Background: GH3 -N-acetylglucosaminidases atypically employ a His-Asp dyad as a catalytic acid base. Results: Enzymes from this GH3 subfamily are phosphorylases rather than hydrolases. Conclusion: Replacement of the Glu acid/base by His avoids Coulombic repulsion with phosphate. Significance: These are the first anomeric stereochemistry-retaining -glycoside phosphorylases to be found.
Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-D-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenol from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to Nacetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. GH3 β-glycoside phosphorylase from a metagenomic library 2 Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.Carbohydrate active enzymes (CAZymes) are the biocatalysts responsible for the assembly, degradation and modification of glycans in biological systems 1 . They are also widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel and bioproduct development [2][3][4][5] . While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive starting materials, such as nucleotide phosphosugars 6 . The high-cost of these materials makes de novo industrial-scale glycan synthesis difficult and usually non-viable.One class of CAZyme that offers a potential solution to the high costs typically associated with enzymatic glycan synthesis is that of the glycoside phosphorylases (GPs), which are increasingly being recognized and used for the biocatalysis and biotransformation of glycans 7-9 . These enzymes ordinarily carry out phosphorolysis by transferring a glycosyl moiety from the nonreducing end of a di-or polysaccharide substrate onto inorganic phosphate, thereby generating a sugar-1-phosphate 10 ( Figure 1A). GPs distinguish themselves from most CAZymes in that the hydrolytic free energy associated with the glycosidic e...
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.
N-Glycosylation
is a fundamental protein modification found in
both eukaryotes and archaea. Despite lacking N-glycans, many commensal
and pathogenic bacteria have developed mechanisms to degrade these
isoforms for a variety of functions, including nutrient acquisition
and evasion of the immune system. Although much is known about many
of the enzymes responsible for N-glycan degradation, the enzymes involved
in cleaving the N-glycan core have only recently been discovered.
Thus, some of the structural details have yet to be characterized,
and little is known about their full distribution among bacterial
strains and specifically within potential Gram-positive polysaccharide
utilization loci. Here, we report crystal structures for Family 5,
Subfamily 18 (GH5_18) glycoside hydrolases from the gut bacterium Bifidobacterium longum (BlGH5_18) and the soil bacterium Streptomyces cattleya (ScGH5_18), which hydrolyze the core
Manβ1–4GlcNAc disaccharide. Structures of these enzymes
in complex with Manβ1–4GlcNAc reveal a more complete
picture of the −1 subsite. They also show that a C-terminal
active site cap present in BlGH5_18 is absent in ScGH5_18. Although
this C-terminal cap is not widely distributed throughout the GH5_18
family, it is important for full enzyme activity. In addition, we
show that GH5_18 enzymes are found in Gram-positive polysaccharide
utilization loci that share common genes, likely dedicated to importing
and degrading N-glycan core structures.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.