Gropp, J.; Stolper, D. A.; Nayak, D. D.

The stable hydrogen and carbon isotopic composition of methane is widely used to determine its sources. Methanogenic growth on methanol generates methane with significantly lower 13C/12C ratios relative to other substrates, which is often used as a marker for this metabolism in environmental samples. The biochemical basis for the unusual isotope effect associated with methanol growth is currently unknown. Here, we grew Methanosarcina acetivorans on methanol and measured the change in the carbon and hydrogen stable isotopic compositions of the methane. We coupled these results with an inverse modeling approach to calculate the kinetic isotopic effects (KIEs) of the rate limiting step, catalyzed by the methanol-specific methyltransferase complex (MTA). Through this process, we estimate the carbon KIE of MTA (13{varepsilon}MTA) as -65.5 {per thousand} and the hydrogen KIE of MTA (2{varepsilon}MTA) as -56 {per thousand}. Next, we show that the 13{varepsilon}MTA contributes substantially to the large isotopic effect observed for methylotrophic methanogenesis on methanol. We also show that mutant strains that express only a single copy of the MTA complex (either MtaC1B1A1, MtaC2B2A1, MtaC3B3A1) have 13{varepsilon}MTA and 2{varepsilon}MTA that are indistinguishable from the wild-type strain. Finally, based on a thermodynamic analysis, we propose that methanol activation by MTA will remain rate-limiting, even at low environmental methanol concentrations, and the large 13{varepsilon}MTA would be expressed in situ as well.