Structural Mechanism for the Temperature-Dependent Activation of the Hyperthermophilic Pf2001 Esterase

Abstract

Lipases and esterases constitute a group of enzymes that catalyze the hydrolysis or synthesis of ester bonds. A major biotechnological interest corresponds to thermophilic esterases, due to their intrinsic stability at high temperatures. The Pf2001 esterase from Pyrococcus furiosus reaches its optimal activity between 70°C and 80°C. The crystal structure of the Pf2001 esterase shows two different conformations: monomer and dimer. The structures reveal important rearrangements in the "cap" subdomain between monomer and dimer, by the formation of an extensive intertwined helical interface. Moreover, the dimer interface is essential for the formation of the hydrophobic channel for substrate selectivity, as confirmed by mutagenesis and kinetic analysis. We also provide evidence for dimer formation at high temperatures, a process that correlates with its enzymatic activation. Thus, we propose a temperature-dependent activation mechanism of the Pf2001 esterase via dimerization that is necessary for the substrate channel formation in the active-site cleft.

Toward the Complete Functional Characterization of a Minimal Bacterial Proteome

“…Of the aforementioned candidates, the predicted structure of gene /0520 aligns especially well to the AlphaFold2 predicted structure of the esterase/α–β hydrolase yqkD from B. subtilis ( 68 ) with over 96% of residues well-aligned, a RMSD of 3.00 Å, and a p -value for similarity greater than 1.00 × 10 –15 (see Figure 3 A and Table 2 ). Providing further support, structural alignment of the AlphaFold2 predicted protein structure for /0520 to a Pfam PF2001 α–β hydrolase from Pyrococcus furiosis (RCSB PDB: 5G59 ( 69 )), that also has esterase-like properties, shows that the hydrolase helical bundle is highly structurally conserved, which can been seen by Qres coloring of the STAMP structural alignment in VMD (see Figure 3 B, C). This catalytic site conservation is additionally corroborated by previous bioinformatic work by Zhang et al 19 via the COFACTOR analysis pipeline where peptidase ester or lipase cosubstrate binding sites are predicted at residues 96 and 166–168 especially.…”

“… 25 The alignment characterizes the structures as being significantly similar to more than 95% of the residues of /0520 being well-aligned to the B. subtilis protein predicted structure. (B) Structural alignment via STAMP of P. furiosis PF2001 hydrolase (yellow, RCSB PDB: 5G59 ( 69 )) to the predicted structures for /0520 from both AlphaFold2 (red) and RoseTTAFold (blue). (C) The alignment with Qres coloring 43 shows that the hydrolase helical bundle is highly conserved (blue-white-red color scheme of decreasing conservation), supporting the assignment of /0520 as an aminopeptidase/esterase/lipase, of the α–β hydrolase superfamily (see ref ( 66 ) and Pfam), and suggesting that PF2001 might play a similar role beyond its confirmed general esterase activity.…”

Structure and Dynamics of an Archeal Monoglyceride Lipase from Palaeococcus ferrophilus as Revealed by Crystallography and In Silico Analysis

“…Temperature was also reported to mediate the opening of Pyrococcus furiosus PF2001 esterase through dimerization and complete remodeling of cap subdomain [ 40 ]. Thus, we tested the PFL activity on a short and a long chain p -nitrophenyl derived substrate at low and high temperatures ( Figure 5 c).…”

Accelerated CO2 Hydration with Thermostable Sulfurihydrogenibium azorense Carbonic Anhydrase-Chitin Binding Domain Fusion Protein Immobilised on Chitin Support

“…For example, lipases undergo a structural shift in the presence of hydrophobic substrates, [29] whilst piezozymes undergo activation at high pressures [30]. Most recently a thermophilic esterase of Pyrococcus furiosus was found to be thermally activated through such changes of conformation resulting in heat-induced formation of dimers [31]. The exact structural causes of thermal activation of Saz_CA are yet to be elucidated and our preliminary data suggest that dimerisation is not a mechanism that is found in Saz_CA activation.…”