In general, it was observed that amino acid polarity and structure of side chain had significant impact on enzyme activity. For example, Pro406Leu and Leu441Pro, where cyclic amino acids were being replaced by aliphatic amino acids, showed severe effect. While, mild to moderate effect was observed where nonpolar but neutral amino acids were replaced by polar but uncharged amino acids and vice versa. However, this classification is very weak and cannot be implemented on all cases. The structural findings are described as follows;
Structural Analysis
The 3D models of all reported PRODH mutations (Supplementary Fig. 1) were superimposed with 3D models of wild-type PRODH protein (Fig. 1). The manual comparison of these models observed remarkable structural differences, which were measured in the form of similarity indices.
Among all the models, highest similarity index of wild-type PRODH protein with mutant was shown by Arg185Gln (73.83%), while the lowest similarity index was shown by mutant Leu441Pro + Leu441Pro/ Arg453Cys and Thr466Met + Thr466Met/Arg453Cys, which was 41.17% (Fig. 2).Complete detail of similarity indices of all the models are summarized in Table 1.
Table 1
Nature of wild-type and Substituted amino acids in mutant PRODH protein.
Mutation | Nature of Mutation | Effect on enzyme activity | Similarity Index of mutant and normal protein | References |
Wild-type amino acid | Substituted Amino Acid |
Class | Polarity | Charge | Class | Polarity | Charge |
Pro8Leu | Cyclic | Nonpolar | Neutral | Aliphatic | Nonpolar | Neutral | Moderate | 69.33% | Jang et al. 2013 |
Arg11Pro | Basic | Basic polar | Positive | Cyclic | Nonpolar | Neutral | Mild | 72.83% | Guilmatre et al. 2010 |
Gln19Pro | Amide | Polar | Neutral | Cyclic | Nonpolar | Neutral | Moderate | 66.11% | Bender et al. 2005 |
Gln19Term | Amide | Polar | Neutral | Termination occurred | Severe | 6.25% | Raux et al. 2007 |
Pro30Ser | Cyclic | Nonpolar | Neutral | Hydroxylic | Polar | Neutral | Mild | 58.50% | Guilmatre et al. 2010 |
Ala58Thr | Aliphatic | Nonpolar | Neutral | Hydroxylic | Polar | Neutral | Moderate | 68.33% | Guilmatre et al. 2010 |
Ala167Val | Aliphatic | Nonpolar | Neutral | Aliphatic | Nonpolar | Neutral | Moderate | 69.17% | Bender et al. 2005 |
Arg185Gln | Basic | Basic polar | Positive | Amide | Polar | Neutral | Mild | 73.83% | Bender et al. 2005 |
Arg185Trp | Basic | Basic polar | Positive | Aromatic | Nonpolar | Neutral | Moderate | 61.33% | Bender et al. 2005 |
Thr275Asn | Hydroxylic | Polar | Neutral | Amide | Polar | Neutral | No detrimental effect | 58.00% | Guilmatre et al. 2010 |
Leu289Met | Aliphatic | Nonpolar | Neutral | Sulfuric | Nonpolar | Neutral | Mild | 43.17% | Bender et al. 2005 |
Pro406Leu | Cyclic | Nonpolar | Neutral | Aliphatic | Nonpolar | Neutral | Severe | 56.83% | Bender et al. 2005 |
Asp426Asn | Acid | Acidic polar | Negative | Amide | Polar | Neutral | Moderate | 52.00% | Bender et al. 2005 |
Val427Met | Aliphatic | Nonpolar | Neutral | Sulfuric | Nonpolar | Neutral | Moderate | 57.67% | Bender et al. 2005 |
Arg431His | Basic | polar | Positive | Aromatic | Basic polar | Positive, | Moderate | 66.33% | Bender et al. 2005 |
