Development of a Substrate and Cofactor Docked Model for the ER of SQTKS

In the next step, different substrates were docked into the active pocket of the ER SQTKS (table 1). On the one hand, this was done to obtain a substrate and cofactor docked model of the ER domain, which could be used as a template for the mutagenesis in silico. On the other hand this was done in order to determine the range of values for different parameters, such as C-H distances and carbonyl position, etc. which could be correlated with productive or non-productive conversion of the substrate by the ER domain. Hence, in the following in silico experiments we aimed to correlate results from in vitro kinetic experiments with the in silico docking results to attempt to determine a range of

productive and non-productive geometric substrate poses from which predictive

geometric parameters could be extracted.58,137 Therefore data was generated which included three-dimensional parameters of the substrates known to be active or inactive with the SQTKS ER.58

Table 1: Model substrates for the determination of the validation parameters (ER SQTKS).

Substrate Observed Substrate

Specificity (kcat/KM)58 A Okay (12.0) B Good (119.0) C Okay (23.6) C Bad (0.0)

The docking was performed by manually overlaying the substrate in the SQTKS ER active site using PyMOL. This was done to simplify the docking in the next step. This method minimized the so-called Grid Box, which has a critical role in the speed of the docking calculations. Molecular docking was done using AutoDock Vina (Chapter 2, section 2.1.2).110,147,148 _{The model was then refined by YASARA. The visualization of}

the different models, after the refinement step was always done in PyMOL. An example is shown with squalestatin triketide pantetheine 85 (E-4S-2,4-dimethylhex-2-enoyl-

pantetheine, Figure 22 and Figure 23) which is known to be both a natural substrate and a good substrate in vitro.

Figure 22: Active site of the ER domain with a mesh surface. In green the cofactor NADPH 11 and in white

the modeled substrate squalestatin triketide pantetheine (E-4S-2,4-dimethylhex-2-enoyl-pantetheine) 85.

Figure 23: Active site of the ER domain. In green the Cofactor NADPH 11 and in white the modeled substrate

squalestatin triketide pantetheine (E-4S-2,4-dimethylhex-2-enoyl-pantetheine) 85.

Since no structural data for the SQTKS ER domain was available, it was not possible to perform a direct validation of the molecular docking of the substrates. For example, the RMSD between the docked substrate and the structural available substrate could not be compared. In addition, validation of the docked substrates according to the lowest energy

conformation is not very reliable (section 2.1.2). The last validation option that could be used would be a cluster of RMSD analysis. Even so, this method is very time consuming.

Hence, a visual validation of the docking was performed based on different criteria, before the values for the different parameters were determined. The criteria of the visual validation included amongst other things the orientation of the substrate towards the cofactor 11.

In the following, the determination of the values for the C-H bond, distance to the active hydride, etc. are shown for squalestatin triketide pantetheine 85 (E-4S-2,4-dimethylhex- 2-enoyl-pantetheine, Fig 24 and 25) and 93a squalestatin tetraketide (6S,4S-2E- dimethyloct-2-enoylpantetheine, Fig 26 and 27) which is known not to be a substrate.The best docking result, which could be obtained with optimization of different parameters of the Grid Box in the docking procedure with AutoDock Vina (section 2.1.2), are shown in Figures 24-27.

In the model of the squalestatin triketide pantetheine 85, the catalytic nicotinamide moiety of the NADPH cofactor 11 is located inside the ER-domain. NADPH 11 is in contact with its binding site, consisting of highly conserved residues (Sequence alignment of the ER, Table 2). The squalestatin triketide pantetheine 85 extends into the protein. The pantetheine part of the substrate extends parallel to the adenine diphosphate locating the thiolester and the β-carbon adjacent to the nicotinamide. The α/β-unsaturated carbonyl of the substrate adopts an s-cis conformation (Figure 25), which places the reactive β-carbon 3.4 Å away from the cofactor's correct/observed reactive 4´-pro-R hydrogen (Fig 24). The Burgi-Dunitz angle substrate-cofactor complex is 61.7° and the dihedral angle 46.6° (table 2). The Burgi-Dunitz angle and the dihedral angle might differ from the optimal angles from literature (e.g. Burgi-Dunitz angle: 107°),149,150 however, enzymes are flexible systems and in crystals and the generated model only snapshot of a certain state is displayed. Hence, in nature the enzyme might change is structural conformation until the right pose with the necessary angle is occupied. In addition, other angles than the optimal angle of 107° have been observed in enzymatic reactions, since the angle in the enzymatic conversion in the SQTKS ER might be also different.151

Table 2: Sequence alignment of the ER (wild type) domain from SQTKS with other fungal ER domains.

