Sensores y transductores

Next, the potential FAD and NADPH binding amino acids of the pea YUC1 protein were predicted (Table 3.4). The T. municipal FMO sequence was selected as a reference due to the high similarity scores given by the binding prediction web servers. The bacteria binding residues included in Table 3.4 were obtained by X-ray diffraction of proteins in bonded solution with ligands at 1.60 ångström (Romero et al., 2016). The pea YUC1 protein has fewer predicted binding residues overall when compared with those present in the bacteria FMO. Two servers were used to

compare and confirm results obtained for PsYUC1. The majority of predicted residues were in agreement and represented highly conserved amino acids clustering to areas defined by the consensus FMO domains. Intriguingly, no amino acids were

predicted for the FMO -identifying motif. The 3-D ligand prediction server (sbg.bio.ic.ac.uk/3dligandsite/) predicted fewer binding residues than did the RaptorX-Binding site (raptorx.uchicago.edu/BindingSite/). Most discrepancies related to interactions of PsYUC1 with the NADPH cofactor. Of interest, several regions of the bacteria and pea sequences, not characterised as core FMO motifs, were predicted as having important binding residues. The section between the GG motif and the first ATG motif had three clusters of amino acids defined as having binding roles. The region between the NADPH binding motif and the second ATG (LATGY) motif had three areas with predicted binding residues. Furthermore, and of relevance to this study, one area downstream of the LATGY motif was predicted to have binding properties.

Table 3.4. Predicted binding amino acids of PsYUC1 compared with bacteria FMO binding residues from crystallographic studies (Romero et al., 2016). Results from two independent ligand prediction servers are included: 3dligandsite and RaptorX- Binding. Residues in bold are present in bacteria and predicted by both servers.

FMO/YUC motifs and secondary structures Function Important residues defined by bacteria FMO*

PsYUC1 predicted residues 3DLigand Site lnE score=23.5 RaptorX p=4.38e-10 FAD motif: Loop-2 and -helix A

Binds the Adenine moiety of FAD cofactor I12, G15, A16, G17, G20, I21 V19, G20, G22 V19, G20, G22, P23, S24 GG motif: Loop-4 and -helix B

May stabilise FAD cofactor G32, E39 L42, E43, R44, S45, S50, L51, W52 E43, R44, S50, L51, W52

-strand 3 Unknown W48, Y53, K57 N54, R55, Y57, L60, K61, L62, H63

Y57, L60,

K61, H63

-helix D Unknown Y83, Y92, E94 K86 K86

-strand 4 and -strand 5 Unknown N109, V112, A115, F117, D118/ W124, V126, T128 Q108, T109, V110 Q108, T109, V110 ATG1 Motif: -strand 6 May provide linkage between FAD and NADPH

L139, V140, A142, G144, I151, P152 A140, T141, G142, E143, N144, A145 A140, T141, G142, E143, P150 FMO/ YUC motif: -helix E and -strand 7 Baeyer-Villiger motif motif found in all FMOs. May contribute to NADPH binding

I154, G156, F160, G162, V165, T167

NADPH motif: Loop-14 and -helix G Binds to NADPH cofactor. Reduction of FAD V181, V183, I184, G185, G187, G190 S187, E190 I182, G183, C184, G185, N186, S187, E190 -strand 9 and -helix J Unknown R209/ P216 S305, P322/ K328, P330 A206, R207, N208 K269 LATGY motif: -strand 16 and loop-27 Present in N-hydro- xylating enzymes. May assist in NADPH linkage A379, T380, G381 Y324 A321, T322, G323, Y324, N327 Loop-28 and 29 Unknown D398, G399, G420 S335 Loop-30 and -helix N

Unknown I444, S449, I452 G362, R365, R366, G367, L368 *PDB #5M0Z (Romero et al., 2016).

The data from the two binding prediction servers can also be visualised in 3-D (Figure 3.8 and 3.9). The majority of the pea -helices and -strands form the ‘nest’ with -helix B forming the ‘floor’. When spun 90 forward and viewed from the top, the ‘floor’ of the ‘bird nest’ can be seen at the centre of the structure (Figure 3.8a). As with AtYUC1, the shorter PsYUC1 sequence (411 bp) folds slightly differentially than the bacteria FMO structure, resulting in fewer -helices and - strands. The predicted binding residues are located within the pocket (Figure 3.8b). These represent the critical binding region present in bacteria FMO (Schlaich et al., 2007). Furthermore, when the whole structure in bonded formation is viewed in space-filling mode, nearly all the predicted binding residues are incorporated, and the FAD cofactor is quenched in the pocket. However, a substantial part of the NADPH ligand remains exposed, potentially to permit the interaction with molecular oxygen, which leads to the formation of the C4-intermediate (Figure 3.8d).

