Exploration of GH94 Sequence Space for Enzyme Discovery Reveals a Novel Glucosylgalactose Phosphorylase Specificity

Abstract

The substantial increase in DNA sequencing efforts has led to a rapid expansion of available sequences in glycoside hydrolase families. The ever-increasing sequence space presents considerable opportunities for the search for enzymes with novel functionalities. In this work, the sequence-function space of glycoside hydrolase family 94 (GH94) was explored in detail, using a combined approach of phylogenetic analysis and sequence similarity networks. The identification and experimental screening of unknown clusters led to the discovery of an enzyme from the soil bacterium Paenibacillus polymyxa that acts as a 4-O-β-d-glucosyl-d-galactose phosphorylase (GGalP), a specificity that has not been reported to date. Detailed characterization of GGalP revealed that its kinetic parameters were consistent with those of other known phosphorylases. Furthermore, the enzyme could be used for production of the rare disaccharides 4-O-β-d-glucosyl-d-galactose and 4-O-β-dglucosyl-l-arabinose. Our current work highlights the power of rational sequence space exploration in the search for novel enzyme specificities, as well as the potential of phosphorylases for rare disaccharide synthesis.

Converting Bulk Sugars into Functional Fibers: Discovery and Application of a Thermostable β-1,3-Oligoglucan Phosphorylase

“…At βOGP followed Michaelis–Menten kinetics under the conditions applied (Table ). While it showed a lower affinity for glucose ( K M = 5.8 mM) than the known βOGPs, it is in line with the affinity for glucose of other β-glucoside phosphorylases from the related GH94 family . In contrast, the turnover number ( k cat = 784 s –1 ) was 450- to 700-fold higher.…”

“…While it showed a lower affinity for glucose (K M = 5.8 mM) than the known βOGPs, 11 it is in line with the affinity for glucose of other βglucoside phosphorylases from the related GH94 family. 23 In contrast, the turnover number (k cat = 784 s −1 ) was 450-to 700fold higher. Altogether, these properties result in an over 20fold increase in catalytic efficiency compared to known βOGPs, 11 and a 60-fold increase compared to TaβGP, 12,14 the only βGP reported to show detectable activity toward Dglucose as acceptor substrate (Table 1).…”

Discovery and Biotechnological Exploitation of Glycoside-Phosphorylases

“…This structure–sequence–function data integration permitted focusing biochemical characterization efforts onto five cluster representatives, to demonstrate the segregation of hydrolases and phosphorylases at the cluster level, and identify new functions, such as the GH130 Man-GlcA phosphorylase [ 112 ]. Finally, we can report the discovery of a new GP activity among GH94 enzymes using an SSN based strategy, 4-O-β- d -glucosyl- d -galactose phosphorylase [ 86 ]. However, the CAZy database and the publicly available metagenomic sequence datasets remain largely untapped, representing gold mines to be explored with such approaches, with the prospect of novel GP discoveries.…”

Efficient synthesis of nigerose by a novel nigerose phosphorylase from Anaerosporobacter mobilis

“…In the past three years, some articles have also reported newly discovered phosphorylases [28][29][30], promising in synthesizing rare sugars. In addition to the one-pot enzymatic preparation method, some articles report phosphorylase cascade reactions to prepare target products [25,[31][32][33].…”