The truncated CYP52A13 is not active

The original aim of this work was to create an enzyme reactor for the synthesis of long-chain α,ω-DCAs. Therefore, we designed constructs to deliver soluble forms of the CPR-a and CYP52A13 that could be introduced in such a reactor. Unfortunately, while we could achieve recombinant production of the proposed truncated form of CYP52A13, this did not result in an oleic acid-converting construct. This can be due to either an incompatibility with the redox partners used, or due to inherent inactivity of CYPtr.

Three alternative redox partners were considered for the delivery of electrons from NADPH to CYPtr; (1) the endogenous CPR from P. pastoris, (2) CPRtr, either recombinantly produced and added to the microsomes as a separate enzyme, or fused to CYPtr, and (3) BMR, fused to CYPtr.

In the first assays, the use of endogenous CPR from P. pastoris as a redox partner did not result in DCA formation, nor was NADPH consumed to a higher extent upon substrate addition. In fact, several examples were found in literature where the low level of endogenous CPR from P. pastoris was able to reduce the recombinant CYP [328]–[333]. However, other CYPs did not show activity when this CPR served as the only redox partner [335] and it is a generally used strategy to overexpress either the homologous or a heterologous CPR when CYP is recombinantly produced in P. pastoris [237], [331], [336]–[338]. Also fusion to the reductase domain of the self-sufficient CYP102A1 did not yield an active protein product. It is documented that changing the redox partner can have a marked influence on the catalytic properties of CYP [264]–[267]. Therefore, it was decided to focus further on using the native redox partner CPR-a.

In a first series of experiments, an attempt was done to produce CPRtr in a soluble and secreted form using the expression host P. pastoris. Indeed, CPRtr could be purified from the medium of the NRRL-Y-11430 strain by IMAC. Herein, secreted CPRtr proved to be active towards the nonphysiological redox partner cytochrome c. However, no increased NADPH consumption was seen upon substrate addition, indicating that the delivery of electrons to CYPtr was not accomplished. On the one hand, this can be ascribed to the low activities obtained. On the other hand, it must be considered that CPRtr might not be able to deliver electrons to CYPtr. It has been reported in literature that the binding site of CPR to either cytochrome c or CYP overlaps [202], negating the hypothesis that truncating CPR prevents interaction with its redox partner.

However, we remind that the very first CPR crystal structure to be resolved, i.e. rat CPR obtained after a limited trypsin digest, was able to reduce cytochrome c but not CYP [191].

Therefore, it cannot be excluded that our CPRtr is unable to reduce our CYPtr. In the course of this PhD study, the structure of a N-terminally truncated CPR-b from C. tropicalis strain 1230, has been resolved [195]. This reductase shares 98.7 % amino acid sequence identity with CPR-a and the activity was similar. He and Chen inferred that the allelic cpr variants from C. tropicalis 1230 were the same as those from C. viswanathii ATCC 20336 [310]. Ebrecht et al showed that

103 this truncated CPR was able to not only reduce cytochrome c, but also CYP (more specifically CYP52A21 from C. albicans) [195]. It must be noted that they only deleted amino acids 2 to 22. One might thus wonder if the truncation applied in this dissertation (36 residues were deleted), led to an inability to interact with CYPtr. We based our design on the report of a truncated CPR from S. cerevisiae. In earlier reports of self-sufficient CYP construction, Shibata et al published a study where this CPR from S. cerevisiae was truncated differentially for its use in a CYP17A1 chimeric construct. They reported that the deletion of up to 53 amino acids all resulted in 90-95 % progesterone conversion, be it with different 17α-hydroxylase activities [269]. This strengthens the hypothesis that indeed our CPRtr cannot only reduce cytochrome c but CYPtr as well.

