Instituto de Agrobiotecnología del Litoral, Universidad Nacional del Litoral, Consejo Nacional de Investigaciones Científicas y Técnicas, Facultad de Bioquímica y Ciencias Biológicas, Santa Fe, Argentina.
Department of Chemistry and Biochemistry, Loyola University Chicago, Chicago, IL, USA; Department of Chemistry, Al Baha University, Al Baha, Saudi Arabia.
Biochimie. 2022 Jan;192:30-37. doi: 10.1016/j.biochi.2021.09.011. Epub 2021 Sep 22.
Until recently, the cyanobacterial phylum only included oxygenic photosynthesizer members. The discovery of Melainabacteria as a group of supposed non-photosynthetic cyanobacteria asked to revisit such scenario. From metagenomic data, we were able to identify sequences encoding putative ADP-glucose pyrophosphorylases (ADP-GlcPPase) from free-living and intestinal Melainabacteria. The respective genes were de novo synthesized and over-expressed in Escherichia coli. The purified recombinant proteins from both Melainabacteria species were active as ADP-GlcPPases, exhibiting V values of 2.3 (free-living) and 7.1 U/mg (intestinal). The enzymes showed similar S values (∼0.3 mM) for ATP, while the one from the intestinal source exhibited a 6-fold higher affinity toward glucose-1P. Both recombinant ADP-GlcPPases were sensitive to glucose-6P activation (A ∼0.3 mM) and Pi and ADP inhibition (I between 0.2 and 3 mM). Interestingly, the enzymes from Melainabacteria were insensitive to 3-phosphoglycerate, which is the principal activator of ADP-GlcPPases from photosynthetic cyanobacteria. As far as we know, this is the first biochemical characterization of an active enzyme from Melainabacteria. This work contributes to a better understanding of the evolution of allosteric regulation in the ADP-GlcPPase family, which is critical for synthesizing the main reserve polysaccharide in prokaryotes (glycogen) and plants (starch). In addition, our results offer further information to discussions regarding the phylogenetic position of Melainabacteria.
直到最近，蓝藻门仅包括产氧光合作用成员。作为一组假定的非光合蓝细菌的 Melainabacteria 的发现要求重新审视这种情况。从宏基因组数据中，我们能够鉴定出来自自由生活和肠道 Melainabacteria 的假定 ADP-葡萄糖焦磷酸化酶 (ADP-GlcPPase) 的序列。分别从头合成和在大肠杆菌中过表达相应的基因。两种 Melainabacteria 物种的纯化重组蛋白均作为 ADP-GlcPPase 发挥活性，表现出 2.3（自由生活）和 7.1 U/mg（肠道）的 V 值。这些酶对 ATP 表现出相似的 S 值（约 0.3 mM），而来自肠道来源的酶对葡萄糖-1P 的亲和力高 6 倍。两种重组 ADP-GlcPPase 均对葡萄糖-6P 激活（A 约 0.3 mM）和 Pi 和 ADP 抑制（I 在 0.2 和 3 mM 之间）敏感。有趣的是，来自 Melainabacteria 的酶对 3-磷酸甘油酸不敏感，而 3-磷酸甘油酸是光合蓝细菌 ADP-GlcPPase 的主要激活剂。据我们所知，这是首次对 Melainabacteria 中活性酶进行的生化表征。这项工作有助于更好地理解 ADP-GlcPPase 家族中变构调节的进化，这对于合成原核生物（糖原）和植物（淀粉）中的主要储备多糖至关重要。此外，我们的结果为有关 Melainabacteria 系统发育位置的讨论提供了进一步的信息。