SSS S S S S .

Fumonisin (1809) NFRDVLLAMGIVEANNLGIGLEGSGVITDVGAGV----TDLQVGDRVF Zearalenone (1688) NFRDVMASMALVPVK--GLGQEASGIVLRTGRDA----THLKPGDRVS Alternapyrone (1185) NFKDVLVALGNLAEN-K-LGVDASGIVTRVGSAV----TNVQVGDRVM

Squalestatin (1923) NFRDVMVAMGQLEES--IMGFEC-GVVRRVGPSS--AGHNIKVGDRVC

Asperfuranone (1853) NFRDVMVAMGQLKER-V-MGLECAGVITRVGAEA-A-AQGFAVGDRVM CSC CC . Fumonisin (1853) YLDDNCFSTRITMSAMRCAKIPSFLSYEEAATMPCVYATVIHSLVDIG Zearalenone (1706) TLDMGTHATVMRADHRVTVKIPDAMSFEEAAAVPVVHTTAYYALVRLA Alternapyrone (1927) TASCDTFATYVRFPAKGAIGVPTGMSFEEAASMPLIFLTAYYALVTAG Squalestatin (1966) ALLGGQWTNTVRVHWHSVAPIPQAMDWETAASIPIVFVTAYISLVKIA Asperfuranone (1897) ALLLGPFSSRARVSWHGVASMPAGMGFADAASIPMIFTTAYVALVQAA CCCCCCCCCC CCCC C . Fumonisin (1901) GLQSGQSVLIHSACGGIGIAAINVCQSIGGVQVYVTVGNQDKVRYLME Zearalenone (1758) KLQRGQSVLIHAAAGGVGQAALQLAN-HLGLVVYATVGSDDKRKLLTD Alternapyrone (1975) GIVAGEKVLIHAAAGGVGQAAIMIAQ-AKGAEIFATVGADTKKQLLIE Squalestatin (2015) RMQAGETVLIHAASGGVGQAAIILAK-HVGAEIFATVGTDEKRDLLIK Asperfuranone (1945) RLSQGQTVLIHAAAGGVGQAAVILAKEYLGAEVFATVGSQEKRDLLIK CC C S . Fumonisin (1954) TFNIPRASIFNSRDTSFREDVLAHTNGRGVDLVLNSLSGELLHASW-E Zearalenone (1810) TYQVSEDHIFNSRDASFAKGIMRVTGGRGVDCVLNSLSGELLRVSW-S Alternapyrone (2027) QYGIPEDHIFSSRDTSFVKGVLRATDGQGVDLVLNSLAGEALRLSWTD Squalestatin (2067) EYKIPDDHIFSSRNALFAKSIRQRTNGKGVDVVLNCLAGGLLQESF-D Asperfuranone (1998) EYGIPDDHIFNSRDSSFAPAALAATAGRGVDCLI---E CCSSS CSSS .

Fumonisin (2006) CVAPYGKMLEIGKRDFIGKAKLSMDIFEANRSFIGIDL---ARFDAAR Zearalenone (1862) CLATFGTFVEIGLRDITNNMLLDMRPFSKSTTFSFINMYTLFEEDPSA Alternapyrone (2080) CLAKFGRFLEIGKADLFANTGLDMKPLLDNKSYIGVNLLDFENNPTPR

Squalestatin (2119) CLADFGRFIEIGKRDIELNHCLNMGMFARSATFTAVDLIAIGRDRSYM

Asperfuranone (2038) VLAPFGHFVEIGKRDLEQNSLLEMATFTRAVSFTSLDMMTLLRQRGDE

S . Fumonisin (2056) CHPLLTRTVQMLEAGHIKPIAPRTTFSAGHIEDSFR Zearalenone (1915) LGDILEEVFKLLGGGILQTPSPMTVYPINQVEDAFR Alternapyrone (2133) AVALWHDTAKMIHDGAIKPIAPLQVFTMAEVEKAFR Squalestatin (2157) FAEALPKIMTLLQEKAIRPVTPISIYKIGDIETAFR Asperfuranone (2091) AHRVLSELARLAGQGIVKPVHPVSVYPMRQVDKAFR Legend:

