Crowther Gregory J, Kosály George, Lidstrom Mary E
Department of Chemical Engineering, University of Washington, Box 355014, Seattle, Washington 98195-5014, USA.
J Bacteriol. 2008 Jul;190(14):5057-62. doi: 10.1128/JB.00228-08. Epub 2008 May 23.
In serine cycle methylotrophs, methylene tetrahydrofolate (H4F) is the entry point of reduced one-carbon compounds into the serine cycle for carbon assimilation during methylotrophic metabolism. In these bacteria, two routes are possible for generating methylene H4F from formaldehyde during methylotrophic growth: one involving the reaction of formaldehyde with H4F to generate methylene H4F and the other involving conversion of formaldehyde to formate via methylene tetrahydromethanopterin-dependent enzymes and conversion of formate to methylene H4F via H4F-dependent enzymes. Evidence has suggested that the direct condensation reaction is the main source of methylene H4F during methylotrophic metabolism. However, mutants lacking enzymes that interconvert methylene H4F and formate are unable to grow on methanol, suggesting that this route for methylene H4F synthesis should have a significant role in biomass production during methylotrophic metabolism. This problem was investigated in Methylobacterium extorquens AM1. Evidence was obtained suggesting that the existing deuterium assay might overestimate the flux through the direct condensation reaction. To test this possibility, it was shown that only minor assimilation into biomass occurred in mutants lacking the methylene H4F synthesis pathway through formate. These results suggested that the methylene H4F synthesis pathway through formate dominates assimilatory flux. A revised kinetic model was used to validate this possibility, showing that physiologically plausible parameters in this model can account for the metabolic fluxes observed in vivo. These results all support the suggestion that formate, not formaldehyde, is the main branch point for methylotrophic metabolism in M. extorquens AM1.
在丝氨酸循环甲基营养菌中，亚甲基四氢叶酸（H4F）是还原态一碳化合物进入丝氨酸循环以在甲基营养代谢过程中进行碳同化的切入点。在这些细菌中，甲基营养生长期间从甲醛生成亚甲基H4F有两条可能的途径：一条途径涉及甲醛与H4F反应生成亚甲基H4F，另一条途径涉及通过依赖亚甲基四氢甲烷蝶呤的酶将甲醛转化为甲酸，并通过依赖H4F的酶将甲酸转化为亚甲基H4F。有证据表明，直接缩合反应是甲基营养代谢过程中亚甲基H4F的主要来源。然而，缺乏使亚甲基H4F和甲酸相互转化的酶的突变体无法在甲醇上生长，这表明这条亚甲基H4F合成途径在甲基营养代谢过程中的生物量生产中应具有重要作用。在嗜甲基甲基杆菌AM1中对这个问题进行了研究。获得的证据表明，现有的氘测定法可能高估了通过直接缩合反应的通量。为了验证这种可能性，结果表明，在缺乏通过甲酸的亚甲基H4F合成途径的突变体中，只有少量同化为生物量。这些结果表明，通过甲酸的亚甲基H4F合成途径主导了同化通量。使用修订后的动力学模型来验证这种可能性，结果表明该模型中生理上合理的参数可以解释体内观察到的代谢通量。这些结果都支持这样的观点，即甲酸而非甲醛是嗜甲基甲基杆菌AM1中甲基营养代谢的主要分支点。
