Structure-based substrate screening for an enzyme
© Xu et al; licensee BioMed Central Ltd. 2009
Received: 19 September 2008
Accepted: 21 August 2009
Published: 21 August 2009
Nowadays, more and more novel enzymes can be easily found in the whole enzyme pool with the rapid development of genetic operation. However, experimental work for substrate screening of a new enzyme is laborious, time consuming and costly. On the other hand, many computational methods have been widely used in lead screening of drug design. Seeing that the ligand-target protein system in drug design and the substrate-enzyme system in enzyme applications share the similar molecular recognition mechanism, we aim to fulfill the goal of substrate screening by in silico means in the present study.
A computer-aided substrate screening (CASS) system which was based on the enzyme structure was designed and employed successfully to help screen substrates of Candida antarctica lipase B (CALB). In this system, restricted molecular docking which was derived from the mechanism of the enzyme was applied to predict the energetically favorable poses of substrate-enzyme complexes. Thereafter, substrate conformation, distance between the oxygen atom of the alcohol part of the ester (in some compounds, this oxygen atom was replaced by nitrogen atom of the amine part of acid amine or sulfur atom of the thioester) and the hydrogen atom of imidazole of His224, distance between the carbon atom of the carbonyl group of the compound and the oxygen atom of hydroxyl group of Ser105 were used sequentially as the criteria to screen the binding poses. 223 out of 233 compounds were identified correctly for the enzyme by this screening system. Such high accuracy guaranteed the feasibility and reliability of the CASS system.
The idea of computer-aided substrate screening is a creative combination of computational skills and enzymology. Although the case studied in this paper is tentative, high accuracy of the CASS system sheds light on the field of computer-aided substrate screening.
Enzyme catalyzes a wide variety of chemical reactions with great efficiency and specificity . Applications of enzymes in industrial catalysis continue to grow because of their considerable advantages . Although the classical approach of cultivating and characterizing isolates on the strain level prior to gene isolation is valid and powerful, it is severely restricted in scope . So capturing the genes of organisms that have evolved as participants in biotopes promises to revolutionize and broaden enzyme applications in the chemical industry . By the analysis of relationships among sequence, structure and activity , the function of newly obtained biocatalysts can be identified. However, broadening enzyme substrate specificity  is still a tough task most of the time.
On the other hand, computer-aided drug design (CADD) , especially different new protein inhibitors design [6–9], has been developed rapidly. Many theories and methodologies have been brought forward in this field [10–12]. More and more new drugs have been designed [13–16] by in silico methods. In view of the similar molecular recognition nature between ligand-target protein system and enzyme-substrate system , molecular docking [17, 18] which was used in CADD to find the binding pattern between ligand and target protein was applied to broaden enzyme mapping of substrates in this study.
Although molecular docking is efficient in predicting energetically favourable poses of ligand , it may be inappropriate for explaining the substrate-enzyme reactions sometimes. Because in some situations, substrates may adopt energetically unfavourable poses which can not be accounted by molecular docking to facilitate the catalytic reactions that are mediated by enzymes . It is especially true when the catalysis step is the actual rate-determining step. So we employed a third screening criterion in our designed screening system to address the problem (see "Distance 2 Check" section of the Method). Although the most accurate way of studying substrate-enzyme reactions is the quantum chemical (QM) level computation , its application in biomacromolecules is too costly to be achieved at present. So molecular docking, a rough but much less costly computational tool, was used to simulate the substrate binding step of the enzyme reaction process. Although the structure-based CASS system developed in the present study seemed simple and coarse, its screening accuracy was unexpectedly high. 223 out of 233 tested compounds were identified correctly for the enzyme by CASS. This suggests that biotechnologists can use the same computational means to reduce their mount of experimental work.
Results and Discussion
Generally speaking, experimental work should be carried out to inspect the accuracy of the in silico screening result. However, all the 233 compounds used in the present study had been fortunately reported by references (references were listed in Additional file 1). By comparing the virtual screening results with the reported experimental observations, we found an unexpected but inspiring result: all 190 compounds accepted by CASS system were confirmed to be catalyzed by the enzyme; 33 out of the 43 rejected compounds were verified as inappropriate substrate of the enzyme; in all, 223 out of the 233 tested compounds were identified correctly by the in silico screening system. Such high accuracy of the method (95.7%) guaranteed not only the feasibility but also the availability of the CASS system.
There were still 10 compounds which were predicted mistakenly by the CASS system. The error would probably come from molecular docking, because substrates may adopt energetically unfavourable poses which can not be accounted by molecular docking to facilitate the catalytic reactions.
