Biology subjects

Stolper, D. A.

Publications and source records attributed to Stolper, D. A..

3 recordsLinked to original sources

bioRxiv2026

Form I and II Rubiscos Exhibit Temperature Dependent Carbon Kinetic Isotope Effects

Nearly all carbon on Earth today is fixed by the enzyme ribulose-1,5-bisphopshate carboxylase/oxygenase ( rubisco), which converts carbon dioxide (CO2) to sugar phosphates. All rubiscos measured thus far display a kinetic isotope effect (KIE) where 12CO2 is fixed at a faster rate than 13CO2. The relationship between rubiscos KIE and the carbon isotope composition of plants, algae, and organic matter is central to many fields in the Earth sciences, plant biology, and biochemistry. Currently, all applications assume that the KIE does not vary with temperature. Here, we examine this assumption experimentally with in vitro KIE measurements of two rubiscos from phylogenetically distinct host organisms and rubisco protein clades - a Form I rubisco from the plant, Spinacia oleracea (spinach) and a Form II rubisco from the bacterium Rhodosprillium rubrum. We that find that both KIEs decrease linearly by [~]4.5{per thousand} from 10-35{degrees}C with statistically indistinguishable slopes. We place these results into biological and geologic contexts by comparing them to observed variations in the carbon isotope composition of modern terrestrial plants and marine organic carbon, the geologic carbon isotope record, and rubiscos biochemistry. We show that the measured temperature dependencies are sufficiently large to impact our interpretations of the enzymatic processes that drive variations in rubisco KIEs, as well as applications of stable carbon isotopes in the Earth and biological sciences. Significance StatementThe carbon isotope composition of plants, algae, and organic matter are interpreted with models that assume the kinetic isotope effect of the carbon-fixing enzyme rubisco is temperature-independent, even though temperature varies by tens of degrees across the Earth today and in the past. Here, we demonstrate that the kinetic isotope effect of rubisco is temperature-dependent, suggesting that some of this isotopic variation may be due to intrinsic enzyme properties alone. In addition, though the rubiscos we measured are from diverse organisms (plant vs. bacteria), their KIEs show statistically indistinguishable temperature dependencies. This data forms the basis for future thermodynamic models on rubisco biochemistry.

biochemistry↗

bioRxiv2026

Methanol-specific methyltransferase isozymes have large carbon kinetic isotope effects that impact methane isotopic signatures

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, or 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. ImportanceMethane is a potent greenhouse gas, and distinguishing between its biological sources is vital for modeling global carbon cycles. Methylotrophic methanogenesis produces methane with a uniquely depleted carbon isotope signature. However, the biochemical mechanisms driving this fractionation have remained unclear. In this study, we identify the methanol-specific methyltransferase (MTA) complex as the primary driver of these large carbon isotope effects. By utilizing Methanosarcina acetivorans mutants, we demonstrate that these effects are consistent across different MTA isozymes. Our results suggest these signatures are intrinsic to the enzyme complex and persist at low substrate concentrations. These findings provide a critical biochemical foundation for using stable isotopes to track microbial methane production in diverse natural ecosystems.

microbiology↗

bioRxiv2024

CRISPR-enabled control of gene expression sets the isotopic composition of microbial methane

The stable isotopic composition of biogenic methane varies substantially in the environment and is routinely used to fingerprint its source. However, the underlying cause of this variation is debated. Here, we experimentally manipulate the growth rate of the model methanogen, Methanosarcina acetivorans, using CRISPR mutagenesis to generate a tunable version of the key and final enzyme in methanogenesis, methyl-coenzyme M reductase (MCR). We demonstrate that the carbon and hydrogen isotopic composition of methane change as a function of MCR expression and growth rate. Using an isotope enabled metabolic model we show that these changes stem from a substrate-independent increase in reversibility of methanogenic enzymes. Overall, these data provide a novel framework for calibrating growth coupled changes in the isotopic composition of biogenic methane.

microbiology↗