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.
Regulation of bacteriophage gene expression involves repressor proteins that bind and downregulate early lytic promoters. A large group of mycobacteriophages code for repressors that are unusual in also terminating transcription elongation at numerous binding sites (stoperators) distributed across the phage genome. Here we provide the X-ray crystal structure of a mycobacteriophage immunity repressor bound to DNA, which reveals the binding of a monomer to an asymmetric DNA sequence using two independent DNA binding domains. The structure is supported by small-angle X-ray scattering, DNA binding, molecular dynamics, and in vivo immunity assays. We propose a model for how dual DNA binding domains facilitate regulation of both transcription initiation and elongation, while enabling evolution of other superinfection immune specificities.
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