Amine-mediated CO2 hydration as a potential carbonic anhydrase alternative in marine Thaumarchaeota

Abstract

Marine Thaumarchaeota are ammonia-oxidizing archaea that make up the predominant group of nitrifiers in the global N cycle and fix bicarbonate (HCO3-) autotrophically via the highly efficient 3-HP/4-HB pathway. Most autotrophs produce intracellular bicarbonate via the ubiquitous enzyme carbonic anhydrase (CA), which catalyzes CO2 hydration. However, the common Thaumarchaeota species Nitrosopumilus maritimus lacks CA, and it is unknown how this organism produces sufficient intracellular bicarbonate to satisfy its anabolic requirement without this enzyme. One hypothesis is the autocatalytic addition of water to CO2 mediated by free intracellular amines, in the form of protein N-termini, via a carbamate intermediate at physiological pH. In order to determine the viability of this bicarbonate pathway as a potential CA alternative in N. maritimus, the reaction kinetics of 12 aqueous primary and secondary amine solutions buffered to pH 7.2 were measured via 13C NMR spectroscopy and compared to a novel chemical kinetic model of intracellular carbon dynamics in N. maritimus. The experimental observations showed that amine reactivity generally increases with increasing pKa of the amine group, but is heavily influenced by the amine structure; primary and secondary amines form carbamate and bicarbonate through a two-step mechanism involving formation and decarboxylation of a carbamate zwitterion, while sterically hindered and tertiary amines form bicarbonate via a base-catalysis mechanism in which the nitrogen abstracts a proton from water to drive CO2 hydroxylation. The measured 1st-order reaction rate constants of carbamate formation and dissociation are 1.1 x 10-2 s-1 and 4.88 x 10-3 s-1 respectively, yielding intracellular fluxes of carbamate and bicarbonate of 4.4 x 10-4 mM s-1 and 1.46 x 10-4 to 1.46 x 10-2 mM s-1, respectively. The bicarbonate-fixing enzyme of the 3-HP/4-HB pathway, acetyl-CoA/propionyl-CoA carboxylase, has a turnover rate of 2.168 x 10-1 mM s-1, and the modeled fluxes required to sustain this rate are 5.067 x 10-1 mM s-1 for carbamate formation and 2.153 x 10-1 mM s-1 for carbamate dissociation/bicarbonate formation. Thus, the flux of bicarbonate supplied by amine-mediated CO2 hydration is insufficient to drive autotrophy in N. maritimus. Future work to determine the reaction kinetics of tertiary amines could shed light on the potential activity of the base-catalysis pathway of amine-mediated CO2 hydration; however, it is suggested that the true hydration mechanism may be driven by the generation of hydroxide ions in situ coupled to redox potentials present in copper-containing oxidoreductase enzymes responsible for electron transfer at the cell membrane that could double as CA alternatives or antecedents.