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

Abstract

Thermus thermophilus proline dehydrogenase (TtProDH) catalyzes the first step in proline catabolism. The thermostable flavoenzyme consists of a distorted triosephosphate isomerase (TIM) barrel and three N-terminal helices: αA, αB, and αC. Using maltose-binding protein (MBP) fused constructs, it has been recently demonstrated that helix αC is crucial for TtProDH catalysis and for tetramerization through positioning of helix α8. Here, the structural features that determine the thermostability of TtProDH are reported. Selective disruption of two ion pairs in the dimerization interface of several MBP-TtProDH variants result in the formation of monomers. The newly created monomers have improved catalytic properties but their melting temperatures are decreased by more than 20°C. Sequence comparison suggests that one of the ion-pairs involved in dimerization is unique for ProDHs from Thermus species. In summary, intermolecular ion-pairs improve the thermostability of TtProDH and a trade-off is made between thermostability and catalytic activity.

Targeted Enzyme Engineering Unveiled Unexpected Patterns of Halogenase Stabilization

“…However, it resembles the biological status of the protein in solution, prior to the ionization event, revealing structural information on the protein of interest . Owing to the ability of native ESI‐MS to preserve noncovalent interactions, characterization of several protein classes has successfully been applied …”

“…[47] Owing to the ability of native ESI-MS to preserve noncovalent interactions, characterization of several protein classes has successfully been applied. [48][49][50] It is known from previous reports that tryptophan halogenases PyrH, RebH and PrnA tend to form dimers, as evident from gel filtration and crystal structures. [45,51,52] Likewise, Thal was found as a dimer in the crystal structure whereas it exists as a monomer in solution.…”

Techniques for Enzyme Purification

“…For hydroquinone dioxygenase from P. fluorescens ACB, we obtained strong indications from gel filtration that this non-heme, iron-dependent enzyme is an α2β2 heterotetramer [33]. With proline dehydrogenase from Thermus thermophilus, we showed that the native enzyme is a homotetramer [40,50] and that dimerization of the protein subunits strongly increases the enzyme thermostability [51]. For 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1, we obtained evidence that the monomermonomer contact of the dimer is stabilized by the binding of a phosphatidylinositol ligand [20].…”

“…Site-directed mutagenesis (F10E/L12E variant) then showed that the hydrophobic N-terminal helix of ProDH is responsible for the self-association process [96]. Truncation of the N-terminal arm of TtProDH (ΔA and ΔAB variants) resulted in highly active tetramers [50], while selective disruption of two ion pairs in the dimerization interface of the enzyme (D205K/E207K variants) resulted in monomer formation (Figure 1.8) [51]. The newly created TtProDH monomer showed excellent catalytic properties but a significant lower thermal stability than the tetramer.…”