A Synthetic Gene Library Yields a Previously Unknown Glycoside Phosphorylase That Degrades and Assembles Poly-β-1,3-GlcNAc, Completing the Suite of β-Linked GlcNAc Polysaccharides

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities. Utilizing an in silico sequence analysis of CAZy family GH94, we discovered and then functionally and structurally characterized the new GP β-1,3-N-acetylglucosaminide phosphorylase. This new GP was sourced from the genome of the cell-wall-less Mollicute bacterium, Acholeplasma laidlawii and was found to synthesize β-1,3-linked N-acetylglucosaminide linkages. The resulting poly-β-1,3-N-acetylglucosamine represents a new, previously undescribed biopolymer that completes the set of possible β-linked GlcNAc homopolysaccharides together with chitin (β-1,4) and PNAG (poly-β-1,6-N-acetylglucosamine). The new biopolymer was denoted acholetin, a combination of the genus Acholeplasma and the polysaccharide chitin, and the new GP was thus denoted acholetin phosphorylase (AchP). Use of the reverse phosphorolysis action of AchP provides an efficient method to enzymatically synthesize acholetin, which is a new biodegradable polymeric material.


AchP Kinetics Analysis
Kinetic parameters for AchP reverse phosphorolysis were determined using the phosphate release method described previously 10 . In brief, phosphate release was coupled to the formation of molybdenum blue, which can be quantified by measuring absorbance at 655 nm. Phosphate concentration was determined using a standard curve ranging between 0 and 10 mM phosphate. Kinetic parameters for AchP were determined with four donor and acceptor combinations: (A) Glc1-P and GlcNAc, (B) Glc1-P and GalNAc, (C) GlcNAc1-P and GlcNAc, and (D) GlcNAc1-P and GalNAc. Donor concentration was held constant at 10 mM while the concentrations of the acceptors were varied (as described below). Reactions were initiated by adding 5 µL of 0.25 mg/mL AchP to 10  Reactions were initiated by adding 0.5 µL AchP (6 mg/mL) then incubating at room temperature for 3 h for those containing Glc1-P and GalNAc1-P and 30 min for those with GlcNAc1-P. Reactions were stopped by diluting 50x with super DHB MALDI matrix (Fluka) then analyzed by MALDI-TOF MS (Bruker Autoflex).
Acholetin two-pot large scale synthesis GlcNAc1-P synthesis: In 60 mL, 1 g GlcNAc, 3 g ATP were dissolved in Tris, pH 7.0 buffer with MgCl 2 . NahK was added and the reaction was incubated at 37°C for 18 h (FIG 3A). Final reaction conditions: GlcNAc (75 mM), ATP (100 mM), MgCl 2 (10 mM), NahK (0.08 mg/mL) and 967 mM Tris, pH 7.0. ATP, ADP, AMP and free phosphate was precipitated by the addition of barium acetate. 6.7 mL of 1 M barium acetate was added, the slurry was centrifuged, then the supernatant was transferred to clean 50 mL Falcon tubes. This barium acetate precipitation process was repeated twice more (FIG 3A). GlcNAc1-P was precipitated from the supernatant by adding 267 mL of ethanol followed by centrifugation. The pellet was then washed with 333 mL acetone then dried under vacuum for 18 h (FIG 3A). Total mass of the dried pellet was 2.66 g, which is composed primarily of GlcNAc1-P (maximum theoretical yield was 1.36 g) and barium acetate.
Acholetin synthesis: In 10 mL, 2.66 g of the GlcNAc1-P/barium acetate product was dissolved in 10 mM HEPES, pH 7.0 buffer with 0.4 mM GlcNAc. The donor/acceptor ratio was set roughly to 1000:1 to maximize the degree of polymerization (DP) of the acholetin product. AchP was added to a final concentration of 0.6 mg/mL to initiate the reaction. After addition of AchP, the reaction began to turn cloudy as the phosphate released from reverse phosphorolysis formed an insoluble barium salt. The reaction was incubated at room temperature for 48 h (FIG 6). The precipitated barium phosphate salt was removed by centrifugation, the supernatant was then incubated at 70 °C for 5 minutes to precipitate the AchP, which was also removed by centrifugation. The product was desalted on either a Sephadex G-25 column with a bed height of 33 cm or a PD-10 Desalting Column (GE Healthcare). The 33 cm column was attached to the AKTA (same as above) and equilibrated with degassed water. 4.4 mL of the acholetin mixture was injected onto the column with a 5 mL/min flowrate and the fraction collector set to collect 2 mL fractions after 20 mL post injection (SUP FIG 7A). Fractions 4-13 were individually lyophilized. The combined mass was 151.5 mg which equates to a 344.3 mg yield from the whole 10 mL reaction. NMR analysis was performed on fraction 4, the fraction containing the highest proportion of high molecular weight acholetin oligomers (SUP FIG 7B). Fraction 4 was dissolved in D 2 O and subjected to 1 H, 13 C, H-H COSY, HSQC and HMBC NMR analyses. The same sample was also reduced by NaBH 4 (followed by quenching with acid, 4x treatment with MeOH and removal of solvent) and subjected to the same NMR analyses to identify the reducing end GlcNAc unit. A second desalting preparation was done by applying 1 mL of the acholetin mixture to a PD-10 Desalting Column according to the manufacturer's instructions with water used as the mobile phase. The desalted 3.5 mL elution fraction was lyophilized then resuspended in water to a final concentration of 50 mg/mL. This material was analyzed by Multi-Angle Light Scattering (MALS).
