The crystal structure of a hyperthermoactive exopolygalacturonase from Thermotoga maritima reveals a unique tetramer

Abstract

a b s t r a c tThe exopolygalacturonase from Thermotoga maritima is the most thermoactive and thermostable pectinase known to date. Here we present its crystal structure at 2.05 Å resolution. High structural homology around the active site allowed us to propose a model for substrate binding, explaining the exo-cleavage activity and specificity for non-methylated saturated galacturonate at the non-reducing end. Furthermore, the structure reveals unique features that contribute to the formation of stable tetramers in solution. Such an oligomerization has not been observed before for polygalacturonases.

Cloning, Expression and Characterization of a Novel Thermophilic Polygalacturonase from Caldicellulosiruptor bescii DSM 6725

“…The three groups are characterized by having different catalytic types (1st group: exo -PGases (EPG; EC 3.2.1.67); 2nd group: exo -PGases (EPGD; EC 3.2.1.82); 3rd: endo -PGases (PG; EC 3.2.1.15)). Our results suggest that ACM61449 and ACM60667 belong to the 1st group, and may exhibit EPG activity, similar to what has been reported for AAD35522 (PelB) [18] or ACL36472 (PecJKR01) [29]. …”

“…The higher thermo-stability of CbPelA that we characterized may be attributed to its low molecular mass (Table 1), which may decrease the solvent-exposed hydrophobic surface area [15]. Furthermore, the C -terminal may play a significant role in enzyme thermo-stability [18]. According to our sequence alignment results (Figure 2), CbPelA has an extended C -terminus.…”

“…Five additional substrate-binding sites (N179, H232, G233, R264, and K266, ACM61499 reference) were conserved in ACM61449 but not in ACM60667, where the residues His, Gly, Arg, and Lys were replaced by Ser, Ala, Ala, and Pro, respectively. Furthermore, the G238-S239 cis -peptide motif, which is believed to play an important role in the anchoring of the oriented galacturonate unit to the catalytic sites [18], was also conserved in ACM61449 but not in ACM60667. All these results suggested that ACM61449 may have catalytic behavior similar to that of ADD35522.…”

“…It is well known that thermophilic enzymes have certain advantages over mesophilic enzymes in that they have a greater ease of purification, higher thermo-stability, and greater chemical resistance, all of which may result in lower costs in industry applications [17]. A number of thermophilic organisms have been discovered in recent years, including a handful of thermophilic pectinases from thermophiles [18–26]. Polygalacturonase has been isolated from Thermotoga maritima , Bacillus sp.…”

“…One of the thermo-active EPGs that was identified was PelB cloned from T. maritime. Its optimal active and melting temperatures are 80 and 105 °C, respectively [18,27]. Another thermophilic pectinase, PecJKR01, was isolated from a soil metagenome sample.…”