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 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.

Dimerization of Proline Dehydrogenase from Thermus thermophilus Is Crucial for Its Thermostability

“…The far‐UV CD spectra of the KK variants of MBP‐ Tt ProDH are very similar (Figure S1A, Supporting Information) and resemble those of the non‐KK variants . In addition, the visible flavin absorption spectra of the KK variants of MBP‐ Tt ProDH (Figure S1B, Supporting Information) are all nearly identical to those of the non‐KK variants . Therefore, the D205K and E207K replacements do not alter the secondary structure of the enzyme, nor the local environment of the flavin isoalloxazine ring.…”

“…All MBP‐ Tt ProDH variants were expressed and purified successfully and with high yields, as we have reported before for different MBP‐ Tt ProDH variants . From 1 L culture, about 200–250 mg of each variant was purified.…”

“…While MBP‐ Tt ProDH WT forms soluble aggregates and tetramers, the EE variant with a more polar N‐terminal helix exclusively forms tetramers . The truncated variants ΔA and ΔAB form tetramers as well, while ΔABC purifies as a dimer . Introducing Lys205 and Lys207 in the α5 interface helices of Tt ProDH clearly changes the oligomerization state of the protein.…”

“…The native and subunit masses of the MBP‐ Tt ProDH KK variants were determined by nano‐flow electrospray ionization mass spectrometry (ESI‐MS), essentially as described earlier . With native ESI‐MS analysis, the quaternary protein structure of the protein variants could be determined, while with ESI‐MS analysis under denaturing conditions the exact molecular mass of the subunits of the MBP‐ Tt ProDH KK variants could be determined.…”

“…Increasing the polarity of helix αA (variant MBP‐ Tt ProDH F10E/L12E [EE]) eliminates the formation of aggregates . In addition, we constructed variants of Tt ProDH in which we sequentially removed one, two, or three N‐terminal helices, resulting in variants ΔA, ΔAB, and ΔABC . While ΔA and ΔAB form active tetramers, ΔABC mainly consists of poorly active dimers.…”