Leu441Pro | Aliphatic | Nonpolar | Neutral | Cyclic | Nonpolar | Neutral | Severe | 65.00% | Bender et al. 2005 |
Gly444Asp | Aliphatic | Nonpolar | Neutral | Acid | Acidic polar | Negative | Severe | 61.50% | Jang et al. 2013 |
Arg453Cys | Basic | Basic polar | Positive | Sulfuric | Nonpolar | Neutral | Severe | 68.00% | Bender et al. 2005 |
Ala455Ser | Aliphatic | Nonpolar | Neutral | Hydroxylic | Polar | Neutral | Mild | 57.83% | Bender et al. 2005 |
Thr466Met | Hydroxylic | Polar | Neutral | Sulfuric | Nonpolar | Neutral | Severe | 51.50% | Bender et al. 2005 |
Ala472Thr | Aliphatic | Nonpolar | Neutral | Hydroxylic | Polar | Neutral | Mild | 57.67% | Bender et al. 2005 |
Gln521Glu | Amide | Polar | Neutral | Acid | Acidic polar | Negative | Severe | 61.00 | Bender et al. 2005 |
Gln521Arg | Amide | Polar | Neutral | Basic | Basic polar | Positive | Enhance activity | 58.67% | Bender et al. 2005 |
Gln526Term | Amide | Polar | Neutral | Termination occurred | Unknown effect | 61.60% | Hu et al. 2014 |
Table 2
Number of bonds and nature of amino acids docked with substrate proline molecule in all reported PRODH protein mutations
Mutation | Number and nature of bonding | Position and nature of bonded amino acid |
| H bond | Alkyl bond | C-H bond | Unfavorable bonds | Polar | Non-Polar | Basic polar | Acidic Polar |
Wild-type | 2 | 1 | | | Tyr548 | Leu527 | Arg563 | |
Pro8Leu | 5 | 4 | 1 | | | Ala496,Val442,Leu527,Gly444 | His498,Arg443 | Glu567 |
Arg11Pro | 4 | 1 | | | | Gly229,Phe301,Trp300 | Arg224 | Asp228 |
Gln19Pro | 2 | 1 | | | | Leu150 | | Glu147,Glu556 |
Gln19Term | Unable to dock with proline molecule due to small truncated protein |
Pro30Ser | 4 | 2 | | | Gln123,Tyr144 | Gly552,Phe187 | Arg217 | Glu121 |
Ala58Thr | 1 | 1 | | | Thr411 | Ala381 | | |
Ala167Val | 3 | 4 | | | | Leu527,Val442,Ala496 | Arg564,His498 | Glu567,Asp178 |
Arg185Gln | 2 | 2 | | | Asn410 | Ala491,Val427 | | Asp426 |
Arg185Trp | 2 | 2 | | 1 | Ser569 | Val46,Pro49 | Arg324 | |
Thr275Asn | 3 | 1 | | 3 | Gln123,Ser210, Tyr144,Thr214 | | Arg217 | |
Leu289Met | 2 | 2 | 1 | | Gln533 | Ala186,Ile534 | Arg579 | Glu500 |
Pro406Leu | 1 | 1 | | | | Leu561 | | Glu60 |
Asp426Asn | 1 | 3 | 1 | | Asn568 | Ala52,Ala565,Phe113 | Arg63 | |
Val427Met | 2 | 3 | | | Tyr560,Tyr548 | Leu527 | Arg563 | Asp380 |
Arg431His | 2 | 1 | 2 | | Asn499,Asn594 | Leu595,Pro599,Gly574 | | |
Leu441Pro | 2 | | 2 | | Thr112 | Leu20,Phe17,Phe113 | | |
Gly444Asp | 2 | 1 | 2 | 1 | Tyr467 | | Arg563 | Asp444,Glu567 |
Arg453Cys | 5 | 1 | | | Tyr551,Gln123 | Pro553 | Arg217 | Glu121 |
Ala455Ser | 1 | 3 | 1 | | Tyr200 | Met555,Phe201,Trp254 | | Glu148 |
Thr466Met | 2 | 2 | | 2 | Asn499 | Val442 | His498,Arg443 | Glu567 |
Ala472Thr | 4 | 3 | | | Tyr446, Tyr548 | Leu527, Ala445 | Arg563,Lys234 | Asp380 |
Gln521Glu | 1 | 3 | | | Tyr144 | Ala237,Leu238,Ile233 | | |
Gln521Arg | 3 | 1 | 2 | | | Met555,Pro559 | Lys207 | Glu147,Glu154, Glu158 |
Gln526Term | 2 | | | | | | Arg217 | Asp122 |
Thr466Met + Thr466Met/Arg453Cys | | | | 1 | 580Gln | | | |
Leu441Pro + Leu441Pro/Arg453Cys | No protein substrate bonding were noted just Van der waal forces were noted |
Active site predication.
