Trimming proline dehydrogenase : protein and cofactor minimization

Proline is one of the proteinogenic amino acids and one of the most abundant amino acids in the cell. Next to serving as one of the non-essential amino acids, proline also has a central role in metabolism. In <strong>Chapter 1</strong>, the different functions of this imino acid are described, as well as the proline metabolic enzymes. The focus is on the enzyme proline dehydrogenase (ProDH), which catalyzes the flavin-dependent conversion of L-proline to Δ1-pyrroline-5-carboxylate (P5C). Malfunctioning of this enzyme has severe implications for human health and has been associated with tumorigenesis and schizophrenia. This thesis deals with the engineering and biochemical characterization of <em>Thermus thermophilus</em> ProDH (<em>Tt</em>ProDH) in order to gain more insight into the structure-function relationship of this thermo-resistant flavoenzyme. <em>Tt</em>ProDH is a membrane-associated protein and recombinant soluble forms of the enzyme have only been obtained in limited amounts. <strong>Chapter 2</strong> describes the heterologous production of <em>Tt</em>ProDH in <em>Escherichia coli</em>. Using maltose-binding protein (MBP) as solubility tag, high yields of active holoenzyme are obtained. The MBP-tag can be efficiently removed from the fusion protein with trypsin, yielding native <em>Tt</em>ProDH. This enzyme is thermotolerant as well as solvent tolerant; however, both fused and clipped <em>Tt</em>ProDH are prone to aggregation. In <strong>Chapter 3</strong>, we show that the hydrophobic N-terminal helix of <em>Tt</em>ProDH is responsible for this non-native self-association. Phe10 and Leu12, located at the protein surface, were replaced by glutamates, generating the F10E/L12E (EE) variant of MBP-<em>Tt</em>ProDH. This more polar variant exclusively forms tetramers and exhibits excellent catalytic features. Specific removal of the MBP-tag of the EE variant is less easy than for WT, as trypsinolysis of the fusion enzyme leads to degradation of <em>Tt</em>ProDH. Since the MBP tag does not influence the spectral and catalytic properties of the enzyme, further experiments were performed with MBP-tagged variants of <em>Tt</em>ProDH. ProDH has a distorted (βα)8 TIM-barrel fold which is conserved throughout the PutA/ProDH family. In contrast, the N-terminal sequence of ProDH is poorly conserved. <em>Tt</em>ProDH contains, next to the distorted TIM-barrel, three N-terminal helices, αA, αB and αC, of which the function is not well understood. In <strong>Chapter 4</strong>, we describe the characterization of helical arm-truncated variants, lacking respectively one (ΔA), two (ΔAB), or three (ΔABC) N-terminal helices. All three variants show flavin properties that are highly similar to EE, indicating no changes in the microenvironment of the flavin isoalloxazine ring. ΔA and ΔAB are highly active tetramers, whereas removal of the complete N-terminal arm (ΔABC) results in poorly active dimers. Furthermore, EE, ΔA and ΔAB rapidly react with the suicide inhibitor <em>N</em>-propargylglycine, while ΔABC is not capable of forming a flavin adduct with <em>N</em>-propargylglycine. This indicates that helix αC has a crucial role in both the oligomerization and activity of <em>Tt</em>ProDH. Closer examination revealed an ionic interaction as well as a hydrophobic patch between helices αC and α8, the latter helix being crucial for substrate recognition. To investigate the functional role of helix αC in further detail, additional enzyme variants were created that disrupt the interactions between both helices. While disrupting the ionic interaction had minor effects, disrupting the hydrophobic patch leads to dimer formation, loss of activity and decreased reactivity with <em>N</em>-propargylglycine. This supports that helix αC is crucial for <em>Tt</em>ProDH catalysis and tetramerization through positioning of helix α8. The quaternary structure of <em>Tt</em>ProDH was investigated in more detail in <strong>Chapter 5</strong>. Two ionic interactions at the dimeric interface were selectively disrupted by changing Asp205 and Glu207 of <em>Tt</em>ProDH variants EE, ΔA, ΔAB and ΔABC into lysines. These KK-variants form monomers (except for EE KK, which forms dimers) and have improved catalytic properties at moderate temperatures compared to their non-KK counterparts. However, their melting temperatures are decreased by more than 20 °C. This indicates that a trade-off is made between thermostability and catalytic activity. In <strong>Chapter 6</strong>, we studied the cofactor binding of <em>Tt</em>ProDH. Flavoenzymes contain 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. To study the cofactor binding of <em>Tt</em>ProDH, we produced MBP-<em>Tt</em>ProDH EE in its apoform using a riboflavin auxotrophic<em> E. coli </em>strain. Reconstitution of the enzyme with either FAD or FMN revealed that MBP-<em>Tt</em>ProDH has no preference for FAD as cofactor. Kinetic parameters of both holo-FAD and holo-FMN are similar, as are the dissociation constants for FAD and FMN release. We show that the holo form of MBP-<em>Tt</em>ProDH, as produced in <em>E. coli</em> TOP10 cells, contains about three times more FMN than FAD. In addition, we obtained the crystal structure <em>Tt</em>ProDH ΔABC, which shows no electron density for an AMP moiety of the cofactor. This indicates the presence of mainly FMN in the enzyme. The capability of <em>Tt</em>ProDH to display equal properties with both cofactors is unique for flavoenzymes, and classification of <em>Tt</em>ProDH as an FAD-containing enzyme should be reconsidered. In <strong>Chapter 7</strong>, we discuss the novel findings described in this thesis and put them in a broader perspective. We have created a minimalist ProDH that is an excellent catalyst, but is deprived of all structural features that are unnecessary for <em>in vitro</em> functioning. Our results expand the knowledge on the structure-function relationship of ProDHs, and give insight into enzyme functionality from an industrial perspective. We also discuss how this knowledge might be used in future studies for a better understanding of the properties of eukaryotic ProDHs, with a special interest in the human enzyme.