Throughout the expression experiments, it became evident that the CPRtr was very unstable, which is likely to be the result of removing the enzyme from its membrane environment. High yields could not be obtained and a high amount of intracellular degradation was observed as well, indicating folding issues and consequently directing the misfolded protein to the cytoplasm for degradation. Furthermore, using the recombinant host co-expressing HAC1, precipitation issues during enzyme purification occurred. In fact, the latter strain led to a significant yield increase and improved removal of the secretion signal. This was concluded based on two results. Firstly, it was observed that only one band appeared with the right MW on blot, whereas two bands were visible after CPRtr production in NRRL-Y-11430. The two bands differed only slightly in MW and it was thought that this resulted from an unprocessed signal sequence. Only one band appears upon HAC1 co-expression, indicating that the signal sequence was processed correctly in that case, which might lead to increased secretion. Indeed, the co-expression of HAC1 has been shown to aid in the signal sequence processing in efforts to produce the adenosine A2A receptor [315] and increased yield of secreted protein has been reported with this strain [339]. Secondly, a significant increase of the absorption peak eluting with 100 mM imidazole was observed in case where no precipitation occurred either during sample preparation preceding IMAC or during sample loading on the HisTrap column. CPRtr produced in the HAC1 co-expressing strain could not be recovered in any case, and thereby proved to be highly unstable. These instability issues appeared to be more pronounced than in case of CPRtr production in NRRL-Y-11430. From this, it was hypothesized that inclusion of the cofactors FAD and FMN became more limiting, whereas in the wild type strain, the lower production rate can be better followed by cofactor inclusion. This hypothesis could theoretically be confirmed by measuring the FAD and FMN concentrations spectrophotometrically and compare these to the CPRtr concentration. The concentration of CPRtr in crude samples can be determined based on the cytochrome c reductase activity [340]. Alternatively, total protein concentration can be measured from a pure CPRtr sample. However, the enzyme was already lost during the first steps of enzyme collection and aforementioned assays are therefore not possible without further optimizations.

104

CPRtr was fused N-terminally to the C-terminus of CYPtr in an effort to create a self-sufficient CYP. By creating a chimer, it was no longer necessary to separately produce CPRtr, which proved to be difficult and where yields, too low for functional assays, were obtained. Clear cytochrome c reducing activity was observed by the CPRtr, present as the reductase domain of the created self-sufficient CYPtr. If the truncated reductase indeed is able to reduce CYPtr as well, as discussed above, than why was oleic acid not converted? Several possible answers can be given to this question. Uncoupling reactions always occur to some extent and these increase when for example the substrate does not fit the binding pocket well or alterations are made concerning the redox partner [176]. Theoretically, the construction of a self-sufficient enzyme should increase coupling efficiency by improving the electron transfer. However, coupling might not be sufficient in this chimer for efficient reduction of CYPtr. The possibility of uncoupling was addressed in this dissertation by assaying whether NADPH was increasingly consumed in presence of oleic acid compared to NADPH consumption in absence of oleic acid by microsomes after recombinant expression. If substrate binding occurs, followed by the delivery of electrons from NADPH to CYPtr via CPRtr, it was expected that an increased NADPH consumption would be observed. If increased NADPH consumption does not match the level of FA hydroxylation, uncoupling occurs through one of the three shunts (autooxidation, peroxide and/or oxidase shunt). However, no such increased NADPH consumption was seen. Taken together with the absence of hydroxylated products in the GC chromatograms, this led to the conclusion that no hydroxylation, nor uncoupling is happening.

Next to these shunt pathways, other uncoupling reactions, independent of substrate binding, have been mentioned in literature [176]. For example, the reduced flavin cofactors of CPR could interact with molecular oxygen, whereby ROS are formed [195], possibly harmful for the recombinant CYP. In fact, such an assay was performed, measuring the formed hydrogen peroxide after complete oxidation of NADPH, and 3.54 % of the added NADPH resulted in hydrogen peroxide formation (data not shown). No excessive uncoupling reactions thus seem to happen. To finally address whether CPRtr can transfer electrons to CYP, one could exploit the fact that the reduced CO-bound form shows a characteristic absorption peak at a wavelength of 450 nm. Hayashi et al showed how the redox partner was able to reduce the heme group by observing an increased 450 nm absorption peak upon NADPH reduction in presence of CO and substrate [341]. Unfortunately, we did not have access to facilities to produce CO-bound CYPtr.