Crystallization, X-ray data collection and structure determination The AchP was screened against several crystallization screens: Crystal Screen, SaltRx, PEG/Ion, Index and PEGRx (Hampton Research), MCSG-1 (Anatrace) and Berkeley Screen 13 . Crystals of AchP were found in 0.05 M ammonium sulfate, 0.05 M Bis-TRIS-HCl pH 6.5 and 30 % (v/v) Pentaerythritol Ethoxylate (15/4_EO/OH). The crystals were obtained after 1 week by the sitting-drop vapordiffusion method with a mixture of 0.2 μL of protein solution and 0.2 μL of reservoir solution. Similar conditions were used for the D513A-AchP crystals and the soaking experiment was performed using a 20 mM GlcNAc-β1,3-GlcNAc solution diluted in crystallization solution for 30 minutes. Crystals of wild-type AchP, D513A-AchP/GlcNAc-β1,3-GlcNAc and AchP/2FGlc-α1-P were placed in a reservoir solution containing 20% (v/v) glycerol, then flash-cooled in liquid nitrogen. The X-ray crystallographic data sets for AchP, D513A-AchP/GlcNAc-β1,3-GlcNAc and AchP/2FGlc-α1-P were collected at the Berkeley Center for Structural Biology beamlines 5.0.2 and 8.2.2 at the Advanced Light Source at Lawrence Berkeley National Laboratory (LBNL). The diffraction data were processed using the program Xia2 14 .
The wild-type AchP crystal structure was determined using selenomethionine (Se-Met)-labeled protein by the single-wavelength anomalous diffraction (SAD) method 15 with phenix.autosol 16 and phenix.autobuild 17 programs within the Phenix suite72,73. The atomic coordinates obtained from the initial SAD data set were used as a search model for molecular replacement using Phaser 20 against a native AchP dataset and to initiate crystallographic refinement and model rebuilding. Structure refinement was performed using the phenix.refine program 19 and manual rebuilding was performed with COOT 21 . Root-mean-square deviations from ideal geometries for bond lengths, angles and dihedrals were calculated with Phenix 18 . The stereochemical quality of the final model of wild-type AchP was assessed by the program MOLPROBITY 22 . The final model of wild-type AchP was used as the initial model for the refinement of D513A-AchP/GlcNAc-β1,3-GlcNAc and AchP/2FGlc-α1-P. Summary of crystal parameters, data collection, and refinement statistics can be found in (SUP
Synthesis of Glc-β1,3-GlcNAc-pNP Glc1-P and GlcNAc were dissolved in buffer (HEPES, pH 7.0), AchP was added and the reaction mixture was incubated at 37 °C. Final reaction conditions: 15 mg/mL Glc1-P and 40 mg/mL GlcNAc, 0.05 mg/mL AchP, 50 mM HEPES. Reaction progress was monitored by TLC (BuOH:MeOH:NH 4 OH:H 2 O 5:4:4:2, p-anisaldehyde staining). After Glc1-P was fully consumed, the enzyme was removed from the reaction mixture by ultrafiltration (MWCO 10 kDa, Sartorius) and product was isolated by gel filtration chromatography (eluent: H 2 O; Bio-gel P2, BioRad). The fraction containing pure product was lyophilized and characterized by MALDI-TOF MS (Bruker Autoflex) and NMR (Bruker AV-400 MHz spectrometer; solvent: D 2 O). Supporting Figure 1: E. coli cell lysate AchP donor and acceptor specificity screen. Cell lysate from E. coli expressing AchP was combined with the indicated donor and acceptor combinations. Activity was monitored by coupling phosphate release from reverse phosphorolysis to the formation of molybdenum blue, which absorbs strongly at 655 nm. Grey indicates corresponding donor/acceptor combination not tested. and Σ i is the sum over all equivalent and symmetry-related reflections. b R factor = Σ |Fobs -Fcalc|/ Σ Fobs. c R free = R factor for 5 % of the data were not included during crystallographic refinement.