Hyperthermophilic Carbamate Kinase Stability and Anabolic In Vitro Activity at Alkaline pH

ABSTRACT 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.

Reconstitution of an Active Arginine Deiminase Pathway in Mycoplasma pneumoniae M129

ABSTRACTSome species of the genusMycoplasmacode for the arginine deiminase pathway (ADI), which enables these bacteria to produce ATP from arginine by the successive reaction of three enzymes: arginine deiminase (ArcA), ornithine carbamoyltransferase (ArcB), and carbamate kinase (ArcC). It so far appears that independently isolated strains ofMycoplasma pneumoniaeencode an almost identical truncated version of the ADI pathway in which the proteins ArcA and ArcB have lost their original enzymatic activities due to the deletion of significant regions of these proteins. To study the consequences of a functional ADI pathway,M. pneumoniaeM129 was successfully transformed with the cloned functionalarcA,arcB, andarcCgenes fromMycoplasma fermentans. Enzymatic tests showed that while theM. pneumoniaeArcAB and ArcABC transformants possess functional arginine deiminase, ornithine carbamoyltransferase, and carbamate kinase, they were unable to grow on arginine as the sole energy source. Nevertheless, infection of a lung epithelial cell line, A549, with theM. pneumoniaetransformants showed that almost 100% of the infected host cells were nonviable, while most of the lung cells infected with nontransformedM. pneumoniaewere viable under the same experimental conditions.

Expression ofSTM4467-Encoded Arginine Deiminase Controlled by theSTM4463Regulator Contributes to Salmonella enterica Serovar Typhimurium Virulence

ABSTRACTArginine deiminase (ADI), carbamate kinase (CK), and ornithine transcarbamoylase (OTC) constitute the ADI system. In addition to metabolic functions, the ADI system has been implicated in the virulence of certain pathogens. The pathogenic intracellular bacteriumSalmonella entericaserovar Typhimurium possesses theSTM4467,STM4466, andSTM4465genes, which are predicted to encode ADI, CK, and OTC, respectively. Here we report that theSTM4467gene encodes an ADI and that ADI activity plays a role in the successful infection of a mammalian host byS. Typhimurium. AnSTM4467deletion mutant was defective for replication inside murine macrophages and was attenuated for virulence in mice. We determined that a regulatory protein encoded by theSTM4463gene functions as an activator forSTM4467expression. The expression of the ADI pathway genes was enhanced inside macrophages in a process that required STM4463. Lack of STM4463 impaired the ability ofS. Typhimurium to replicate within macrophages. A mutant defective inSTM4467-encoded ADI displayed normal production of nitric oxide by macrophages.