Data_Sheet_1_Carbohydrate Metabolism in Bacteria: Alternative Specificities in ADP-Glucose Pyrophosphorylases Open Novel Metabolic Scenarios and Biotechnological Tools.PDF

We explored the ability of ADP-glucose pyrophosphorylase (ADP-Glc PPase) from different bacteria to use glucosamine (GlcN) metabolites as a substrate or allosteric effectors. The enzyme from the actinobacteria Kocuria rhizophila exhibited marked and distinctive sensitivity to allosteric activation by GlcN-6P when producing ADP-Glc from glucose-1-phosphate (Glc-1P) and ATP. This behavior is also seen in the enzyme from Rhodococcus spp., the only one known so far to portray this activation. GlcN-6P had a more modest effect on the enzyme from other Actinobacteria (Streptomyces coelicolor), Firmicutes (Ruminococcus albus), and Proteobacteria (Agrobacterium tumefaciens) groups. In addition, we studied the catalytic capacity of ADP-Glc PPases from the different sources using GlcN-1P as a substrate when assayed in the presence of their respective allosteric activators. In all cases, the catalytic efficiency of Glc-1P was 1–2 orders of magnitude higher than GlcN-1P, except for the unregulated heterotetrameric protein (GlgC/GgD) from Geobacillus stearothermophilus. The Glc-1P substrate preference is explained using a model of ADP-Glc PPase from A. tumefaciens based on the crystallographic structure of the enzyme from potato tuber. The substrate-binding domain localizes near the N-terminal of an α-helix, which has a partial positive charge, thus favoring the interaction with a hydroxyl rather than a charged primary amine group. Results support the scenario where the ability of ADP-Glc PPases to use GlcN-1P as an alternative occurred during evolution despite the enzyme being selected to use Glc-1P and ATP for α-glucans synthesis. As an associated consequence in such a process, certain bacteria could have improved their ability to metabolize GlcN. The work also provides insights in designing molecular tools for producing oligo and polysaccharides with amino moieties.

本研究探讨了不同细菌来源的ADP-葡萄糖焦磷酸化酶（ADP-Glc PPase）利用葡萄糖胺（GlcN）代谢物作为底物或变构效应剂的能力。来自放线菌科科里菌属（Kocuria rhizophila）的酶在将葡萄糖-1-磷酸（Glc-1P）和三磷酸腺苷（ATP）转化为ADP-葡萄糖的过程中，对GlcN-6P的变构激活表现出显著且独特的敏感性。这种特性亦见于已知唯一能呈现此激活特性的红球菌属（Rhodococcus spp.）的酶。GlcN-6P对其他放线菌（如链霉菌属Streptomyces coelicolor）、厚壁菌门（如瘤胃球菌属Ruminococcus albus）和变形菌门（如根瘤农杆菌属Agrobacterium tumefaciens）的酶的影响则相对较小。此外，我们还研究了在不同来源的ADP-Glc PPase在各自的变构激活剂存在下，以GlcN-1P为底物时的催化能力。在所有情况下，Glc-1P的催化效率比GlcN-1P高1至2个数量级，唯独来自地温菌属（Geobacillus stearothermophilus）的无调控异四聚蛋白（GlgC/GgD）例外。Glc-1P底物偏好的解释基于基于土豆块茎酶的晶体结构构建的恶臭假单胞菌属（A. tumefaciens）的ADP-Glc PPase模型。底物结合域位于α-螺旋的N端附近，该螺旋具有部分正电荷，因此更倾向于与羟基而非带电荷的伯胺基团相互作用。结果表明，尽管ADP-Glc PPase被选用于α-葡萄糖聚糖的合成而使用Glc-1P和ATP，但其在进化过程中使用GlcN-1P作为替代底物的能力可能已经发生。在此过程中，某些细菌可能提高了其代谢GlcN的能力。该研究还为设计用于生产含氨基基团的寡糖和多糖的分子工具提供了见解。