Structural Characterization of Sphingomonas sp. KT-1 PahZ1-Catalyzed Biodegradation of Thermally Synthesized Poly(aspartic acid)

Abstract

Poly(aspartic acid) (PAA) is a green alternative to nonbiodegradable poly(carboxylates) and has applications in both industrial and biomedical settings. PAA is synthesized by heating monomeric aspartic acid to yield a polysuccinamide that can be ring-opened to yield thermal PAA composed of 30% α-amide and 70% β-amide linkages. Here, we report the first X-ray crystal structure of a PAA hydrolase from the bacteria Sphingomonas sp. KT-1 (PahZ1 KT-1 ) which functions to degrade synthetic PAA to oligo(aspartic acid) by selective cleavage of β-amide linkages. The structure was solved to 2.45 Å and shows a dimeric assembly where each monomer maintains an α/β hydrolase fold with a prominent, positively lined trough responsible for binding the anionic polymeric substrate. The putative catalytic sites of each monomer lie at the surface of the enzyme on opposite faces. The dimeric interface, as supported by small-angle X-ray scattering/multi-angle light scattering data, is primarily hydrophobic and is further stabilized by flanking hydrogen bonds. Molecular dynamics simulations support the previously determined specific cleavage of only the β-amide linkage through a conformational change that aligns the substrate with the active site Ser. These data provide a scaffold for further understanding the mechanism of PAA hydrolysis and opens the opportunity for using protein engineering to catalyze the biodegradation of other xenobiotics.

Sphingomonas sp. KT-1 PahZ2 Structure Reveals a Role for Conformational Dynamics in Peptide Bond Hydrolysis

“…The solvation free energy gain is estimated as Δ G i = −21.8 kcal mol –1 (−91.2 kJ mol –1 ), where the strongly negative value here indicates the presence of a hydrophobic interface. By comparison, estimates of overall dimer interface area and solvation free energy gain have been reported for the PahZ1 KT‑1 dimer as 626.2 Å 2 and −7.3 kcal mol –1 , respectively, consistent with reports of increased thermal stability for PahZ2 KT‑1 versus PahZ1 KT‑1 . , …”

“…To produce the substrate for PahZ2 KT‑1 , thermally synthesized poly­(aspartic acid), tPAA, was solubilized to 100 mg/mL in 50 mM HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.0. Subsequently, the tPAA was digested overnight at 37 °C with 0.5 mg/mL PahZ1 KT‑1 . The sample was then incubated at 70 °C to precipitate the PahZ1 KT‑1 , and the supernatant containing the digested tPAA (dPAA) served as the substrate for PahZ2 KT‑1 in all gel permeation chromatography (GPC) assays.…”

“…This serine protease was shown to have a dimeric assembly and displays a positively lined trough of residues that directs the polyanionic tPAA substrate toward the catalytic active site allowing for endolytic cleavage. 36 Here, we further build upon our mechanistic understanding of tPAA degradation with the crystal structure of PahZ2 KT-1 which was solved to 1.85 Å. PahZ2 KT-1 is also a dimer but is structurally related to M28 metallopeptidases, a family of dinuclear Zn(II)dependent exopeptidases. 37 PahZ2 KT-1 subunits are similar to two other well-characterized M28 family members, carboxypeptidase G2 and DapE, containing both a dimerization and a catalytic domain that harbors two Zn binding sites.…”

“…Recently, we determined the first structure of a poly­(aspartic acid) hydrolase, PahZ1 KT‑1 . This serine protease was shown to have a dimeric assembly and displays a positively lined trough of residues that directs the polyanionic tPAA substrate toward the catalytic active site allowing for endolytic cleavage . Here, we further build upon our mechanistic understanding of tPAA degradation with the crystal structure of PahZ2 KT‑1 which was solved to 1.85 Å. PahZ2 KT‑1 is also a dimer but is structurally related to M28 metallopeptidases, a family of dinuclear Zn­(II)-dependent exopeptidases .…”

“…Subsequently, the tPAA was digested overnight at 37 °C with 0.5 mg/mL PahZ1 KT-1 . 36 The sample was then incubated at 70 °C to precipitate the PahZ1 KT-1 , and the supernatant containing the digested tPAA (dPAA) served as the substrate for PahZ2 KT-1 in all gel permeation chromatography (GPC) assays.…”