Abstract
Proline dehydrogenase (ProDH) is a ubiquitous flavoenzyme that catalyzes the oxidation of proline to Δ1-pyrroline-5-carboxylate. Thermus thermophilus ProDH (TtProDH) contains in addition to its flavin-binding domain an N-terminal arm, consisting of helices αA, αB, and αC. Here, we report the biochemical properties of the helical arm truncated TtProDH variants ΔA, ΔAB, and ΔABC, 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. ΔA and ΔAB are highly active tetramers that rapidly react with the suicide inhibitor N-propargylglycine. Removal of the entire N-terminal arm (ΔABC) results in barely active dimers that are incapable of forming a flavin adduct with N-propargylglycine. Characterization of V32D, Y35F, and V36D variants of ΔAB established that a hydrophobic patch between helix αC and helix α8 is critical for TtProDH catalysis and tetramer stabilization.
Highlights
Proline dehydrogenase (ProDH; EC 1.5.5.2) is a ubiquitous enzyme involved in proline catabolism.ProDH catalyzes the flavin-dependent oxidation of L-proline to ∆1 -pyrroline-5-carboxylate (P5C).After P5C hydrolysis, the resulting glutamic semialdehyde (GSA) is oxidized to glutamate through the action of ∆1 -pyrroline-5-carboxylate dehydrogenase (P5CDH; EC 1.2.1.88) (Scheme 1)
In these multi-functional enzymes, called proline utilization A (PutA), the C-terminus of ProDH is fused to P5CDH, allowing for the channeling of the P5C/GSA intermediate between the enzymes [4,5,6,7,8,9]
From 1 L of culture about 200–250 mg of each variant was purified, yields that we have before for the Previously, we demonstrated that removal of the fusion tag described with trypsin does not heterologous production of maltose-binding protein (MBP)-Thermus thermophilus ProDH (TtProDH)
Summary
Proline dehydrogenase (ProDH; EC 1.5.5.2) is a ubiquitous enzyme involved in proline catabolism. ProDH from Thermus thermophilus (TtProDH) contains an N-terminal arm consisting of three αA, αB, and αCan TtP5CDH terminus is fused to maltose-binding protein (MBP) [17] and that the recombinant enzyme when does not Previously, we showed that TtProDH. To investigate the functional impact of the N-terminal arm of TtProDH in catalysis and oligomerization in closer detail, we constructed MBP-fused variants, lacking, respectively, one (ΔA), two (ΔAB), or three (ΔABC) N-terminal helices. Oligomerization in closer detail, we constructed MBP-fused variants, lacking, respectively, one (∆A), two (∆AB), or three (∆ABC) N-terminal helices. From 1 L of culture about 200–250 mg of each variant was purified, yields that we have before for the Previously, we demonstrated that removal of the fusion tag described with trypsin does not heterologous production of MBP-TtProDH [17,19].
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