Functional Impact of the N-terminal Arm of Proline Dehydrogenase from Thermus thermophilus.

ABSTRACT: Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to ?¹-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices ?A, ?B, and ?C. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ?A, ?AB, and ?ABC, produced with maltose-binding protein as solubility tag. All three truncated variants show similar spectral properties as TtProDH, indicative of a conserved flavin-binding pocket. ?A and ?AB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (?ABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ?AB established that a hydrophobic patch between helix ?C and helix ?8 is critical for TtProDH catalysis and tetramer stabilization.

SUBMITTER: Huijbers MME 

PROVIDER: S-EPMC6017737 | biostudies-literature | 2018 Jan

REPOSITORIES: biostudies-literature