. Identical within HR-PKS ER domains . Highly conserved within HR-PKS ER domains

. Mutagenesis resiude C = Cofactor binding; S = Substrate binding

In addition, in Scheme 20 the stereochemical course for the reduction of 79 catalyzed by the isolated SQTKS ER domain is shown (Chapter 1.11).58 Previous studies demonstrated that for the highly stereoselective transfer of the 4´-pro-R hydrogen of NADPH 11, the cofactor must be rigidly located in the active site. Furthermore, the transfer of the hydride to the 3-carbon of the substrate is also highly stereoselective, indicating that the substrate must take a single conformation relative to NADPH, which was in the previous studies

determined for the substrate as the s-cis conformation.58 Our calculations for the good substrate are similar to the results found in previous studies.58

Scheme 22: Stereochemical course of the reduction catalyzed by the SQTKS isolated ER domain.

Figure 24: Active site of the ER domain. Cofactor NADPH 11 (green) and the modeled substrate

squalestatin tetraketide (4S-2E-dimethylhex-2-enoylpantetheine) 85 (white). The hydride at the cofactor are marked. A, Back view; B, Front view.

Figure 25: Modeled substrate squalestatin tetraketide (6S,4S-2E-dimethyloct-2-enoylpantetheine) 85

(white). Shown in red the s-cis geometry of the α/β-unsaturated carbonyl.

In the model of the squalestatin tetraketide pantetheine 93a the catalytic nicotinamide moiety of the NADPH cofactor 11 is also located inside the ER-domain. NADPH 11 is in contact with its conserved binding site, consisting of highly conserved residues (see Sequence alignment of the ER, Table 2). The squalestatin tetraketide pantetheine 93a extends with the side-chain into the protein. The pantetheine part of the substrate extends parallel to the adenine diphosphate locating the thiolester and the β-carbon adjacent to the nicotinamide. The α/β-unsaturated carbonyl of the substrate adopts a different pose to the triketide 85. An s-trans conformation is observed (Figure 27), which places the reactive β-carbon 3.2 Å away from the cofactor's reactive 4´-pro-R hydrogen (Fig 26). The Burgi- Duniz angle substrate-cofactor complex is 100° and the dihedral angle at 72.3° is also higher than for the triketide 85 (table 2).

Figure 26: Active site of the ER domain. Cofactor NADPH 11 (green) and the modeled inhibitor squalestatin

Figure 27: Modeled inhibitor squalestatin tetraketide (6S,4S-2E-dimethyloct-2-enoylpantetheine) 93a

(white). Shown in red the s-trans geometry of the α/β-unsaturated carbonyl.

These parameters: distance of the C-2 position to the NADPH hydride; orientation of the substrate towards the NADPH; the geometry of the α/β-unsaturated carbonyl; the Burgi- Dunitz angle and the dihedral angle were reconsidered for the determination of whether the substrate is, or is not, held in a productive conformation. This is summarized for all three substrates in Table 3. Overall, a geometry of the α/β-unsaturated carbonyl in s-cis conformation and a dihedral angle with <65° are the best predictors for a good substrate.

Table 3: Summary of the validation parameters for the different model substrates.

C-H distance Si/Re- Face Geometry - α/β- unsaturated carbonyl Burgi- Dunitz angle Dihedral angle 3.5 Å Re- Face s-cis 46.6° 63.8° 3.4 Å Re- Face s-cis 61.7° 46.6° 4.7 Å Re- Face s-cis 151° 42.8° 3.2 Å Si-Face s-trans 100° 72.3°

3 In Silico Mutagenesis Studies of the SQTKS ER Domain

3.1 Introduction

The generation of the model of SQTKS ER was described in chapter 2. Validation of this model protein by various methods suggested that the binding of cofactor and substrates is chemically reasonable. We therefore considered that these models could form a valid basis for the design of further experiments with the aim of engineering the substrate selectivity of the ER domain of HR-PKS. There have been almost no reports of the successful rational engineering of HR-PKS, but based on the results from several in vitro studies of isolated HR-PKS domains, combined with the modelling described in the previous chapter, we considered that such experiments should now be possible.