19F NMR studies on γ-butyrobetaine hydroxylase provide mechanistic insights and suggest a dual inhibition mode† †Electronic supplementary information (ESI) available. See DOI: 10.1039/c9cc06466d

19F and 1H NMR studies on fluorine labelled γ-butyrobetaine hydroxylase provide mechanistic insight into substrate and ligand binding, suggesting cooperativity between two monomers.


Author Contributions
R.L.K. carried out all the synthetic aspects of the work and most of the assay and 19 F NMR work; he also carried out protein preparations. A.M.R. suggested and pioneered the 19 F labelling approach used, prepared wildtype and variant proteins, carried out thermal shift and MS studies, and guided NMR experimental and inhibitor design. A.K carried out NMR studies involving non-fluorinated protein leading to evidence for cooperativity. J.J.A.G.K. carried out kinetic assays, and inhibitor and binding studies by 1 H NMR. T.D.W.C. supervised NMR aspects of the work. All authors analysed data. C.J.S. supervised the project and with J.J.A.G.K and R.L.K. wrote drafts of the manuscript, which was ultimately approved by all authors.

Reagents for molecular biology
Materials and reagents were from New England Biolabs (NEB), Invitrogen, Bio-Rad, Roche, or Simga-Aldrich, unless otherwise stated. Water was initially purified using a Millipore Elix ® Reverse Osmosis system, then using a Millipore Milli-Q ® Synthesis system, with a 0.22 μm filter. IPTG and ampicillin solutions were sterilized before use with a 0.2 μm filter from Minisart ® , Sartorius Stedim Biotech.
The psBBOX DNA sequence used was: Red letters highlight site of mutagenesis.

Cell lysis and protein purification 1
A frozen cell pellet was placed in an ice cooled glass beaker equipped with a stirrer bar. The His column binding buffer was added to the cell pellet (5 × weight of frozen cells), followed by 1 mg of DNAse I and a protease inhibitor tablet (Roche). Stirring was continued until the cell pellets had thawed. The resultant suspension was subjected to sonication (10 seconds on 10 seconds off pulse sequences for a total of 10 minutes); using a Vibra Cell VCX 500 sonicator equipped with a 13 mm probe. The resultant suspension was centrifuged (14,000 rpm, 20 mins, 4 °C); the supernatant was then collected, and filtered using a 0.4 μm Omnipore™ filter (Millipore U.K.).

F labelling of psBBOX-Y201C
The reported procedure for BFA labelling was used.

Figure S11. 19 F NMR analysis of isoquinoline inhibitors shows sensitivity to differing ligand structures within a series.
Monitoring binding of FG2216 analogues to psBBOX* by 19 F NMR. Peak shapes obtained from individual titrations of isoquinoline-based inhibitors to psBBOX*-Zn (II) complex are displayed. (Only final 1 mM inhibitor results are displayed; apo-psBBOX* and psBBOX*-Zn (II) spectra are shown for reference.

Figure S12 Use of 19 F NMR to analyse binding of reported GBB substrate and product analogues to psBBOX*-Zn(II).
Resulting signals obtained from two separate titrations of Lcarnitine and D-carnitine to psBBOX*-Zn (II) followed by 2OG addition (A). Although Lcarnitine (but not D-carnitine) was previously determined not to be a substrate for wild-type psBBOX within the assay detection limits, 1 19 F NMR experiments with psBBOX* and L-or Dcarnitine, show that 2OG may bind to these complexes. Synthetic analogue P-GBB and GBBNF, are substrates for psBBOX, resulting in the formation of the phospha analogue and fluorinated analogue of L-carnitine respectfully. Their binding in presence of 2OG is observed (B and C).

Synthesis Materials and methods
All chemicals, reagents, and solvents were from Sigma-Aldrich (Dorset, UK) and used without purification. HPLC grade solvents were used for reactions, chromatography, and work-ups.
Aqueous solutions were made using de-ionized water.
The resulting mixture was stirred at room temperature for 3 hours until complete removal of both benzoyl and ethyl ester moieties was observed by LCMS. The reaction mixture was concentrated in vacuo, then lyophilised and purified by reverse phase flash column chromatography using a 50 g Biotage® SNAP Ultra C18 column (1:99 to 0:100 MeCN(+0.1% TFA) /H 2 O(+0.1% TFA). Fractions containing the product were assayed by TLC and LCMS before being collected and lyophilised to yield 6 a pale yellow oil (293 mg, 1.12 mmol, 74%).