Proline dehydrogenase from Thermus thermophilus does not discriminate between FAD and FMN as cofactor

Abstract

Flavoenzymes are versatile biocatalysts containing either FAD or FMN as cofactor. FAD often binds to a Rossmann fold, while FMN prefers a TIM-barrel or flavodoxin-like fold. Proline dehydrogenase is denoted as an exception: it possesses a TIM barrel-like fold while binding FAD. Using a riboflavin auxotrophic Escherichia coli strain and maltose-binding protein as solubility tag, we produced the apoprotein of Thermus thermophilus ProDH (MBP-TtProDH). Remarkably, reconstitution with FAD or FMN revealed that MBP-TtProDH has no preference for either of the two prosthetic groups. Kinetic parameters of both holo forms are similar, as are the dissociation constants for FAD and FMN release. Furthermore, we show that the holo form of MBP-TtProDH, as produced in E. coli TOP10 cells, contains about three times more FMN than FAD. In line with this flavin content, the crystal structure of TtProDH variant ΔABC, which lacks helices αA, αB and αC, shows no electron density for an AMP moiety of the cofactor. To the best of our knowledge, this is the first example of a flavoenzyme that does not discriminate between FAD and FMN as cofactor. Therefore, classification of TtProDH as an FAD-binding enzyme should be reconsidered.

Two-Component FAD-Dependent Monooxygenases: Current Knowledge and Biotechnological Opportunities

“…When the loop closes the respective binding site, the adenine moiety remains solvent-exposed. We see these effects also in proline dehydrogenase from Thermus thermophilus , an FAD-dependent oxidoreductase that can also act with FMN [ 250 ].…”

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

“…This is not the case for ΔA KK and ΔABC KK (Table S1, Supporting Information). For ΔABC, we have already shown that the protein is C‐terminally truncated after purification from E. coli . ΔABC KK has the same truncation (removal of 22 amino acids, sequence 288‐RRIAERPENLLLVLRSLVSGLE‐309); however, this does not occur for all proteins in the enzyme population.…”

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

“…These data support that there are no major structural changes and that deletion of the N -terminal helices does not significantly alter the microenvironment of the flavin-binding site. A recent crystallographic study confirmed that ΔABC (residues 38–279; PDB 5M42) has a similar overall structure as TtProDH (PDB 2G37) with an rmsd value of 0.338 Å (for 221 Cα atoms of A chains) [ 1 , 18 ].…”

“…Previously, we showed that TtProDH is overproduced in Escherichia coli ( E. coli ) when its N -terminus is fused to maltose-binding protein (MBP) [ 17 ] and that the recombinant enzyme does not discriminate between FAD and FMN as cofactor [ 18 ]. Replacing Phe10 and Leu12 in helix αA with Glu residues (further referred to as MBP-TtProDH variant EE) successfully released MBP-TtProDH from non-native self-association, yielding homogeneous tetramers [ 19 ].…”