The unique salt bridge network in GlacPETase: a key to its stability

Abstract

The extensive accumulation of polyethylene terephthalate (PET) has become a critical environmental issue. PET hydrolases can break down PET into its building blocks. Recently, we identified a glacial PET hydrolase GlacPETase sharing less than 31% amino acid identity with any known PET hydrolases. In this study, the crystal structure of GlacPETase was determined at 1.8 Å resolution, revealing unique structural features including a distinctive N-terminal disulfide bond and a specific salt bridge network. Site-directed mutagenesis demonstrated that the disruption of the N-terminal disulfide bond did not reduce GlacPETase's thermostability or its catalytic activity on PET. However, mutations in the salt bridges resulted in changes in melting temperature ranging from -8°C to +2°C and the activity on PET ranging from 17.5% to 145.5% compared to the wild type. Molecular dynamics simulations revealed that these salt bridges stabilized the GlacPETase's structure by maintaining their surrounding structure. Phylogenetic analysis indicated that GlacPETase represented a distinct branch within PET hydrolases-like proteins, with the salt bridges and disulfide bonds in this branch being relatively conserved. This research contributed to the improvement of our comprehension of the structural mechanisms that dictate the thermostability of PET hydrolases, highlighting the diverse characteristics and adaptability observed within PET hydrolases.IMPORTANCEThe pervasive problem of polyethylene terephthalate (PET) pollution in various terrestrial and marine environments is widely acknowledged and continues to escalate. PET hydrolases, such as GlacPETase in this study, offered a solution for breaking down PET. Its unique origin and less than 31% identity with any known PET hydrolases have driven us to resolve its structure. Here, we report the correlation between its unique structure and biochemical properties, focusing on an N-terminal disulfide bond and specific salt bridges. Through site-directed mutagenesis experiments and molecular dynamics simulations, the roles of the N-terminal disulfide bond and salt bridges were elucidated in GlacPETase. This research enhanced our understanding of the role of salt bridges in the thermostability of PET hydrolases, providing a valuable reference for the future engineering of PET hydrolases.

Keywords: PET hydrolase; molecular dynamics; salt bridges; structure; thermostability.

Fig 1

Overall structure of GlacPETase. (A) The two polypeptide chains in an asymmertric unit of GlacPETase. Chain A is colored pink and Chain B green. (B) The alignment of Chain A and Chain B with a root mean square deviation (RMSD) for Cα of 0.215 Å. (C) The overall structure of GlacPETase is shown as a cartoon model. The catalytic triad (green, top) and disulfide bond (yellow, bottom) are shown as sticks. (D) The catalytic triad is composed of Ser147, His227, and Asp196. Ser147 serves as the nucleophile and is located 2.8 Å away from the base His227. The acid Asp196 is located 2.7 Å away from His227 to provide stabilization.

Fig 2

The N-terminal disulfide bond of GlacPETase and its functional validation. (A) The N-terminal disulfide bond of GlacPETase. The structure of the disulfide bond is depicted as a stick model and the omitted electron density map (gray mesh) of the residues involved in the disulfide bond is contoured at 2.0 σ. (B) The melting temperatures of GlacPETase and its mutants C38S and C58S. (C) The relative activities of GlacPETase and its mutants C38S and C58S. (D) The average root mean square fluctuation (RMSF) for Cα of residues 36–62 in the molecular dynamics simulation for WT and its mutants C38S and C58S. (E) Porcupine plot of residues 36–62 in the molecular dynamics simulation for WT and its mutants C38S and C58S. Cysteine involved in forming disulfide bonds, or serine after mutation, was represented using pink sticks. Regions with high fluctuations (high RMSF) were displayed in a thicker, teal color, while areas with low fluctuations (low RMSF) were shown in blue.

Fig 3

The salt bridge distribution of GlacPETase. (A) The nine pairs of salt bridges in GlacPETase. Each pair of salt bridges is marked with a different color. (B) The salt bridge connected by D196 and H227 was colored yellow with a distance of 2.7 Å. (C) The salt bridge connected by R238 and D229 was colored cyan with a distance of 3.2 Å. (D) The salt bridge connected by R271 and E275 was colored green with a distance of 3.9 Å. (E) The salt bridge connected by R127 and E124 was colored blue with a distance of 3.4 Å. (F) The salt bridge connected by K78 and E79 was colored gold with a distance of 2.9 Å. (G) The salt bridge connected by K115 and E111/D159 was colored magenta with a distance of 3.0 Å and 2.8 Å respectively. (H) The salt bridge connected by R53 and E86 was colored deep pink with a distance of 3.4 Å. (I) The salt bridge connected by R280 and E282 was colored light pink with a distance of 2.9 Å.

Fig 4

The melting temperature and PET hydrolysis activity of GlacPETase and its mutants associated with salt bridges. (A) The melting temperature of GlacPETase and its mutants. (B) The relative activities of GlacPETase and its mutants. The asterisks (* and **) indicate significant (P < 0.05 and P < 0.01, respectively) differences between salt bridge mutants and wild type.

Fig 5

Molecular dynamics simulations analysis of GlacPETase and salt bridge mutants. The average RMSF for Cα of residues surrounding each pair of salt bridges were marked with boxes and circles of the same color (A–G).

Fig 6

Phylogenetic analysis of PET hydrolase-like proteins. (A) The phylogenetic tree of 68 identified PET hydrolases and their homologs. The identified PET hydrolases were marked with red color and GlacPETase was marked with blue color. Boxed sequences were shown in panel B. (B) The distribution of residues involved in disulfide bonds and salt bridges in homologs within the same branch of GlacPETase. The disulfide bonds and salt bridges conserved strictly in homologs were colored with a gray background. The hosts of these homologs were also displayed.