Figure 3.8. The PsYUC1 protein architecture and the FAD and NADPH binding amino acids predicted in silico with Phyre2. a) Viewed from above, the PsYUC1 protein is presented in ribbon mode. The binding pocket is encircled, and the predicted binding residues are in dark blue. b) The predicted amino acids presented in space-filling mode. c) The FAD and NADPH ligand in space-filling mode and green. d) The whole structure in space-filling mode. Generated with Phyre2.

When viewed with the RaptorX binding prediction tool, the two principal ligands are placed as expected (Figure 3.9). From the top of the structure, NADPH interacts with the secondary structure on the left side of the protein (Figure 3.9a). Focussing on the interactions of PsYUC1 with the cofactor FAD (Figure 3.9b), the core residues of the FAD binding motif interact with the purine ring of FAD. The amino acids predicted to bind at the tail-end of the protein (Loop-30 and a-helix N) can be clearly seen coming in contact with the central region of the FAD cofactor (Figure 3.9c).

Figure 3.9. The PsYUC1 protein architecture and the FAD and NADPH binding amino acids predicted in silico with RaptorX-Binding site. A) NADPH in the binding pocket. B) FAD and predicted binding amino acids. C) Close-up of FAD cofactor in the binding pocket. The FAD binding motif region and the C-terminus Loop-30 and -helix N predicted binding residues are boxed. Generated with the RaptorX-Binding site.

Next, the potential amino acids preferential interactions with the FAD and NADPH cofactors were then accessed in silico (raptorx.uchicago.edu/BindingSite/) (Figure 3. 10). Amino acids tended to cluster in groups which were primarily associated with one cofactor or the other. However, two regions were predicted to bind with both cofactors. Seven regions of the sequence were principally targeting the FAD cofactor. The majority were in the first half of the protein sequence with one C- terminally located cluster. Three areas centrally located targeted NADPH.

Figure 3.10. Predicted binding residues of the AtYUC1, PsYUC1 and the crd mutant proteins. Previously described FMO motifs are in boxes. Amino acids in red are predicted to interact with FAD and residues in blue with NADPH. Amino acids in red circles are predicted to be important binding residues but have not been

previously defined as such. The mutated residues in the crd mutants are marked in yellow. Sequence alignment generated with ClustalW and binding residues

predicted with 3-D Ligand site (sbg.bio.ic.ac.uk/3dligandsite/) and RaptorX-Binding (raptorx.uchicago.edu/BindingSite/).

FAD GG 2--60 AT4G32540.1_YUC1 MESHPHNKTDQTQHIILVHGPIIIGAGPSGLATSACLSSRGVPSLILERSDSIASLWKSK PsYUC1_Crd ---MDPFKQKPKSLFIHGPIIVGAGPSGIAVAACLSEQGVPSLILERSDCIASLWQNR crd_1_JI2460 ---MDPFKQKPKSLFIHGPIIVGAGPSGIAVAACLSEQGVPSLILERSDCIASLWQNR crd-2_short_whip ---MDPFKQKPKSLFIHGPIIVGAGPSGIAVAACLSEQGVPSLILERSDCIASLWQNR crd-4_pss ---MDPFKQKPKSLFIHGPIIVGAGPSGIAVAACLSEQGVPSLILERSDCIASLWQNR -3- D--- 4--- 120 AT4G32540.1_YUC1 TYDRLRLHLPKHFCRLPLLDFPEYYPKYPSKNEFLAYLESYASHFRIAPRFNKNVQNAAY PsYUC1_Crd TYDRLKLHLPKHFCELPLMSFPQDFPMYPTKHQFISYMESYADQFGIRPRFNQTVVTAEF crd_1_JI2460 TYDRLKLHLPKHFCELPLMSFPQDFPMYPTKHQFISYMESYADQFGIRPRFNQTVVTAEF crd-2_short_whip TYDRLKLHLPKHFCELPLMSFPQDFPMYPTKHQFISYMESYADQFGIRPRFNQTVVTAEF crd-4_pss TYDRLKLHLPKHFCELPLMSFPQDFPMYPTKHQFISYMESYADQFGIRPRFNQTVVTAEF ATG1 FMO 180 AT4G32540.1_YUC1 DSSSGFWRVKTHDNTEYLSKWLIVATGENADPYFPEIPGRKKFSGGKIVHASEYKSGEEF PsYUC1_Crd DPSSEIWNVKTLDGFQYSSPWLVVATGENAEPVIPKIHGMEHFHG-PVVHTCDYKSGSQY crd_1_JI2460 DPSSEIWNVKTLDGFQYSSPWLVVATDENAEPVIPKIHGMEHFHG-PVVHTCDYKSGSQY crd-2_short_whip DPSSEIWNVKTLDGFQYSSPWLVVATGENAEPVIPKIHGMEHFHG-PVVHTCDYKSGSQY crd-4_pss DPSSEIWNVKTLDGFQYSSPWLVVATGENAEPVIPKIHGMEHFHG-PVVHTCDYKSGSQY crd-1