The wild-type PRODH protein's active site prediction revealed three major active sites. Among the three largest active sites, the second largest pocket was found to contain amino acids involved in substrate binding (proline). Leu527, Tyr548 and Arg563 are among the substrate interacting amino acids. The complete description of amino acids and its position, present in these three largest pockets, are summarized in supplementary table 1. The top three largest active site pockets of PRODH protein are illustrated in Fig. 3. It was also observed that residues in which substitution resulted in severe effect on activity of proline dehydrogenase enzyme were mostly present in the 2nd largest active site pocket. These residues include Leu441, Gly444, Arg453, Thr466 and Gln521.While, residue Pro406, exhibiting severe effect on enzyme activity, was present in the 1st largest active pocket of PRODH protein.
Protein-Protein Docking.
Protein-protein docking was carried out between wild-type and all the mutant PRODH protein with their close interactor ALDH4A1 protein and remarkable differences in the interacting sites of wild-type and mutant PRODH proteins were observed. Docking revealed that wild-type PRODH protein interacts with ALDH4A1 protein through 12 different residues i.e.Arg598, Arg451, Ala252, Arg225, Lys234, Ser26, Gln29, Trp254, Leu20, Ser248, Thr22 and Gln246 via 16 interactive forces (15 hydrogen bond and 1 unfavorable bond). However, among all the mutant PRODH proteins, Arg431His interacted with ALDH4A1 protein by 24 bonds (23 hydrogen and 1 unfavorable bond) via 19 different residues. While lowest interaction was shown by Thr466Met + Thr466Met/Arg453Cys protein, wherein the mutant protein interacted with ALDH4A1 through 5 bonds (4 hydrogen and 1 unfavorable bond) involving 4 different residues. Diagrammatic representations of all the protein-protein interaction between normal and mutant PRODH protein with close interactor are shown in Supplementary Fig. 3 (a,b,c).
Protein-Substrate docking.
To better understand the interaction mechanism of proline, a substrate, with the wild type as well as mutant proteins, protein-substrate dockings were also performed. Wild-type PRODH was interacting with proline molecule by 3 bonds (1 hydrogen and 2 alky bond) via three residues i.e. Arg563, Tyr548 and Leu527. Highest protein-substrate interaction was shown by Pro8Leu variant, wherein the mutant protein showed interaction with proline molecule by 10 bonds (5 hydrogen, 4 alky and 1 unfavorable bond) through 7 different residues. However, the lowest protein-substrate interaction was shown by Gln19Ter variant. Wherein the mutant Gln19Ter protein was unable to interact with the proline molecule due to short shortened structure. All the interacting residues of mutant PRODH proteins with proline molecule were different as compared to wild-type. 2D representations of all the protein-substrate interaction between wild-type and mutant PRODH protein with proline molecule are in shown in Fig. 4 & supplementary Fig. 2. Similarly, compound mutant i.e. Thr466Met + Thr466Met/Arg453Cys protein was interacting with proline molecule by only 1 bond (unfavorable Donor Donor Bond) through a single residue, and compound mutant Leu441Pro + Leu441Pro/Arg453Cys protein was unable to dock with proline molecule as shown in Fig. 2.