Zhang Shuyi, Qian Xiao, Chang Shannon, Dismukes G C, Bryant Donald A
403C Althouse Laboratory, Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park PA, USA.
Waksman Institute of Microbiology, Rutgers, The State University of New Jersey, Piscataway NJ, USA.
Front Microbiol. 2016 Dec 9;7:1972. doi: 10.3389/fmicb.2016.01972. eCollection 2016.
For nearly half a century, it was believed that cyanobacteria had an incomplete tricarboxylic acid (TCA) cycle, because 2-oxoglutarate dehydrogenase (2-OGDH) was missing. Recently, a bypass route via succinic semialdehyde (SSA), which utilizes 2-oxoglutarate decarboxylase (OgdA) and succinic semialdehyde dehydrogenase (SsaD) to convert 2-oxoglutarate (2-OG) into succinate, was identified, thus completing the TCA cycle in most cyanobacteria. In addition to the recently characterized glyoxylate shunt that occurs in a few of cyanobacteria, the existence of a third variant of the TCA cycle connecting these metabolites, the γ-aminobutyric acid (GABA) shunt, was considered to be ambiguous because the GABA aminotransferase is missing in many cyanobacteria. In this study we isolated and biochemically characterized the enzymes of the GABA shunt. We show that -acetylornithine aminotransferase (ArgD) can function as a GABA aminotransferase and that, together with glutamate decarboxylase (GadA), it can complete a functional GABA shunt. To prove the connectivity between the OgdA/SsaD bypass and the GABA shunt, the gene from sp. PCC 6803 was heterologously expressed in sp. PCC 7002, which naturally lacks this enzyme. Metabolite profiling of seven sp. PCC 7002 mutant strains related to these two routes to succinate were investigated and proved the functional connectivity. Metabolite profiling also indicated that, compared to the OgdA/SsaD shunt, the GABA shunt was less efficient in converting 2-OG to SSA in sp. PCC 7002. The metabolic profiling study of these two TCA cycle variants provides new insights into carbon metabolism as well as evolution of the TCA cycle in cyanobacteria.
近半个世纪以来，人们一直认为蓝藻的三羧酸（TCA）循环不完整，因为缺少2-氧代戊二酸脱氢酶（2-OGDH）。最近，发现了一条经由琥珀酸半醛（SSA）的旁路途径，该途径利用2-氧代戊二酸脱羧酶（OgdA）和琥珀酸半醛脱氢酶（SsaD）将2-氧代戊二酸（2-OG）转化为琥珀酸，从而在大多数蓝藻中完成了TCA循环。除了最近在少数蓝藻中发现的乙醛酸分流外，连接这些代谢物的TCA循环的第三种变体——γ-氨基丁酸（GABA）分流的存在一直存在争议，因为许多蓝藻中缺少GABA转氨酶。在本研究中，我们分离并对GABA分流的酶进行了生化特性分析。我们发现，N-乙酰鸟氨酸转氨酶（ArgD）可以作为GABA转氨酶发挥作用，并且与谷氨酸脱羧酶（GadA）一起，可以完成一个功能性的GABA分流。为了证明OgdA/SsaD旁路与GABA分流之间的连通性，将来自集胞藻属sp. PCC 6803的该基因在天然缺乏这种酶的集胞藻属sp. PCC 7002中进行了异源表达。对与这两条琥珀酸途径相关的7个集胞藻属sp. PCC 7002突变株进行了代谢物分析，证实了它们的功能连通性。代谢物分析还表明，与OgdA/SsaD分流相比，GABA分流在集胞藻属sp. PCC 7002中将2-OG转化为SSA的效率较低。对这两种TCA循环变体的代谢分析研究为蓝藻的碳代谢以及TCA循环的进化提供了新的见解。
