<link>https://pages.htgaa.org/2026a/jessee-svoboda/projects/individual-final-project/phac_context_template/index.html</link><description>PhaC Enzyme Engineering — LLM Context Document Version: v1.0 Date: [DATE] Engineer: [YOUR NAME] Project goal: [ONE SENTENCE SUMMARY, e.g. “Engineer Class I PhaC to incorporate 3HHx at >15 mol%”] 1. Enzyme Family Background 1.1 Classification Class Subunit structure Size Native substrate preference Example organism I Single subunit ~65 kDa scl (C3–C5): 3HB, 3HV, 3HP Cupriavidus necator H16 II Single subunit ~60 kDa mcl (C6–C14): 3HHx, 3HO, 3HD Pseudomonas aeruginosa III Heterodimer (PhaC + PhaE) ~40+40 kDa scl Allochromatium vinosum IV Heterodimer (PhaC + PhaR) ~40+40 kDa scl Bacillus megaterium Class I and II share ~50% sequence identity; Class III/IV are more distantly related Class I/II are the primary engineering targets for substrate specificity work 1.2 Reaction chemistry Catalyzes polymerization of (R)-3-hydroxyacyl-CoA thioesters into PHA Ping-pong (double displacement) mechanism: Acylation: acyl group transferred to catalytic Cys, CoA released Transacylation: acyl group transferred to growing polymer chain Lipase-like α/β hydrolase fold Catalytic triad: Cys – His – Asp C. necator PhaC1 (Cn) reference numbering: C319, D480, H508 1.3 Substrate scope terminology Term Chain length Key monomers Notes scl C3–C5 3HP, 3HB, 3HV Most Class I enzymes mcl C6–C14 3HHx, 3HO, 3HD, 3HDD Most Class II enzymes lcl >C14 3HHxD+ Very rare Broad/mixed C3–C14 scl + mcl Rare, high engineering value Specialty varies 3H4MV, 3H2MB, aromatic Non-standard monomers 1.4 Why substrate specificity is structurally interesting Substrate-binding tunnel geometry determines acyl chain length tolerance Residues within ~5–10 Å of catalytic Cys are primary selectivity determinants mcl selectivity often results from removal of steric clash (smaller residues), not addition of new contacts — counterintuitive but well-supported Electrostatic environment affects CoA-thioester positioning Dimerization interface indirectly influences active site geometry (Class I/II) 2. Structural Information 2.1 Available experimental structures PDB ID Enzyme Class Resolution Notes 5T6O C. necator PhaC1 I [X] Å Primary Class I reference 4QO9 Chromobacterium sp. USM2 PhaC I [X] Å [ID] [Enzyme] [Class] [Res] [Notes] 2.2 AlphaFold models UniProt accession Organism Class pLDDT (overall) Confidence notes [ACCESSION] [ORG] [I/II] [score] [e.g. low in N-term, residues 1–40] 2.3 Key structural regions (Using C. necator PhaC1 residue numbering as reference)</description><generator>Hugo</generator><language>en</language><atom:link href="https://pages.htgaa.org/2026a/jessee-svoboda/projects/individual-final-project/phac_context_template/index.xml" rel="self" type="application/rss+xml"/></channel></rss>