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首页> 外文期刊>Applied Microbiology >Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types
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Phosphoryl Transfer from α-d-Glucose 1-Phosphate Catalyzed by Escherichia coli Sugar-Phosphate Phosphatases of Two Protein Superfamily Types

机译：两种蛋白质超家族类型的大肠杆菌糖磷酸磷酸酶催化α-d-葡萄糖1-磷酸的磷酸基转移

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摘要

The Cori ester α-d-glucose 1-phosphate (αGlc 1- P ) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes αGlc 1- P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of αGlc 1- P as a phosphoryl donor substrate. The agp gene encodes a periplasmic αGlc 1- P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation ( k _(cat) for phosphoryl transfer from αGlc 1- P to water, 40 s~(?1)) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys~(189) and Cys~(195). Enzymatic phosphoryl transfer from αGlc 1- P to water in H_(2)~(18)O solvent proceeded with complete ~(18)O label incorporation into the phosphate released, consistent with catalytic reaction through O-1–P, but not C-1–O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and d-glucose 6-phosphate was converted with a k _(cat) similar to that of αGlc 1- P . By examining phosphoryl transfer from αGlc 1- P to an acceptor substrate other than water (d-fructose or d-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized αGlc 1- P mainly for phosphoryl transfer to water. By applying d-fructose in 10-fold molar excess over αGlc 1- P (20 mM), enzymatic conversion furnished d-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from αGlc 1- P .
机译：Cori酯α-d-葡萄糖1-磷酸（αGlc1-P）是细胞碳水化合物代谢的高能中间体。它的糖苷磷酸单酯部分可引发αGlc1-P引发的糖基和磷酸基转移反应中的灵活利用。在这项研究中，表征了两种在结构和机制上都与大肠杆菌不同的糖磷酸磷酸酶，以利用αGlc1- P作为磷酰基供体底物。 agp基因编码属于组氨酸酸性磷酸酶家族的周质αGlc1-P磷酸酶（Agp）。 Had13来自卤代酸脱氢酶样磷酸酶家族。 Agp的胞质表达（在大肠杆菌Origami B中）提供了一种功能性酶制剂（k_（cat），用于将磷酰基从αGlc1-P转移到水中，时间为40 s〜（？1）），质谱显示Cys〜（189）和Cys〜（195）之间没有游离的半胱氨酸和天然的分子内二硫键。在H_（2）〜（18）O溶剂中从αGlc1- P到水的酶促磷酸基转移反应，是将〜（18）O标记完全掺入释放的磷酸盐中，这与通过O-1–P而不是C的催化反应一致-1–O，键断裂。两种酶的水解酶活性均不限于糖苷磷酸单酯底物，并且以与αGlc1-P相似的k _（cat）转化了d-葡萄糖6-磷酸。通过检查从αGlc1-P到除水（d-果糖或d-葡萄糖）以外的受体底物的磷酰基转移，我们发现Agp表现出明显的合成活性，这与Had13不同，Had13主要利用αGlc1-P进行磷酰基向水的转移。 。通过以相对于αGlc1-P（20 mM）10倍摩尔过量的方式施用d-果糖，酶促转化提供了以55％的总收率作为主要产物的d-果糖1-磷酸。 Agp是一种很有前景的生物催化剂，可用于αGlc1-P的转磷酸作用。

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