Structure-Function Relationship Of The Polygalacturonase PG8fn With High Specific Activity

Pectin, a negatively charged polysaccharide complex of high molecular weight, is mainly distributed in the middle lamella and primary cell walls of terrestrial plants, and accounts for one-third of the plant dry weight. It is composed of linear polymers consisting of covalently α-1,4-linked D-galacturonic acid residues, which can be methyl-esterified to varying degrees in a nonrandom fashion. Because of the complexity of pectin, its degradation is facilitated by a battery of pectinases, including polygalacturonase, pectate lyase, rhamnogalacturonase, pectin methylesterase, and pectin acetylesterase. Pectinolytic enzymes are widely used in the fruit juice, paper, and textile industries as well as the extraction of oils. One of the most studied and widely used commercial pectinases is polygalacturonase. To understand the catalytic mechanism, it is of importance to reveal the structure and function realtionships of polygalacturonases.A novel polygalacturonase encoding gene PG8 fn was cloned from thermophilic fungus Achaetomium sp. Xz8, overexpressed in Pichia pastoris, and characterized in the present study. Recombinant PG8 fn is distinguished from other enzyme counterparts by its high specific activity of 28,122 U/mg towards polygalacturonic acid, thus representing an aideal material for the study of molecular catalysis mechanism of polygalacturonase. This study intends to explore the structure and function relationship of PG8 fn. The enzyme was engineered for stability improvement without the sacrifice of activity by using a rational design approach. The roles of substrate binding site, second binding site and non-active site residue located on T3 loop were studied systematically and the crucial role of T3 loop in the catalytic process was revealed. Moreover, the information was used to guide the improvement of PG63.A structure-based rational design approach was used to improve the thermostability of PG8 fn via the optimization of charge-charge interactions. By using the enzyme thermal stability system(ETSS), two residues—Asp267 and Asp322—were inferred to be crucial contributors to thermostability. Single(D267A and D322R) and double(D267A/D322R) mutants were then generated, and their properties were compared with the wild type. All mutants showed improved thermal properties, in the order D267A/D322R>D322R>D267A. In comparison with PG8 fn, D267A/D322 R showed the most pronounced shifts in Tmax, T50 and Tm of 10 °C, 17 °C, and 10.2 °C upward, respectively, with the t1/2 extended by 8.4 h at 50 °C and 45 min at 55 °C. Another distinguishing characteristic of the D267A/D322 R mutant was its catalytic activity, which was comparable to that of the wild type(23,000 ± 130 U/mg versus 28,000 ± 293 U/mg). Molecular dynamics simulation studies indicated that mutations at sites Asp267 and Asp322 lowered the overall root mean square deviation(RMSD) and consequently increased the protein rigidity. This study reveals the importance of charge-charge interactions in protein conformation and provides a viable strategy for enhancing protein stability. The most prominent characteristic of this method is to retain the enzyme’s activity when enhancing enzyme stability.Structure analysis indicated that PG8 fn has a flexible T3 loop that folds partly above the substrate in the active site, and forms a hydrogen bond with substrate though the highly conserved residue Asn117. The catalytic roles of Asn117 was then determined. Molecular dynamics simulation performed on the mutant N117 A revealed the loss of the hydrogen bond formed by the hydroxyl group at O3 of GalpA at the subsite +1 and the crucial ND2 of Asn117 and the consequent detachment and rotation of the substrate away from the active site, and that on N117 Q caused the substrate to drift away from its place due to the longer side chain. In line with the simulations, site-directed mutagenesis at this site showed that this position is very sensitive to amino acid substitutions. Except for the altered Km values from 0.32(wild type PG8fn) to 0.75–4.74 mg/ml, all mutants displayed remarkably lowered kcat(~3–20,000 fold) and kcat/Km(~8–187,500 fold) values and significantly increased △(△G) values(5.92–33.47 kJ/mol). Taken together, Asn117 in the GH28 T3 loop has a critical role in positioning the substrate in a correct way close to the catalytic residues. The results revealed the important role of Asn117 on T3 loop in substrate binding to ensure efficient catalytic reactions.The amino acid sequence of PG8 fn shared the highest identity of 79.6% with the structure-resolved endo-polygalacturonase