Fig 1.
Detection of apiose in bacteria.
GC-MS analysis of alditol-acetate derivatives from methanolic, chloroform and cell pellet fractions of G. roseus (A) and X. pisi (B). Standard (Std) contains authentic xylose and apiose. The region of the total ion count for xylose (Xyl) and apiose (Api) alditol-acetate derivatives is expanded. (B) and (C) are MS fragmentation patterns for standard Xyl and Api, respectively. * indicates unidentified residue.
Fig 2.
Phylogenetic analysis of proteins involved in the synthesis of UDP-apiose (UAS) and UDP-xylose (UXS). Amino acid sequences used are the C-terminal region of Escherichia coli ArnA (WP_032205568.1) that forms UDP-4-keto-arabinose, Ralstonia solanacearum UDP-4-keto-pentose/UDP-xylose synthase (RsU4kpxs, WP_011001268.1), UXSs from bacteria (Sinorhizobium meliloti, ACY30251.1) mammal (human & Mus musculus, NP_079352.2 & NP_080706.1), fungi (Rhizopus microsporus, CEI96046.1) and plant (Arabidopsis UXS3; NP_001078768.1). The bacterial UAS-like sequences used are from Candidatus entotheonella, Geminicoccus roseus, Xanthomonas pisi and Yangia pacifica (ETX00953.1, WP_084506503.1, WP_084725965.1 and WP_066111466.1). Other UASs used are from green algae (Netrium digitus, AOG75413.1), from hornwort (Megaceros vincentianus, AOG75412.1), from liverwort (Marchantia paleacea, AOG75410.1) from moss (Physcomitrella patens, AOG75414.1), and from angiosperms (Arabidopsis thaliana & Zostera marina, KMZ68719.1 & NP_180353.1). Bacterial UAS are outlined by a red box. Alignment was made using Clustal Omega [24–26] and the tree generated using Dendroscope [20].
Fig 3.
Activity of recombinant bacterial UDP-apiose synthase (bUAS) by in microbe assay.
Analysis of in microbe nucleotide sugars by HILIC-LC-ESI-MS/MS. (A) top panel elution of standard (Std): UDP-GlcA, UDP-Xyl and UDP-arabinopyranose (UDP-Arap); Nucleotide sugars were extracted from E. coli cells induced to express genes encoding CeUAS, GrUAS, XpUAS or empty vector as the control (bottom panel). [M-H]- ions diagnostic for UDP-pentose (m/z 535.0, solid line), UDP-hexuronic acid (m/z 579.0, dashed line) and Park’s nucleotide (m/z 595.6, dotted line) are displayed. Park’s nucleotide is a UDP-MurNAc-pentapeptide that is used as an internal standard for nucleotide-sugar detection as it is abundantly made in E. coli. The m/z signal for CeUAS and XpUAS is amplified by a factor of 10. (B) Second stage MS fragmentation data for the peaks at the indicated retention times; Left column 11.0 min and right column 12.0 min. MS/MS ions at m/z 323.0, 211.0, 403.0 are consistent with predicted fragmentation of a UDP-sugar into [UMP-H]-, [Ura-2H]-, and [UDP-H2O-H]-, respectively.
Fig 4.
Activity of purified recombinant bUAS proteins.
(A) Nickel-purified proteins from E. coli cells induced to express CeUAS, GrUAS, XpUAS and empty vector control with expected sizes of CeUAS, GrUAS and XpUAS: 44.2, 42.9 and 45.1 kDa, respectively. (B) MS/MS for the m/z 535.0 peaks of UDP-Api (left column, elution time 11.0 min) and UDP-Xyl (right column, 12.0 min). MS/MS ions at m/z 323.0, 211.0, 403.0 are consistent with predicted fragmentation of a UDP-sugar into [UMP-H]-, [Ura-2H]-, and [UDP-H2O-H]-, respectively.
Fig 5.
1H NMR spectra of purified recombinant bUAS reactions.
Selected regions of 1H NMR spectra diagnostic for the products and intermediates generated by incubation of UDP-GlcA and NAD+ with the purified recombinant UASs from bacteria. Anomeric region between 5.50 and 5.75 ppm for the H1 protons of UDP-GlcA (G), UDP-Api (A) and UDP-Xyl (X) products and UDP-4-keto-Xyl (K) intermediate are shown. NMR region (5.95 and 6.15 ppm) diagnostic for UDP and NAD+ cofactor is included. NMR spectral traces from top to bottom show UAS activity of CeUAS, GrUAS, XpUAS and empty vector control. Peaks labeled N correspond to H5 and H6 protons of NAD+. ### indicates a mixture of ribose (R) and uracil (U) proton peaks of UDP from substrate and products. For additional chemical shift assignments see S1 Table.
Fig 6.
UAS reaction mechanism based on real time NMR analysis of recombinant GrUAS activity.
NMR spectra of the UDP-apiose synthase activity at 37 oC showing conversion of the substrate (UDP-GlcA), to intermediate (UDP-4-keto-Xyl), products (UDP-Api and UDP-Xyl ~ 2:1 ratio) and degradation product (apiofuranosyl-1,2-cylcic phosphate, Ac). The proton NMR spectrum of the sugar anomeric regions (H-1”s, between 5.5 and 6.1 ppm) of substrate, intermediate and products is shown. Only select time-resolved spectra are displayed to prevent overcrowding of peaks. ### indicates a mixture of ribose (R) and uracil (U) proton peaks of UDP from substrate and products. For additional chemical shift assignments see S1 Table.
Fig 7.
The effects of temperature and pH on the activity of recombinant GrUAS. (A) Maximum activity of GrUAS is at 37°C. (B) Maximum activity of GrUAS is in Tris-HCl at a pH of 8.1. (C) Size-exclusion chromatography suggests recombinant GrUAS is active as dimer. The relative activity (indicated by closed diamonds) was determined by HPLC. The molecular weight of the enzyme in solution is based on the relative elution times of standard protein markers (indicated by open circles).
Table 1.
Enzymatic properties of recombinant GrUAS.
Table 2.
Effect of nucleotide sugars and nucleotides on recombinant GrUAS activity.
Fig 8.
Detection of bUAS transcripts and UDP-apiose.
In vivo indication for the functional activity of bUAS genes and enzymes. (A) RT-PCR analyses showing bUAS transcripts of G. roseus and X. pisi. (B) HILIC-LC-ESI-MS/MS analysis of aq-methanolic (MeOH:chloroform:H2O; 40:40:20, v/v/v) extracts. Negative mode [M-H]- ions diagnostic for UDP-pentose (m/z 535.0, solid line; amplified by a factor of 10), UDP-hexuronic acid (m/z 579.0, dashed line) and Park’s nucleotide (m/z 595.6, dotted line) are displayed.