The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) cycle in thermoacidophilic Crenarchaeota, like Metallosphaera sedula and Sulfolobus tokodaii, employs AMP-forming Acyl-CoA synthetases of the ANL superfamily to activate its key carboxylate intermediates. This is an energetically costly mechanism that consumes two ATP equivalents, a critical distinction from the energy-saving ADP-forming (ACD) enzymes found in Thaumarchaeota. To define the structural principles of this "AMP-forming solution, " we targeted three specific crenarchaeal enzymes: M. sedula 4-hydroxybutyryl-CoA synthetase (Msed₀406), M. sedula 3-hydroxypropionyl-CoA synthetase (Msed₁456), and S. tokodaii 3-hydroxypropionyl-CoA synthetase (Stok₃HPCS). This work describes the protocols we developed for their heterologous expression and purification. While Msed₀406 and the thermostable Stok₃HPCS were successfully purified, Msed₁456 proved to be highly unstable and prone to degradation. Using a combined in vitro and in silico approach, we demonstrate this instability is an intrinsic feature linked to its catalytic mechanism. AlphaFold 3 models, validated by our purification data, reveal that while both Msed₁456 and Stok₃HPCS are homotetramers, the thermostable Stok₃HPCS buries a massive interface (14, 360 Ų) while the unstable Msed₁456 buries a minimal interface (7, 097 Ų). This flexibility is essential for the ANL mechanism, which we identify as a "phosphate-gated latch". We show the gamma-phosphate of ATP tethers the N- and C-terminal domains; its hydrolysis upon adenylation "un-latches" the enzyme, permitting a massive ~125° domain rotation to the thioesterification state. This work defines the AMP-forming solution to carboxylate activation, providing a critical counterpart to the rigid, energy-efficient ACD enzymes and offering foundational knowledge for engineering novel carbon-fixation pathways. .
Johnson et al. (Mon,) studied this question.