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.

Recent structural studies of the bacteriophage T7 DNA replication system have shed light on how multiple proteins assemble to copy two antiparallel DNA strands. In T7, acidic C-terminal tails of both the primase-helicase and single-stranded DNA binding protein bind to two basic patches on the DNA polymerase to aid in replisome assembly, processivity, and coordinated DNA synthesis. Although these electrostatic interactions are essential for DNA replication, the molecular details for how these tails bind the polymerase are unknown. We have determined an X-ray crystal structure of the T7 DNA polymerase bound to both a primer/template DNA and a peptide that mimics the C-terminal tail of the primase-helicase. The structure reveals that the essential C-terminal phenylalanine of the tail binds to a hydrophobic pocket that is surrounded by positive charge on the surface of the polymerase. We show that alterations of polymerase residues that engage the tail lead to defects in viral replication. In the structure, we also observe dTTP bound in the exonuclease active site and stacked against tryptophan 160. Using both primer/extension assays and high-throughput sequencing, we show how mutations in the exonuclease active site lead to defects in mismatch repair and an increase in the level of mutagenesis of the T7 genome. Finally, using small-angle X-ray scattering, we provide the first solution structures of a complex between the single-stranded DNA binding protein and the DNA polymerase and show how a single-stranded DNA binding protein dimer engages both one and two copies of DNA polymerase.

Poly(aspartic acid) (PAA) is a biodegradable synthetic polymer that is easily produced through heating aspartic acid followed by the subsequent addition of sodium hydroxide. Polymer applications range from drug delivery and biomimetics to hygiene products. Partial biodegradation of PAA is accomplished using poly(aspartic acid) hydrolase‐1 (PAAH‐1) which selectively cleaves a specific cross‐link. E. coli expressions of recombinant PAAH‐1 showed the production of significant amounts of protein which could be purified using a single Ni‐NTA column. Analysis of the PAAH‐1 sequence using the Phyre2 Protein Fold Recognition Server resulted in a 33% similarity to a “putative” poly(3‐hydroxybutyrate) depolymerase belonging to a family of enzymes that hydrolyze carboxylic ester bonds; however, no known structure of PAAH‐1 has been reported. Protein crystallization screening at the Hauptman‐Woodward Research Institute resulted in numerous protein crystallization hits being identified. Crystallization experiments were optimized in‐house and the structure was determined at 2.45Å using molecular replacement. The progress of PAAH‐1 characterization and its structure will be discussed.Support or Funding InformationNSF‐IUSE Grant: DUE 1611988This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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