An aspect that has not been covered, is the involvement of cytochrome b5. Three possible contributions of this enzyme have been reported, i.e. cytochrome b5 has an allosteric effect, it delivers the second electron to CYP or both electrons are delivered by cytochrome b5 [139].

Functional assays performed by Eschenfeldt et al proved that CYP52A13 and its native redox partner CPR-a actively convert oleic acid when recombinantly produced in insect cells. No cytochrome b5 was added or co-expressed [24]. Therefore, it was hypothesized that this third enzyme partner was not required for activity. However, addition of this enzyme or co-expression might still be valuable. For example it has been reported to positively influence the coupling efficiency of chimer constructs in several cases [271].

105 The fact that the reductase domains showed to be active, together with the absence of substrate-induced uncoupling, would lead to the conclusion that CYPtr is not active. It might be asked whether the truncated form is correctly folded and present as a holo-enzyme and if the substrate is able to bind in the active site in presence of the unprocessed secretion signal. These questions as well could be further addressed by means of spectrophotometric assays. Upon reduction of and CO binding to the heme-iron, the Soret peak of the substrate-free enzyme shifts from 418 nm to about 450 nm. This only occurs if cysteine thiolate is retained as the proximal ligand, thereby proving correct folding. Furthermore, substrate binding could be assessed by spectrophotometrically measuring the Soret peak shift occurring upon substrate binding. The Soret peak shifts to 390 nm upon conversion of the low spin state to the high spin state of the heme-iron. This happens when the substrate binds and thereby displaces the axial water ligand [177].

The location of CYPtr needs further consideration as the truncated enzyme contains an α-factor secretion signal, thereby directing the enzyme post-translationally to the ER for subsequent processing and secretion through COPII vesicles. However, no secretion was observed using P. pastoris NRRL-Y-11430 and the enzyme was found in the microsomal fraction. Normally, class II CYPs are anchored in the ER membrane via the N-terminal transmembrane helix at the cytosolic side. However, this anchor was removed in CYPtr and replaced by the α-factor secretion signal aiming for translocation inside the ER lumen. Is CYPtr retained at the cytosolic side? The protein is translocated post-translationally into the ER lumen. If the protein is already folded in the cytoplasm, this translocation is blocked and the CYPtr would thereby be present at the cytoplasmic side. A hydrophobic patch, consisting of the N-terminal part of the catalytic domain and the F-G loop, was reported to enable additional membrane interaction. As the F-G loop was kept intact due to involvement in substrate binding, membrane interaction might still occur through this hydrophobic patch. Alternatively, CYPtr might be retained in the ER lumen [342]. This would be an alternative reason why both the endogenous CPR from P. pastoris and the recombinant CPRtr would not be able to reduce CYPtr. Furthermore, glycosylation already starts in the ER lumen, which might be a reason for the seemingly higher MW observed in Figure 3.8. In case of the chimeric construct, it was hypothesized that the enzyme was not translocated across the ER membrane into the ER lumen. On the one hand, the chimer was retrieved in the soluble supernatant around the expected MW upon washing microsomes with a high salt buffer. On the other hand, reducing activity was observed for the chimeric construct upon addition of cytochrome c and NADPH. Based on the chimeric construct results, it was hypothesized that the lack of oleic acid conversion could be attributed to an inactive CYPtr enzyme.

The different questions that were asked throughout the discussion are shown in light of the catalytic mechanism in Figure 3.41.

106

Figure 3.41: Catalytic mechanism including the different questions that were asked throughout this discussion.

4.2 The yield of CYP52A13 in a nontruncated form is too low for functional