Evaluation of fluoropyruvate as nucleophile in reactions catalysed by N-acetyl neuraminic acid lyase variants: scope, limitations and stereoselectivity

The stereochemical course of aldolase-catalysed reaction between fluoropyruvate and aldehydes is described.

cells were harvested by centrifugation (9 000 g, 20 mins) and the pellet was re-suspended in washing buffer using a homogeniser. The cells were lysed and the cell debris was collected by centrifugation (30 000 g, 45 mins). The supernatant was loaded onto chelating sepharose resin (pre-equilibrated with the wash buffer), in a 50 mL Falcon tube. The suspension was placed on a roller for 1 hr. The suspension was then centrifuged (4000 g, 5 mins, 4 °C) the supernatant was removed, washing buffer added (30 ml) and the suspension placed on a roller for 15 mins followed by centrifugation (4000 g, 5 mins). Contaminating proteins were removed by washing the resin a further 3 times with washing buffer (roll for 15 mins, followed by centrifugation at 4000 g for 5 mins). Elution buffer was added to the resin and placed on a roller for 1 hr. The suspension was centrifuged (4000 g, 5 mins) and the eluted His tagged NAL enzyme dissolved in the supernatant was decanted from the resin. The resin was then washed for a second time with elution buffer and rolled (15 mins) followed by centrifugation (4000 g, 5 mins) and the supernatant was collected. The eluted NAL was then dialysed (12 hr, 4 °C) Tris-HCl dialysis buffer. The following morning, the dialysis tubes containing eluted NAL were then transferred to fresh dialysis buffer (50 mM Tris/HCl, 50 mM NaCl, pH 7.5) and left to dialyse (4 hr, 4 °C). The dialysed solution was then sterile filtered into falcon tubes and stored at 4 °C. For longer-term storage, the NAL was dialysed into ammonium acetate buffer and freeze-dried. Freeze-dried protein was re-dissolved into a suitable buffer depending on the experiment required.

SDS Page
Protein purity was determined by SDS page. The composition of the running gel and stacking gel are given in Table S1. The ladder was provided by Fermentas and the gel was stained (Methanol (50% v/v), acetic acid (10% v/v), Coomassie Brilliant Blue (0.25% v/v) and Water (39.75% v/v)) and destained (Methanol (50% v/v), acetic acid (10% v/v), and Water (40% v/v)).

Concentration of NAL
Concentration of NAL solutions were carried out using 15 ml centrifuge filters (Regenerated cellulose 10 000 NMWL) purchased from Amicon Ultra -IS. Centrifuge filters were prepared by washing with water, followed by centrifugation (2187 g, 10 min) then three times with buffer (depending on which buffer the protein to be concentrated is dissolved into) followed by centrifugation (2187 g, 10 min). The protein solution was then transferred to the filter and centrifuged (2187 g) until the desired volume/concentration was achieve. 19

Monitoring reactions catalysed by NAL variants
For each experiment 0.1 mmol of alkene was cleaved by standard ozonolysis conditions.

Protein crystallisation and complex production
Wild-type saNAL crystals were produced using the following conditions: 100mM Tris/HCl (pH 7.0-8.5), 200 mM NaCl, polyethylene glycol (PEG) 3350 (16-28% wt/vol). Crystals were grown by hanging drop vapour diffusion and yielded crystals in 7-10 days. The enzyme-fluoropyruvate complex was produced by soaking the wild-type saNAL crystals in mother liquor supplemented with fluoropyruvate (100 mM) and 15% (v/v) PEG 400 for 1 min before being sequentially transferred to mother liquor with 5% increments in PEG 400 concentration. The final soak contained the mother liquor supplemented with fluoropyruvate (100 mM) and 25%(v/v) PEG 400. Crystals were then flash-cooled in liquid nitrogen prior to data collection.

Data collection and refinement
Data collection was carried out on beamline I04 at Diamond Light Source. The data set was collected from a single crystal at 100 K. Integration and scaling of data was carried out by XDS 2 and SCALA. 3,4 The structure of the wild-type saNAL enzyme in complex with fluoropyruvate was solved by molecular replacement in Phaser using the structure of wild-type saNAL (PDB ID:4AHP) as the search model. REFMAC5 5 was used for refinement of the data and after each refinement cycle model building was performed in COOT. 6 Coordinates and restraint library files for the lysine residue covalently bound to fluoropyruvate (HETcode: KPF) were generated using the PRODRG server and were manually edited. 7 The model was validated using the Molprobity server. 8 Values given in parentheses correspond to those in the outermost shell of the resolution range. c Rpim -precision indicating (multiplicity-weighted) Rmerge, relative to all I+ or I-. d R free was calculated with 5% of the reflections set aside randomly. e Based on the ideal geometry values of Engh & Huber (ref. 9). f Ramachandran analysis using the program MolProbity (ref. 10). The percentage of residues in the regions of the plot is indicated. The side-chain of Tyr111 is in close proximity to Leu142, Thr143 and Phe110 from an adjacent chain causing the phi and psi angles of Tyr111 to lie in an unfavoured region of the Ramachandran plot.

General method for ozonolysis
Unless otherwise stated, ozonolysis was carried out under the following conditions. The alkene was dissolved in methanol (0.1 mmol of alkene per 0.5 ml of MeOH), cooled to -78°C, purged with O 2 for at least 10 min and then exposed to ozone. Once a blue colour was observed, excess ozone was purged from the reaction with O 2 . Me 2 S (0.15 ml per 0.1 mmol of alkene) was added and the reaction was allowed to stir under nitrogen until all peroxides were quenched (starch-iodide paper).

Preparation of Specific Compounds
General procedure for enzymatic syntheses with wild-type NAL N-Acetyl-D-mannosamine (1.11 g, 5 mmol)

General method for enzymatic synthesis of fluorinated dipropylamides
The alkene was cleaved under standard ozonolysis conditions (see General method).
Methanol was removed in vacuo and the crude mixture was re-dissolved in 50 mM tris buffer (1.2 ml per 0.1 mmol aldehyde) to which sodium fluoropyruvate (0.5-1 eq.) was added. The pH was adjusted to 7.4 by addition of NaOH (2 M) followed by addition of NAL variant (in 50 mM tris buffer, 2-4 mg per 0.1 mmol aldehyde). The reaction was allowed to stir under nitrogen for 24 hr. The mixture was frozen, thawed and filtered through Celite® and the product was isolated by ion exchange chromatrography using Dowex® resin   Figure S2).  -190.5 -194.5 -201.9 -207.4