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Recombinant formate dehydrogenase from the acetogen Clostridium carboxidivorans strain P7 T , expressed in Escherichia coli, shows particular activity towards NADH-dependent carbon dioxide reduction to formate due to the relative binding affinities of the substrates and products. The enzyme retains activity over 2 days at 4°C under oxic conditions. F ormate dehydrogenases (FDHs) catalyze the interconversion of CO 2 and formic acid through an oxidoreductive process ( Fig. 1) (1). Consequently, when catalyzing CO 2 reduction, they are of interest for the sequestration of CO 2 and for the production of formic acid as a stabilized form of hydrogen fuel and as a source of commodity chemicals. In many bacteria and eukaryotes, FDHs catalyze the final step of catabolic processes in which formate is oxidized to CO 2 (2). The ability of certain members of this class, such as FDH from Candida boidinii in particular, to efficiently regenerate NADH in conjunction with formate oxidation has been a research focus (3).Acetogens are known to possess a number of pathways distinct from those found in the other species. FDHs present in acetogens are known to take part in a carbon fixation metabolic pathway producing acetate (the Eastern branch of the Wood-Ljungdahl pathway), in which the first step involves reduction of CO 2 to formate (4). Several FDHs are known to catalyze CO 2 reduction under appropriate conditions (5-9). Those enzymes from acetogenic and related anaerobes, such as Moorella thermoacetica and Clostridium pasteurianum, are better than other FDHs as reduction catalysts but also show similar catalytic efficiency toward formate oxidation and are considered highly oxygen labile, requiring anaerobic expression and purification as well as anoxic assay conditions (10, 11). Clostridium carboxidivorans strain P7 T (equivalent to ATCC BAA-624 T and DSM 15243 T ) was isolated from the sediment of an agricultural settling lagoon after enrichment with CO as the substrate and is an obligate anaerobe that can grow autotrophically with H 2 and CO 2 or CO (fixing carbon via the Wood-Ljungdahl pathway) (12, 13). Therefore, when the gene of a selenocysteine-containing formate dehydrogenase H (FDH H ) from the acetogen Clostridium carboxidivorans strain P7T was first identified, it was suggested that FDH H would catalyze the conversion of CO 2 to formate (14, 15). Here we report the first production of FDH H and its catalytic preference for CO 2 reduction, as well as its tolerance for oxic conditions. Cloning, expression, and purification of FDHs. The overexpression and purification of recombinant FDH H from the Clostridium carboxidivorans strain P7 T (FDH H _CloCa) was carried out, along with that of NAD ϩ -dependent recombinant FDH from Candida boidinii (FDH_CanBo), in order to compare expression and activity of formate dehydrogenases that take part in distinct metabolic pathways. The DNA sequences for the FDH H _CloCa (UniProt E2IQB0) and FDH_CanBo (UniProt O13437) genes were codon optimized for expression in Escherichia coli (com...

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 and, the previously unreported enzymes from the hyperthermophiles and, the thermophiles and, and the mesophile 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). and 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 kinase is efficiently converted 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. 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.

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