The smallest active carbamoyl phosphate synthetase was identified in the human gut archaeon Methanobrevibacter smithii

The genome of *Methanobrevibacter smithii*, a key archaeon in the human gut, encodes a complex gene system for carbamoyl phosphate synthetase (CPSase) that includes both full-length and truncated synthetase subunits. These enzymes, which metabolize ammonia, may play a critical role in regulating ammonia assimilation in *M. smithii*, influencing the metabolism of gut bacteria and potentially impacting host obesity. In this study, we isolated and characterized a small CPSase homolog (41 kDa) from *M. smithii*. The gene was cloned, overexpressed in *Escherichia coli*, and the recombinant enzyme was purified in a single step. Structural analysis using chemical cross-linking and size-exclusion chromatography revealed that the enzyme exists as homodimers and tetramers, consistent with the dimeric mechanism typical of CPSase enzymes.

This small enzyme, designated MS-s, catalyzed the synthesis of H3B-120 carbamoyl phosphate from ATP, bicarbonate, and ammonia, performing the same ATP-dependent partial reactions seen in full-length CPSases. Kinetic analysis showed that the enzyme has high affinity for both ATP and ammonia. Sequence comparisons, molecular modeling, and kinetic studies suggest that MS-s corresponds to one of the two synthetase domains present in full-length CPSases, responsible for ATP-dependent phosphorylations during the three-step carbamoyl phosphate synthesis. MS-s is the smallest active CPSase discovered to date, and it appears to be specialized for carbamoyl phosphate metabolism within methanogens.