CluPG1 from Colletotrichum lupini(PDB:2IQ7), but showed one fold increase in specific activity. The amino acids in catalytic pockets are highly similar, with the exception of three residues, two of them were far from the catalytic center, located in N-terminal and C-terminal, respectively. The last one located on T3 loop is Lys121(PG8fn), corresponding to Thr121 in CluPG1. When Thr121 of CluPG1 was replaced by Lys, mutant T121 K showed significant increased specific activity(22,300 U/mg), which is consistent with that of PG8 fn. Thus, Lys121 was the key residue for PG8 fn with high specific activity. Site-saturation mutagenesis was also performed to replace Lys121 of PG8 fn with various amino acids to undermine the molecular mechanism. As results, all mutants except for K121 F and K121 W showed significantly altered Km and kcat values. Compared to wild-type PG8 fn, mutants K121 F and K121 W had decreased Km values and improved catalytic efficiency by 44% and 105%, respectively. Molecular dynamics simulation studies indicated that the residue substitution at position 121 changed the binding conformation of enzyme complex. Moreover, it interacted with GalpA at the subsite-4, thus belongs to the second binding site of polygalacturonase. This second binding site located on T3 loop of PG8 fn might account for its high specific activity distinguished from other counterparts.The effect of non-active site residue located on T3 loop on PG8 fn catalysis was explored after confirmed that T3 loop have played an improtant role in catalytic efficiency. Locating on T3 loop at the entrance of tunnel(T1), Thr113, was chosen for site-directed mutagenesis according to the distinct residue analysis and tunnel identification. The experimental results indicated that the catalytic efficiency of PG8 fn depends on the side-chain structure of residue at position 113, following the order of Arg and Lys(positively charged) > Ser, Gln and Thr(polar) > Gly, Ala and Ile(nonpolar) > Glu(negatively charged). Tunnel cluster analysis demonstrated that substitution with Arg caused increased occupancy rates of tunnels T2 and T3. Molecular dynamics simulations illustrated that residue substitution at position 113 enhanced the flexibility of T3 loop by beaking a salt bridge and influenced the interaction between enzyme and substrate. The functional role of position 113 was also verified in another GH28 polygalacturonase, PG63. The results revealed the non-active site residue located on T3 loop could affect the catalytic efficiency by changing the flexibility of T3 loop. This polar non-active site located on T3 loop might explain for the high specific activity of PG8 fn on the other hand.A rational design was used to improve the catalytic efficiency of PG63 based on the cation-π interactions on T3 loop of PG8 fn. By combining the sequence analysis, structure modeling, molecular dynamics simulations and occupancy rate calculation, eight mutagenesis sites having the potential to form cation-π interactions were identified in PG63. In comparison to the wild-type, mutant H58 Y that formed the double dentate mode of cation-π interaction located on T3 loop showed improved catalytic efficiency ~144 fold and the increased Tm of ~3.9 °C. Molecular dynamics simulations illustrated that mutant H55 Y contained the double dentate mode of cation-π interaction(Arg89-Trp90/Arg89-Tyr58) located on T3 loop increased the rigidity of the conserved His158 located on T3 loop 2, which further recruits the reducing ends of oligogalacturonic acids into the active site tunnel and initiates new hydrolysis reactions, to influence the catalytic efficiency by increased reaction rate and catalytic constant. The double dentate mode of cation-π interaction located on the T3 loop also contributed to the high specific activity of PG8 fn.In summary, this study has explored the structure-function relationship of highly active polygalacturonase PG8 fn. Firstly, the thermostability of PG8 fn was improved while the enzyme activity was retained at the same level via the optimization of charge-charge interactions, which provides a viable strategy for enhancing protein stability. Secondly, the effect of substrate binding site, second binding site, non-active site residue and cation-π interaction located on T3 loop on PG8 fn catalysis were analyzed systematically, the roles of active loop in enzyme catalysis reaction was revealed and the molecular mechanisms of PG8 fn with high specific activity was clarified. Therefore, this study casts new insight into the role of active loop in enzyme catalysis reaction, which has an important scientific significance for enzyme improvement.