2 Modelling Studies of the SQTKS ER Domain
5.8 Development of a Substrate and Cofactor Docked Model for the KR of TENS
It was possible to generate the KR domain of TENS using Swiss-Model as well as to integrate the cofactor NADPH 11 into the model. With respect to the cofactor, it is now possible to roughly identify the active pocket in PyMOL. Therefore, a PDB file was generated which included three-dimensional parameters of the respective 2R-methyl- acetoacetylpantetheine 135a, which is known to be the correct substrate for the KR
domain of SQTKS and mFAS (Chapter 1.11). Since TENS is also a HR-PKS this stereoisomer was chosen for the docking experiments.
The subsequent docking steps and validation criteria followed the usual standard operation protocol (Chapter 2.6) and will not be described again here in detail. The best docking results were obtained by optimization of different parameters of the Grid Box in the docking procedure with AutoDock Vina (section 2.1.2, Figure 61 and 62).
The docked model of the substrate 135a is shown in figures 61 and 62. The active site of the TENS KR domain with a mesh surface is shown in Figure 61. In addition, the cofactor
11, substrate 135a, the catalytic tyrosine and the LDD-motif are displayed. KR domains
control the stereochemistry of the β-hydroxyl group of a polyketide by the direction that the polyketide enters the active site in relation to the NADPH cofactor (Chapter 1.11.1).185
Hence, the first structural feature which is conserved in KR domains is the entrance of the substrate into the domain. In B-type KR domains, such as SQTKS or mFAS, if the phosphopantetheine arm enters from the right side it will encounter the LDD motif (Figure 61, Chapter 1.11.1).185 The LDD motif prevents the substrate from slipping behind the lid helix.185,62
Figure 61: Mesh surface of the KR domain with the Cofactor, substrate 135a, the conserved LDD-motif and
In Figure 62, for a better display only the cofactor 11 and the substrate 135a are shown. In the model, the catalytic nicotinamide moiety of the NADPH cofactor 11 is located inside the KR-domain.
Figure 62: Acitve site of the KR domain; Green, the cofactor 11; Blue, the substrate 135a; Grey LLD-motif
(L2292, R2293, D2294); Pink, catalytic residue Y2353.
The 2R-methyl-acetoacetylpantetheine 135a extends into the protein (Figure 62), from the right site and is located near the cofactor NADPH 11. This places the reactive β- carbon 3.8 Å away from the cofactor's correct/observed reactive 4´-pro-S hydrogen (Fig 62). The substrate is orientated towards the cofactor with the expected Si-face of the 3- oxo group facing the NADPH 4´-pro-S hydrogen. The Burgi-Dunitz angle for the substrate-cofactor complex is 83° and the dihedral angle 37.5°.
The conserved LDD-motif was also observed. However, the substrate is not in direct contact with the LDD-motif (Figure 61) which was to be expected since the chosen diketide substrate is still an early intermediate of the biosynthesis. Later longer intermediates of the biosynthesis might interact with the LDD-motif.
Furthermore, an α-helix, which is in contact with the substrate at the opening of the active site, was observed (Figure 61). This α-helix will be referred to as substrate
binding helix in the following and will be discussed in detail in sections 6.3 and 6.6-7.
Finally, the catalytical residues, which were determined by Reid and Keating-Clay
et. al., were observed near the substrate.60,185,62 Y2353, from the catalytic triad, is located 4.8 Å away from the substrate 3-oxo group. Furthermore, an angle of 84.2° was observed
for the possible hydrogen bond between the tyrosine and the 3-oxo group. However, this angle differs from the optimal angle of 120°.179 Keatinge-Clay et. al. did not determine an optimal bond length between the catalytic residues and the substrate. Hence, the only factor, which is not particularly good in the TENS model, is the angle of the hydrogen bond between the tyrosine and the substrate. In this case, the geometry of the substrate would have to change with respect to catalytic residue. However, the KR domains may also exist in different conformations. Therefore, in vivo it is quite possible that the KR domain would slightly change its conformation with could orient the substrate differently to reach the optimal angles.
The results of the in silico docking which placed the substrate 135a towards the cofactor with the expected Si-face of the 3-oxo group facing the NADPH 4´-pro-S hydrogen, results in similar stereochemistry of the reduction as observed for other B1 type KR domains, such as SQTKS and corresponds with the known stereochemical course of mFAS (Chapter 1.11, 1.11.1 and Scheme 26).56,57 The actual stereochemistry
of the TENS KR domain is not known, but the results give a good evidence that it should be similar to other HR-PKS and mFAS. Overall, a representative substrate and cofactor- docked model of the KR of TENS was developed.
5.9 Conclusion
Structural information of the single or mutli-domains are necessary for understanding mechanism of the programming of HR-PKS and the subsequent rational engineering of these domains. Here, the enzyme domains that are of interest for engineering and thus modelling are the C-MeT and KR domain of TENS.
Our first aim was to build and validate models of the TENS KR and C-MeT domains that ultimately met with quality criteria to perform in silico studies. The validation of the generated models of the KR and C-MeT domain were done similarly to the ER domain of SQTKS (Chapter 5.3-5.7). One of the validation parameters was the QMEAN score of Swiss-Model. The QMEAN score verified that it was possible to create a detailed model of each respective domain, which was suitable for docking studies. Further, the respective cofactor NADPH 11 or SAM 35 was docked into the domain and showed that this docks in a sensible way preserving known protein-cofactor contacts. In addition, it was shown that the KR domain exposes the correct 4'-pro-S hydrogen known to be transferred during the reduction reaction.56,57 The docking experiments placed the
with the expected Si-face of the 3-oxo group facing the NADPH 4´-pro-S hydrogen. The subsequent hydride transfer would result in the correct stereochemical product, which corresponds to the known stereochemical course in the mFAS and SQTKS KR domains.56 It should be noted, however, that the actual stereochemical course of the TENS KR is not known. Hence, for the KR domain of TENS it was possible to predict the substrate- binding pocket and to perform docking experiments, which indicated that the docked substrate has the likely correct stereochemical orientation in the active site.
In the case of the C-MeT model, the distance of the cofactor to the substrate was in a suitable parameter range. It was not possible to generate a substrate-cofactor docked model for TENS C-MeT, which would be representative. However, it was possible to roughly predict the active site of the C-MeT domain.
The templates chosen for the modeling had a high resolution of 2.1 Å (C-MeT) and 1.4 Å (KR). Overall the QMEAN, the inflexible structure, the good substrate docking and the good template resolution, indicate that the models generated for KR and C-MeT of the TENS were good enough for further study.
Overall, the models should be suitable to model a chimeric model of TENS and mFAS in silico. For the KR domain, the model should be suitable to investigate the influence of the domain swaps on the active site. These models shall be used to understand the molecular basis of the methylation and chain-length programming in silico.