Functional Characterization of UDP-apiose Synthases from Bryophytes and Green Algae Provides Insight into the Appearance of Apiose-containing Glycans during Plant Evolution

Abstract

Apiose is a branched monosaccharide that is present in the cell wall pectic polysaccharides rhamnogalacturonan II and apiogalacturonan and in numerous plant secondary metabolites. These apiose-containing glycans are synthesized using UDPapiose as the donor. UDP-apiose (UDP-Api) together with UDPxylose is formed from UDP-glucuronic acid (UDP-GlcA) by UDP-Api synthase (UAS). It was hypothesized that the ability to form Api distinguishes vascular plants from the avascular plants and green algae. UAS from several dicotyledonous plants has been characterized; however, it is not known if avascular plants or green algae produce this enzyme. Here we report the identification and functional characterization of UAS homologs from avascular plants (mosses, liverwort, and hornwort), from streptophyte green algae, and from a monocot (duckweed). The recombinant UAS homologs all form UDP-Api from UDP-glucuronic acid albeit in different amounts. Apiose was detected in aqueous methanolic extracts of these plants. Apiose was detected in duckweed cell walls but not in the walls of the avascular plants and algae. Overexpressing duckweed UAS in the moss Physcomitrella patens led to an increase in the amounts of aqueous methanol-acetonitrile-soluble apiose but did not result in discernible amounts of cell wall-associated apiose. Thus, bryophytes and algae likely lack the glycosyltransferase machinery required to synthesize apiose-containing cell wall glycans. Nevertheless, these plants may have the ability to form apiosylated secondary metabolites. Our data are the first to provide evidence that the ability to form apiose existed prior to the appearance of rhamnogalacturonan II and apiogalacturonan and provide new insights into the evolution of apiose-containing glycans.

Synthesis of UDP-apiose in Bacteria: The marine phototroph Geminicoccus roseus and the plant pathogen Xanthomonas pisi

“…Real time 1 H NMR spectroscopic analysis of the products formed when GrUAS reacts with UDP-GlcA ( Fig 6 and S1 Table ) confirmed that UDP-Api is the first product formed. GrUAS produces UDP-Api and UDP-Xyl in a ratio of ~1.7: 1.0, which is similar to characterized plant UASs [ 29 – 31 ]. The NMR study with GrUAS also confirmed that some of the UDP-Api is degraded and converted to the apiofuranosyl-1,2-cyclic phosphate during the in vitro reaction ( Fig 6 ); this instability of UDP-apiose is a known phenomenon [ 29 , 31 , 32 ].…”

“…The recombinant GrUAS is most active in 50 mM Tris-HCl, pH 8.0–8.5, at 37°C ( Fig 7A and 7B ) and exists in solution as a dimer with a predicted size of 84 kDa ( Fig 7C ). Table 1 shows that GrUAS has a Km of 251 μM similar to that for Spirodela UAS [ 31 ], and a Kcat/ Km of 60.2 nM s -1 , while recombinant Arabidopsis AXS/UAS1 has a reported Km of 7 μM and Kcat/ Km of 43 nM s -1 [ 30 ]. Previous studies have shown that UAS is inhibited by certain nucleotides and nucleotide sugars, especially UDP-Xyl and UDP-GalA [ 30 , 31 ].…”

“…Table 1 shows that GrUAS has a Km of 251 μM similar to that for Spirodela UAS [ 31 ], and a Kcat/ Km of 60.2 nM s -1 , while recombinant Arabidopsis AXS/UAS1 has a reported Km of 7 μM and Kcat/ Km of 43 nM s -1 [ 30 ]. Previous studies have shown that UAS is inhibited by certain nucleotides and nucleotide sugars, especially UDP-Xyl and UDP-GalA [ 30 , 31 ]. Table 2 demonstrates that under our assay conditions UDP-Xyl and UDP-GalA inhibited GrUAS activity by 9% and 77%, respectively.…”

“…A fraction (1 μl) of each of the alditol-acetate derivative samples was analyzed by gas-liquid chromatography (GLC; 7890A, Agilent, Santa Clara, CA, USA) equipped with a mass selective detector (EI-MS, Agilent 5975C) and separated over a RTx-2330 fused silica column (Restek, Bellefonte, PA, USA) as previously described [ 31 ]. Alditol-acetate derivatives of standard apiose, rhamnose, fucose, ribose, arabinose, xylose, mannose, glucose, and galactose (50 μg each) were prepared under the same conditions as samples.…”

“…Nucleotide sugars from E . coli harboring the expression plasmids were harvested as described [ 31 , 36 ]. BL21-derived E .…”