About

Propsed FAD-dependent Oxidative Cyclization

1. Hydride transfer to FAD forms a tertiary carbocation that arranges the ring closure
2. Tyr484 abstracts a proton from CBGA to form an alkoxide nucleophile

• Mutations at Tyr484 abolish catalysis (catalytic base)
• Mutations at His114 and Cys176 abolish catalysis (covalent FAD bound residues)
• Mutations at Tyr417 and His292 greatly reduce K_cat (non-covalent FAD bound residues)

BRENDA: EC 1.21.3.7

DOI: 10.1007/978-3-319-54564-6_8

Active-site + FAD + substrate

TODO

Combined rendering of all minimization and equilibration runs descibed in exp.00/01-Assembly.ipynb. CBGA(-) (1.61 SwissDock pose 1.61) is shown at the entance of the active site (backbone H-bond in green, FAD as space-fill).

In [exp.01], a minimized CBGA(-) conformer was placed by hand in the active site and run through the same minimization steps in [exp.00]. More importantly, Tyr484 was replaced with a tyrosine phenolate (charge -1.0) in the QC runs only (the phenol was used in minimization runs).

A Tyr484 phenolate is proposed to be the catalytic base, but phenolate is very unstable, so I find it implausible that this species exists without interaction from neighboring side chains.

Short clip of MD run from [exp.01] before a segfault broke it.

Add model components

• O2
• NAG
• HOOH

Write system temp/energy during equilibration

• I'm unsure if the temp of the system in the amber sims

Fix nonstandard residues

• Curently all His residues are HIE (except FAD-bound His)

Fix FAD linkage residues

• Bound His114 is cuently parameterized as HID
  • Might actually be an OK approximation
  • Adding some params from 4-HNE might be best
    • Haven't checked for differences compared to HID
    • http://research.bmh.manchester.ac.uk/bryce/amber
      • FRCMOD
      • OFF
• I dont think Cys176 needs to be CYX
  • I think CYX just tells Amber to delete the proton
    • No noticeable problems with CYS so far

Fix protons

• Unsure of His114 protonation state
  • It curently exists as HID (neutral)
• Protonate His292
  • Replace with HIP
• Protonations should reflect:
  • pH 5.0
  • Expected H-bonds