Expanding the Substrate Scope of a Bacterial Nucleotidyltransferase via Allosteric Mutations

Abstract

Bacterial glycoconjugates, such as cell surface polysaccharides and glycoproteins, play important roles in cellular interactions and survival. Enzymes called nucleotidyltransferases use sugar-1-phosphates and nucleoside triphosphates (NTPs) to produce nucleoside diphosphate sugars (NDP-sugars), which serve as building blocks for most glycoconjugates. Research spanning several decades has shown that some bacterial nucleotidyltransferases have broad substrate tolerance and can be exploited to produce a variety of NDP-sugars in vitro. While these enzymes are known to be allosterically regulated by NDP-sugars and their fragments, much work has focused on the effect of active site mutations alone. Here, we show that rational mutations in the allosteric site of the nucleotidyltransferase RmlA lead to expanded substrate tolerance and improvements in catalytic activity that can be explained by subtle changes in quaternary structure and interactions with ligands. These observations will help inform future studies on the directed biosynthesis of diverse bacterial NDP-sugars and downstream glycoconjugates.

Systematic Enzymatic Synthesis of dTDP‐Activated Sugar Nucleotides

“…Now, kinases have been identified for the phosphorylation of several common monosaccharides, such as D‐GalNAc, D‐GlcNAc, D‐Gal, D‐GlcA, D‐GalA, D‐Man, and L‐Fuc [4] . Recently, Lupoli and co‐workers reported that a nucleotidyltransferase from Salmonella enterica (SeRmlA) could recognize diverse phosphorylated monosaccharides, [41] indicating the potential for application in the large‐scale preparation of dTDP‐sugars.…”

Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity

“…With synthetic access to l -S-1Ps, we evaluated the substrate scope of engineered RmlA. As expected, incubation of RmlA* with α-Glc-1P, α-Man-1P, or β- l -Fuc-1P and dTTP (2 mM each) led to formation of the corresponding dTDP-sugars (Figure A). However, when the synthetic β- l -S-1Ps ( 3 – 5 ) or corresponding α- l- S-1Ps were incubated with dTTP under similar conditions, only β- l -Man-1P ( 4 ) was activated (Figure A,B), as confirmed by MS analysis (Figure C).…”

“…Expanding on past work, − we recently demonstrated that engineering of SALTY RmlA’s allosteric site leads to enhanced substrate promiscuity . Incorporation of the allosteric mutation E256D in a known active site mutant (Y146F) carrying the stabilizing alteration D104N also led to improvements in catalytic activity.…”

Biocatalytic Approaches to Building Blocks for Enzymatic and Chemical Glycan Synthesis

“…Modification to the allosteric site permits enhanced activity and expanded substrate scope. Adapted and reproduced from ref ( 111 ). Copyright 2022 American Chemical Society.…”

“…As an alternative to bump-and-hole modifications of the active site, rational mutation of the allosteric site of the nucleotidyltransferase RmlA was recently demonstrated, leading to expanded substrate tolerance and improvements in catalytic activity. 111 RmlA is responsible for catalyzing the formation of dTDP- d -Glc from Glc-1-phosphate and deoxythymidine triphosphate in vivo. The catalytic efficiency of RmlA is limited by a negative feedback inhibition loop, through binding of dTDP- l -Rha to the allosteric site ( Figure 3 B).…”