In the present study, the idea of computer-aided enzyme substrate screening (CASS) was introduced, designed and applied successfully to CALB. 223 out of 233 compounds were identified correctly by this in silico screening system. Such high accuracy of the method guaranteed both the feasibility and the reliability of the CASS system. Although the idea of structure-based computer-aided substrate screening sounds wonderful, its application seems more difficult than lead screening in CADD because of three main operational difficulties: (1) how to determine the 3-D structure of enzyme; (2) how to define the screening criteria to ensure availability and accuracy; (3) how to apply the screening criteria to computer software. In this light, there is still a long way to go. However, conformational and geometrical checks which were used in this study suggest clues. Our further work will revolve around the application of the CASS system to a lipase which was discovered by our own group recently . We hope to broaden the substrate mapping of it with less experimental work, meanwhile we would also check again the method designed in this study.
Design of the computer-aided substrate screening system
Building the structures of the tested compounds
All 233 compounds which were used as tested compounds were built by Builder (InsightII, version 2000 release, Accelerys), and energy minimised by Discover (InsightII, version 2000 release, Accelerys) using the CVFF force field. Their coordinates were stored in Additional file 2.
Structure of enzyme and binding site
CALB was studied in the present study because it had been widely used in the academic world as well as in industry as an efficient biocatalyst for asymmetric transformation of sec-alcohols and related compounds  due to its high activity, stability and selectivity in both aqueous and organic solutions .
So far there were six crystal structures of CALB in PDB databank. And the ligand free enzyme (PDB code 1TCA) was used as the starting point in this study. The two N-acetyl-D-glucosamine (NAG) moieties in the structure were removed. Hydrogen atoms were added to the enzyme and water molecules. The catalytic histidine, His 224, was defined as protonated. Then an iterative series of energy minimizations were performed on the water hydrogen, enzyme hydrogen, and full water molecules. Finally, the whole system was energy minimized.
Docking engine – Affinity
Mechanism based restricted docking
X-ray diffraction of CALB indicated that its active site was made up of two pockets. One of them was for acyl part of the ester (acyl pocket) and the other for alcohol part (alcohol pocket) . It seemed that the size of the acyl pocket was larger than that of alcohol pocket . So we proposed a hypothetic conformational rule that the larger part of the substrate might bind into the larger binding pocket of the enzyme. And it may be used as the first screening criterion in CASS if it was proved correct.
Result of the MD simulation proved the correctness of the proposed conformational rule (see table s1 of Additional file 4), so it was used as the first screening criteria of the CASS system and named "Conformational Check".
Distance 1 Check
Once the substrate went into the binding site with correct conformation, more detailed criteria were needed to filter the substrate binding results. Two geometrically important distances were adopted in CASS. One was distance 1 which referred to distance between the oxygen atom of the alcohol part of the ester (if the substrate was an amine or thiol ester, the oxygen atom was replaced by nitrogen and sulfur atom) and the hydrogen atom of imidazole of His224 (figure 5). Distance 1 was considered as an important parameter to discriminate the enatioselectivity of secondary alcohols . A shorter distance may suggest greater affinity between enzyme and substrate. So compounds which passed the "Distance 1 Check" were identified directly as the substrates of the enzyme without any further check. Compounds which failed the "Distance 1 Check" would be further checked by "Distance 2 Check".
Affinity was an energy-driven docking engine. During the docking process in this study, values of vdW potential energy were always much larger than the values of electrostatic potential energy. So the values of atom vdW radius [33, 34] were used to determine the standard value of Distance 1. Distance 1 referred to distance between H atom and O (or N, S) atom in all the tested compounds. So 2.78 Å, the sum of 1.58 Å (the average vdW radius of the O, N, S) and 1.2 Å (the vdW radius of H), was used as the standard value of "Distance 1 Check".
Distance 2 Check
In some cases, Affinity may find no energy favorably binding conformations and would just give the energy unfavorably binding conformations. This was allowable in our substrate screening system. Because some compounds would take the energy unfavorable binding patterns to facilitate the reaction of compounds with biocatalyst. To address the problem, "Distance 2 Check" was adopted. It referred to distance between the carbon atom of the carbonyl group of the candidate compound and the oxygen atom of hydroxyl group of Ser105 (figure 5). And it was a subsidiary screening criterion of" Distance 1 Check" to guarantee the sensitivity and availability of the in silico system. A shorter distance was believed to better facilitate the nucleophilic attack of Ser105 to the carbonyl group of compounds. Only compounds which failed the "Distance 1 Check" could be further subjected to the "Distance 2 Check". 3.12 Å (sum of the atom vdW radius of C and O) was used as the standard value, because "Distance 2 Check" contains C and O atom in all compounds.
List of abbreviations
Candida antarctica lipase B
Computer-aided drug design
Computer-aided substrate screening
Van der Waals
N-hexylphosphonate ethyl ester
Root mean square deviation.
The research is founded by National Basic Research Program of China (973 Program) 2009CB724703.
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