Isotopic discrimination and kinetic parameters of RubisCO from the marine bloom-forming diatom, Skeletonema costatum

The cosmopolitan, bloom-forming diatom, Skeletonema costatum, is a prominent primary producer in coastal oceans, fixing CO2 with ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO) that is phylogenetically distinct from terrestrial plant RubisCO. RubisCOs are subdivided into groups based on sequence similarity of their large subunits (IA-ID, II, and III). ID is present in several major oceanic primary producers, including diatoms such as S. costatum, coccolithophores, and some dinoflagellates, and differs substantially in amino acid sequence from the well-studied IB enzymes present in most cyanobacteria and in green algae and plants. Despite this sequence divergence, and differences in isotopic discrimination apparent in other RubisCO enzymes, stable carbon isotope compositions of diatoms and other marine phytoplankton are generally interpreted assuming enzymatic isotopic discrimination similar to spinach RubisCO (IB). To interpret phytoplankton delta C-13 values, S. costatum RubisCO was characterized via sequence analysis, and measurement of its K-CO2 and V-max, and degree of isotopic discrimination. The sequence of this enzyme placed it among other diatom ID RubisCOs. Michaelis-Menten parameters were similar to other ID enzymes (K-CO2 = 48.9 +/- 2.8 mu M; V-max = 165.1 +/- 6.3 nmol min(-1) mg(-1)). However, isotopic discrimination (epsilon = [(12)k/(13)k - 1] x 1000) was low (18.5 parts per thousand; 17.0-19.9, 95% CI) when compared to IA and IB RubisCOs (22-29 parts per thousand), though not as low as ID from coccolithophore, Emiliania huxleyi (11.1 parts per thousand). Variability in epsilon-values among RubisCOs from primary producers is likely reflected in delta C-13 values of oceanic biomass. Currently, delta C-13 variability is ascribed to physical or chemical factors (e.g. illumination, nutrient availability) and physiological responses to these factors (e. g. carbon-concentrating mechanisms). Estimating the importance of these factors from delta C-13 measurements requires an accurate epsilon-value, and a mass-balance model using the epsilon-value for S. costatum RubisCO is presented. Clearly, appropriate epsilon-values must be included in interpreting delta C-13 values of environmental samples.