Hyperthermophilic Carbamate Kinase Stability and Anabolic
In Vitro
Activity at Alkaline pH
Authors: Hennessy James E. , Latter Melissa J. , Fazelinejad Somayeh , Philbrook Amy , Bartkus Daniel M. , Kim Hye-Kyung , Onagi Hideki , Oakeshott John G. , Scott Colin , Alissandratos Apostolos , Easton Christopher J. 
Published: November 2017

Carbamate kinases catalyze the conversion of carbamate to carbamoyl phosphate, which is readily transformed into other compounds. Carbamate forms spontaneously from ammonia and carbon dioxide in aqueous solutions, so the kinases have potential for sequestrative utilization of the latter compounds. Here, we compare seven carbamate kinases from mesophilic, thermophilic, and hyperthermophilic sources. In addition to the known enzymes from
Enterococcus faecalis
and
Pyrococcus furiosus
, the previously unreported enzymes from the hyperthermophiles
Thermococcus sibiricus
and
Thermococcus barophilus
, the thermophiles
Fervidobacterium nodosum
and
Thermosipho melanesiensis
, and the mesophile
Clostridium tetani
were all expressed recombinantly, each in high yield. Only the clostridial enzyme did not show catalysis. In direct assays of carbamate kinase activity, the three hyperthermophilic enzymes display higher specific activities at elevated temperatures, greater stability, and remarkable substrate turnover at alkaline pH (9.9 to 11.4).
Thermococcus barophilus
and
Thermococcus sibiricus
carbamate kinases were found to be the most active when the enzymes were tested at 80°C, and maintained activity over broad temperature and pH ranges. These robust thermococcal enzymes therefore represent ideal candidates for biotechnological applications involving aqueous ammonia solutions, since nonbuffered 0.0001 to 1.0 M solutions have pH values of approximately 9.8 to 11.8. As proof of concept, here we also show that carbamoyl phosphate produced by the
Thermococcus barophilus
kinase is efficiently converted
in situ
to carbamoyl aspartate by aspartate transcarbamoylase from the same source organism. Using acetyl phosphate to simultaneously recycle the kinase cofactor ATP, at pH 9.9 carbamoyl aspartate is produced in high yield and directly from solutions of ammonia, carbon dioxide, and aspartate.

IMPORTANCE
Much of the nitrogen in animal wastes and used in fertilizers is commonly lost as ammonia in water runoff, from which it must be removed to prevent downstream pollution and evolution of nitrogenous greenhouse gases. Since carbamate kinases transform ammonia and carbon dioxide to carbamoyl phosphate via carbamate, and carbamoyl phosphate may be converted into other valuable compounds, the kinases provide a route for useful sequestration of ammonia, as well as of carbon dioxide, another greenhouse gas. At the same time, recycling the ammonia in chemical synthesis reduces the need for its energy-intensive production. However, robust catalysts are required for such biotransformations. Here we show that carbamate kinases from hyperthermophilic archaea display remarkable stability and high catalytic activity across broad ranges of pH and temperature, making them promising candidates for biotechnological applications. We also show that carbamoyl phosphate produced by the kinases may be efficiently used to produce carbamoyl aspartate.