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Enzymes of Ancient Metabolic Pathways (dm)

It is unknown how the Archaea fix sulfur or synthesize cysteine. We report the purification and characterization of O-acetyl-serine sulfhydrylase (OASS) from acetate-grown Methanosarcina thermophila, a moderately thermophilic methanoarchaeon. The purified OASS contained pyridoxal 5’-phosphate and catalyzed the formation of L-cysteine and acetate from O-acetyl-L-serine and sulfide. The N-terminal amino acid sequence has high sequence similarity with other known OASS enzymes from the Eukarya and Bacteria domains. The results of this study provide the first evidence, of any kind, for a sulfur-fixing enzyme in the Archaea domain. The results also provide the first biochemical evidence for an enzyme with the potential for involvement in cysteine biosynthesis in the Archaea.

Carbonic anhydrases catalyze the reversible hydration of CO2 and are ubiquitous in highly evolved eukaryotes. We report that carbonic anhydrase is widespread in the Archaea and Bacteria domains and is an ancient enzyme. The occurrence in chemolithoautotrophic species occupying deep branches of the universal phylogenetic tree suggests a role for this enzyme in the proposed autotrophic origin of life. We also report on the first known plant-type (b class) carbonic anhydrase in the Archaea. The Methanobacterium thermoautotrophicum DH cab gene was hyperexpressed in Escherichia coli and the heterologously produced carbonic anhydrase was purified 13-fold to apparent homogeneity. These results show that b carbonic anhydrases extend not only into the archaea domain but also into the thermophilic prokaryotes.

Formation of this important ancient enzyme and its NiFeS active site requires cysteine. Through genetic, physiological and biochemical studies we determined that in Methanosarcina thermophila cysteine biosynthesis occurs through the serine pathway. The two genes required for this pathway form an operon in M. thermophila. O-acetyl-serine sulfhydrylase (OASS), which catalyzes the final step of the pathway, exhibits positive cooperativity and is expressed under growth conditions requiring cysteine biosynthesis. The last two genes of the homoserine pathway do not seem to be present in the genome, and the enzymes are not expressed under growth conditions requiring cysteine biosynthesis.