NADPH ---9--- 240 AT4G32540.1_YUC1 RRQKVLVVGCGNSGMEISLDLVRHNASPHLVVRNTVHVLPREILGVSTFGVGMTLLKCLP PsYUC1_Crd KNKKVLVIGCGNSGMEVSLDLCRHNALPHLVARNTVHMLPRDIFGFSTFGVAMTLNKWLP crd_1_JI2460 KNKKVLVIGCGNSGMEVSLDLCRHNALPHLVARNTVHMLPRDIFGFSTFGVAMTLNKWLP crd-2_short_whip KNKKVLVIGCGNSGMEVSLDLCRHNALPHLVARNTISSVSCYFKFSILSQSSLLSRKSYA crd-4_pss KNKKVLVIGCGNSGMEVSLDLCRHNALPHLVARNTVHMLPRDIFGFSTFGVAMTLNKWLP crd-2

-J- 300 AT4G32540.1_YUC1 LRLVDKFLLLMANLSFGNTDRLGLRRPKTGPLELKNVTGKSPVLDVGAMSLIRSGMIQIM PsYUC1_Crd LKLVDKFLLLVSSFFLGNTNHYGIKRPKTGPIELKLATGKTPVLDVGQIAQIKSGNIKVM crd_1_JI2460 LKLVDKFLLLVSSFFLGNTNHYGIKRPKTGPIELKLATGKTPVLDVGQIAQIKSGNIKVM crd-2_short_whip TDDVTSIEVA--- crd-4_pss LKLVDKFLLLVSSFFLGNTNHYGIKRPKTGPIELKLATGKTPVLDVGQIAQIKSGNIKVM ATG2 (LATGY) 360 AT4G32540.1_YUC1 EGVKEITKKGAKFMDGQEKDFDSIIFATGYKSNVPTWLQGGDFFTDDGMPKTPFPNGWRG PsYUC1_Crd EGVKEITRNGAKFMDGQEKEFEAIILATGYKSNVPSWLKGSDFFTKDGMPKTPFPHGWKG crd_1_JI2460 EGVKEITRSGAKFMDGQEKEFEAIILATGYKSNVPSWLKGSDFFTKDGMPKTPFPHGWKG crd-2_short_whip --- crd-4_pss EGVKEITRNGAKFMDGQEKEFEAIILATGYKSNVPSWLKEVIFSLKMECRKHPFLMDGKE crd-4

Loop-30 414 AT4G32540.1_YUC1 GKGLYTVGFTRRGLLGTASDAVKIAGEIGDQWRDEIKGSTRNMCSSRFVFTSKS--- PsYUC1_Crd EQGLYTVGFTRRGLHGTYFDAIKISEDITSQWKTVKSKSCSDSHIINLISTTTLNNV crd_1_JI2460 EQGLYTVGFTRRGLHGTYFDAIKISEDITSQWKTIKSKSCSDSHIINLISTTTLNNV crd-2_short_whip --- crd-4_pss SKDCIR---

The uncharacterised clusters of amino acids defined as binding to the FAD and NADPH cofactors were placed on the 3-D PsYUC1 protein structure to assess whether the predictions were physically plausible. The five predicted areas, despite not being associated with known FMO domains, are located in the binding pocket and could therefore have the capacity to interact with the relevant ligands (Figure 3.11). The residues defined as being FAD-associated are in regions that are likely to interact with the FAD cofactor. FAD tightly fits in the Eastern portion of the protein and is likely to come in contact with -strand 4, -helix D and Loop-30. NADPH locates in the Western side of the binding pocket increasing the likelihood of interaction with -strand 9. Furthermore, and strengthening the case of the predictions being credible, -strand 3, predicted to contain amino acids binding to both cofactors, is located at the junction where FAD and NADPH meet.

Figure 3.11. Uncharacterised PsYUC1 regions predicted as containing important binding residues. The protein is shown as a ribbon model. Potential novel binding regions are highlighted. Structure generated with Swiss-Model server.