumulation of sedoheptulose 1,7-bisphosphate when a haemolysate was incubated with ribose 5-phosphated showed that the F-type pathway is not a complete description of the system of reactions. Transaldolase was demonstrated to be essential for the normal metabolism of sugar phosphates by haemolysates. The effects of the heat-inactivation of transaldolase on the metabolism of sugar phosphates were accurately predicted by the F-type model.The relevance of attempting to describe the reaction of the non-oxidative HMS as a distinct 'pathway' or 'cycle' is discussed.Two major roles of the hexose monophosphate shunt are to generate reducing equivalents in the form of NADPH, and pentose sugars for nucleotide synthesis. The first function is carried out by the oxidative reactions : the non-oxidative Correspondence to P. W. Kuchel,

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Synthesis and n.m.r. characterization of intermediates in the l-type pentose phosphate cycle

Cited by 17 publications

References 11 publications

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Synthetic Utility of Yeast Hexokinase. Substrate Specificity, Cofactor Regeneration, and Product Isolation

Comparison of computer simulations of the F‐type and L‐type non‐oxidative hexose monophosphate shunts with ³¹P‐NMR experimental data from human erythrocytes

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Synthesis and n.m.r. characterization of intermediates in the l-type pentose phosphate cycle

Cited by 17 publications

References 11 publications

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Characterization of a thermotolerant ROK-type mannofructokinase from Streptococcus mitis: application to the synthesis of phosphorylated sugars

Synthetic Utility of Yeast Hexokinase. Substrate Specificity, Cofactor Regeneration, and Product Isolation

Comparison of computer simulations of the F‐type and L‐type non‐oxidative hexose monophosphate shunts with 31P‐NMR experimental data from human erythrocytes

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Comparison of computer simulations of the F‐type and L‐type non‐oxidative hexose monophosphate shunts with ³¹P‐NMR experimental data from human erythrocytes