Biology subjects

Capra, N.

Publications and source records attributed to Capra, N..

3 recordsLinked to original sources

bioRxiv2026

Structural and Oligomeric Characterization of Substrate- and Product-selective Nylon Hydrolases

Enzymatic degradation of synthetic polymers has attracted broad interest because it offers environmental and manufacturing advantages compared to traditional mechanical and chemical breakdown approaches. Enzymes are highly specific and reaction conditions are generally aqueous and require low pressure and temperature, resulting in lower energy consumption and lower chemical waste production. Here we report the biochemical and structural characterization of three newly discovered enzymes capable of nylon hydrolysis: Nyl10, Nyl12 and Nyl50. Using solution characterization techniques, we found that the enzymes adopt a single oligomeric state consistent with a tetramer over a wide range of concentrations. X-ray crystallographic structures of all three enzymes support the association into tetramers. Comparison of ligand-bound X-ray crystal structures of Nyl10 and Nyl12 with the previously determined structure of Nyl50 identified key structural determinants involved in ligand binding. Noticeably, a flexible loop found in several polyamide degrading enzymes is observed to flip towards (closed conformation) and away (open conformation) from the active site upon ligand binding. Analysis of adduct and surrogate substrate-bound enzyme complex structures provide a model for substrate binding directionality. Finally, activity assays showed that both Nyl10 and Nyl12 can hydrolyze ester bonds, and that Nyl12 has the highest activity toward PA66, identifying it as the best candidate for protein engineering for efficient nylon hydrolysis.

biochemistry↗

bioRxiv2025

Regioselective biosynthesis of oligoamides as precursors for sequence-controlled co-polyamides

Polyamides are important natural and synthetic polymers, best exemplified by proteins and nylons respectively. Proteins demonstrate that novel polymers with emergent properties can be generated by combining diverse monomers in precisely defined sequences. However, commercial polyamides represent only a small fraction of the potential diversity in polyamide sequences, due to the synthetic challenges of sequence-controlled polymerization. Amide synthetases have been shown to synthesize a broad array of nylon-relevant diads, but the generation of novel sequenced copolyamides requires enzymes capable of acting with longer and more diverse substrates. In this study, we demonstrated that NRPS-independent siderophore (NIS) synthetases, represented by DesD, can ligate oligomeric substrates. A simultaneous enzyme cascade using DesD enabled the synthesis of an oligotriad directly from unprotected substrates. Moreover, the regioselectivity of DesD allowed the selective synthesis of a sequenced amide tetrad, the precursor to a novel sequenced-defined polyamide with properties superior to nylon 66. This study establishes a direct biocatalytic route for the facile synthesis of sequenced oligoamides from unprotected bifunctional substrates, opening new possibilities to synthesize sequenced copolyamides.

biochemistry↗

bioRxiv2024

Identification and characterization of substrate- and product-selective nylon hydrolases

Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases. SignificanceNylons are common industrial polyamides with few recycling options. As an alternative to mechanical or chemical recycling, enzymes may provide a selective and energy-efficient route to deconstruct nylons from mixed waste. Several nylon hydrolases have been identified, most notably NylC, but these enzymes are all closely related and demonstrated similar activity and substrate range. In this work, we investigated a diverse set of enzymes and showed that nylon hydrolase activity is common, providing new insights into the evolution of microbial nylon hydrolysis. Unlike NylC, several enzymes demonstrated unprecedented substrate selectivity, preferentially hydrolyzing Nylon 6,6 compared to Nylon 6. These enzymes can be used to understand substrate selectivity in nylon hydrolysis and to engineer enzymes for nylon recycling from mixed waste.

biochemistry↗