Fig 3.

HPLC analysis of products formed by 3-hydroxypropionyl–CoA synthetase and acrylyl-CoA reductase (AcuI). Acrylyl-CoA was produced in situ by recombinant Sulfolobus tokodaii 3-hydroxypropionyl–CoA synthetase (A to C). (A) The reaction mixture contained 100 mM Tris-Cl (pH 8.0), 10 mM MgCl₂, 3 mM ATP, recombinant S. tokodaii 3-hydroxypropionyl–CoA synthetase, and 0.1 mM CoA; (B) 1 min after the addition of 20 mM acrylate; (C) after 2 additional minutes of incubation; (D) 1 min after the addition of 1 mM NADPH; (E) 1 min after the addition of 0.3 μg of purified recombinant AcuI; (F) after 3 additional minutes of incubation. In panel C, the peaks denoted by the dotted arrows were considered the CoA adduct of acrylyl-CoA (30), which were not used as the substrates by AcuI. Free CoA and propionyl-CoA were identified by coelution with standard compounds (not shown).