Fig 1.
A. A proposed pathway for the formation of UDP-QuiNAc in Bacillus cereus ATCC 14579. The enzyme encoded by Bc3750, UDP-GlcNAc C4,6-dehydratase (Abbr. Pdeg), converts UDP-GlcNAc to UDP-4-keto-6-deoxy-GlcNAc. At steady state, the UDP-4-keto-sugar form (K) is converted non-enzymatically to a hydrated form W. The enzyme encoded by Bc3749 (Abbr. Preq) is a UDP-4-keto-sugar C4”-reductase and UDP-D-QuiNAc. B. Organization of the two-genes operon and flanking regions in B. cereus ATCC 14579.
Table 1.
Comparative analysis of Bacillus cereus ATCC 14579 proteins Pdeg (Bc3750) and Preq (Bc3749) with known genes encoding UDP-sugar 4,6-dehydratase (A) and 4-reductase (B) from bacterial sp.
Table 2.
NMR data for the sugar moiety of UDP-4-keto-6-deoxy-GlcNAc (W) and UDP-QuiNAc (Q).
Fig 2.
SDS—PAGE analysis of the B. cereus ATCC 14579 purified recombinant Bc3750 (Pdeg) and Bc3749 (Preq) proteins involved in the biosynthesis of UDP-QuiNAc.
Protein standards are shown on the right in kDa. The final elution fraction (E7) of purified recombinant proteins from affinity column is shown for Bc3749 (lane 1) and Bc3750 (lane 2).
Fig 3.
Analysis of recombinant enzyme reaction Pdeg by UV-HPLC and LC-ESI-MS-MS.
A. UDP-GlcNAc standard reaction is shown. B. UDP-GlcNAc C4,6-dehydratase reaction, is the conversion of UDP-GlcNAc to UDP-4-keto-6-deoxy-HexNAc. The broad peak (K and W) denotes 4-keto and 4-hydrated-keto form of the UDP-4-keto-6-deoxy-sugar. Boxed top panel shows the product ions, K and W, m/z 587.99 and 605.99, respectively. MS-MS analysis of parent ions K and W gave fragment ions at m/z 402.9, 384.9 and 304.9 that are consistent with [UDP-H]-, [UDP-H2O-H]-, and [UMP-H2O-H]-, respectively. (Boxed the second and third panel) C. Pdeg negative control reaction carried out with unrelated protein.
Fig 4.
Analysis of Pdeg recombinant enzyme reaction products by 1H-NMR indicates formation of hydrated-UDP-4-keto-6-deoxyl-D-GlcNAc.
The product peak of the Pdeg reaction was collected and analyzed at 600 MHz NMR. Full proton spectrum of the Pdeg product hydrated-UDP-4-keto-6-deoxy-D-GlcNAc. Expanded proton spectra between 3.8 and 4.4 ppm that shows the sugar ring. The short line above NMR ‘peaks’ denotes specific chemical shifts belonging to a UDP-4-keto-6-deoxy-D-GlcNAc. Symbol(#) denotes column contamination and symbol (*) denotes DSS.
Fig 5.
Time-resolved 1H-NMR analysis of Pdeg reaction showing the conversion of UDP-GlcNAc to hydrated-4-keto-UDP-suagr.
Spectra were collected for the first 120 min of the reaction that was conducted at 25°C and included Pdeg and 1 mM of UDP-GlcNAc (time 0). Four selected regions of the UDP-4-keto-6-deoxy-GlcNAc formed over time, can be observed with a diagnostic UH-6 (A), anomeric proton (B), acetyl methyl proton (C), and 6-deoxy proton H6’ (D). Proton signals of UDP-GlcNAc and product, UDP-4-keto-6-deoxy-HexNAc, are labeled as GUH-6, GH-1”, GNAc-H” for the substrate UDP-GlcNAc, WUH-6, WH-1”, WNAc-H” and WH-6” for the hydrated form, respectively.
Fig 6.
Analysis of Preq recombinant enzyme reaction by UV-HPLC and LC-ESI-MS-MS.
A. Pdeg reaction shows the conversion of UDP-GlcNAc to UDP-4-keto-6-deoxy-HexNAc before recombinant Preq addition. B. Following Pdeg reaction, purified Preq was added and reacted with UDP-4-keto-6-deoxy-GlcNAc in the presence of NADPH to give product Q, UDP-QuiNAc, with m/z 590 and ms/ms of 402.94, 384.93, and 304.98 (Boxed top and second panel) C. Preq negative control reaction carried out with unrelated protein.
Fig 7.
Analysis of Preq reaction product by 1H-NMR indicates formation of UDP-QuiNAc.
The product of the Preq reaction (peak Q, in Fig 6 Panel B) was collected and analyzed at 600 MHz NMR. Full proton spectrum of HPLC-collected product UDP-QuiNAc. Expanded proton spectra between 3.2 and 4.4 ppm that shows the QuiNAc sugar ring. The short line above NMR ‘peaks’ denotes specific chemical shifts belonging to a UDP-QuiNAc.
Fig 8.
TOCSY spectrum of UDP-QuiNAc shows the coupling among QuiNAc sugar ring protons.
Fig 9.
Time-resolved 1H-NMR analysis of Preq reaction showing the conversion of hydrated-UDP-4-keto-6-deoxy-GlcNAc to UDP-QuiNAc.
Spectra were collected for the first 60 min of the reaction that was conducted at 25°C and included Preq and 1 mM NADPH after Pdeg reaction. Four selected regions of the UDP-QuiNAc formed over time, can be observed with a diagnostic anomeric proton QH-1” (A), 6-deoxy proton QH-6” (B), appearance of QH-4”. Proton signals of UDP-QuiNAc are labeled as QH-1”, QH-6”, and QH-4” and disappearance of WH-2” and WH-5” (D). Note that the chemical shift of the anomeric proton of UDP-QuiNAc (QH-1”) is very close to the proton of UDP-4-keto-6-deoxy-GlcNAc (slightly shifted). WH-1” is the hydrated form of UDP-4-keto-6-deoxy-GlcNAC.
Table 3.
Enzymatic properties of recombinant Bc3750-His6 (Pdeg) and Bc3749-His6 (Preq).