Engineering PsNCA to shift regiospecificity from α-N to ε-N carbamoyl hydrolysis, delivered via MMP-14-responsive DOT...

IF a computationally-guided active-site variant of recombinant Pseudomonas sp. ON-4a N-carbamoyl-L-amino acid amidohydrolase (PsNCA-ε), engineered via structure-informed directed evolution to shift regiospecificity from the α-carbamoyl to the ε-carbamoyl position of lysine residues within peptide chains, is co-encapsulated at 200–500 nM with 100 μM MnCl₂ inside MMP-14-cleavable DOTAP cationic liposomes (incorporating a GPLGIAGQ peptide-lipid conjugate as a pericellular MMP-responsive release trigger) and applied for 72–96 hours to type I collagen-rich extracellular matrix deposited by primary dermal fibroblasts from aged C57BL/6J mice (18–24 months),

THEN a ≥25% reduction in homocitrulline (HCit) and hydroxyhomocitrulline (HHCit) per 1,000 collagen amino acids quantified by LC-MS/MS with isotope-labeled internal standards will be observed, accompanied by ≥2°C elevation in collagen denaturation temperature by differential scanning calorimetry and ≥15% increase in pyridinoline/deoxypyridinoline cross-link density by HPLC-fluorescence, relative to vehicle-treated aged controls,

BECAUSE the following causal chain links enzyme engineering, smart delivery, and collagen biochemistry:

1. Wild-type PsNCA is catalytically incompetent for the intended substrate. The active site of EC 3.5.1.87 from Pseudomonas sp. ON-4a is architecturally optimized to recognize the free α-carboxylate of small N-carbamoyl amino acids for substrate orientation; the enzyme neither recognizes the ε-carbamoyl moiety of lysine side chains nor tolerates the steric bulk of a peptide backbone flanking the target nitrogen (Feasibility Assessment, Section "Critical Feasibility: Substrate Specificity and Steric Constraints," citing Martinez-Rodriguez et al., J. Mol. Biol., 2002, and Ogawa et al., J. Biosci. Bioeng., 2000, as reported in the evidence set). Engineering a variant (PsNCA-ε) with an enlarged, electrostatically reconfigured substrate-binding pocket — modeled to eliminate the α-carboxylate anchoring residues and introduce contacts with the peptide backbone nitrogen flanking the ε-carbamoyl — is therefore a necessary prerequisite rather than an optional refinement.

2. HCit and HHCit accumulate irreversibly in aged type I collagen and functionally disrupt cross-linking. Carbamylation converts the positively charged ε-amino group of lysine and hydroxylysine into a neutral ureido group (homocitrulline/hydroxyhomocitrulline), competitively blocking lysyl oxidase-mediated enzymatic cross-linking, generating non-physiological cross-links, and increasing MMP resistance (Feasibility Assessment, Section "Target Pathology," citing Gorisse et al., Proc. Natl. Acad. Sci. USA, 2016, as reported in the evidence set). This damage accumulates linearly with chronological age in skin collagen and is causal — not merely correlative — to tissue mechanical aging, making it a repair target rather than a preventive one.

3. **Standard DOTAP liposomes fail as extracellular enzyme...

SENS category: GlycoSENS