Activity prediction of substrates in NADH-dependent carbonyl reductase by docking requires catalytic constraints and charge parameterization of catalytic zinc environment

Abstract

Molecular docking of substrates is more challenging compared to inhibitors as the reaction mechanism has to be considered. This becomes more pronounced for zinc-dependent enzymes since the coordination state of the catalytic zinc ion is of greater importance. In order to develop a predictive substrate docking protocol, we have performed molecular docking studies of diketone substrates using the catalytic state of carbonyl reductase 2 from Candida parapsilosis (CPCR2). Different docking protocols using two docking methods (AutoDock Vina and AutoDock4.2) with two different sets of atomic charges (AM1-BCC and HF-RESP) for catalytic zinc environment and substrates as well as two sets of vdW parameters for zinc ion were examined. We have selected the catalytic binding pose of each substrate by applying mechanism based distance criteria. To compare the performance of the docking protocols, the correlation plots for the binding energies of these catalytic poses were obtained against experimental Vmax values of the 11 diketone substrates for CPCR2. The best correlation of 0.73 was achieved with AutoDock4.2 while treating catalytic zinc ion in optimized non-bonded (NBopt) state with +1.01 charge on the zinc ion, compared to 0.36 in non-bonded (+2.00 charge on the zinc ion) state. These results indicate the importance of catalytic constraints and charge parameterization of catalytic zinc environment for the prediction of substrate activity in zinc-dependent enzymes by molecular docking. The developed predictive docking protocol described here is in principle generally applicable for the efficient in silico substrate spectra characterization of zinc-dependent ADH.

Inversion of cpADH5 Enantiopreference and Altered Chain Length Specificity for Methyl 3‐Hydroxyalkanoates

“…In our docking protocol, all the residues which are within 3 Å from the mutated positions were treated as flexible to take into account conformational changes introduced by the mutation, while the rest of the protein was treated as rigid. In case of the double variant L119M/W286S unreliable results were obtained with a rigid ADH cavity (receptor) as reported in our previous publication . The introduction of flexibility to some of the residue sidechains which are in the vicinity of mutated amino acids therefore is essential to further understand the effect of substitutions.…”

“…As an example, the W286S variant has a 0.28 kcal mol −1 increase in binding energy and in D3 distance to 4.07 Å. The distance D3 between the terminal carbonyl oxygen of Me‐( S )‐3‐OHC 4 ( 1 a ) and the side chain of catalytical S46 is shorter than 3 Å which explains the decrease of the activity of the W286S variant toward Me‐( S )‐3‐OHC 4 ( 1 a ). The increased size of the small binding pocket also reduces the oxidation activity toward Me‐( S )‐3‐OHC 4 ( 1 a ).…”

“…The catalytic zinc ion activates the hydroxyl group of the substrate Me3‐OHC 6 .Thus the distance between the catalytic zinc ion and the hydroxyl group of substrates (termed D1 in Figure. 3), which should be shorter than 2.5 Å, plays a crucial role in initiation of the catalytic activity . Figure shows that there is no interaction between the catalytic zinc ion and 3‐hydroxyl group of Me3‐OHC 6 ( 1 d ) due to a steric hindrance in the presence of W286 in WT (D1; 4.626 Å and binding energy; −5.25 kcal mol −1 ).…”

“…The cpADH5 WT had no activity toward Me3‐OHC 6 ( 1 d ) and the L119M variant had moderate activity toward Me3‐OHC 6 ( 1 d ) ( V max : 0.37 U mg − ). In order to improve cpADH5 activity toward Me3‐OHC 6 a mechanism‐based substrate docking study was performed as described in Experimental Section to identify amino acid positions for site saturation mutagenesis (SSM) . Based upon our previous experimentation with cpADH5, the substrate binding pocket is known to be composed of two parts (a large and a small binding pocket, Figure ).…”

“…Molecular docking was carried out by using the dock run.mcr macro in YASARA Structure Version 13.9.8 with the optimized atomic charges and Van der Waals parameters for zinc ions and coordinating residues . Docking of selected 3‐hydroxy fatty acid methyl ester substrates to cpADH5 WT and resulting variants have been performed in order to generate a first molecular understanding of the structure‐function relationships of cpADH5 ability to oxidize 3‐hydroxy fatty acid methyl ester.…”