Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

Abstract

Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine-and purine-based NTPs as well as non-native D-and L-sugars (both ␣-and ␤-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.

Distinct Substrate Specificity and Catalytic Activity of the Pseudoglycosyltransferase VldE

“…Therefore, we prepared all five possible NDP-valienols and evaluated the substrate preference of VldE – whether or not it can process other NDP-valienols. GDP-, ADP-, and CDP-valienols were prepared from valienol 1-phosphate and their corresponding nucleotidyl triphosphates (GTP, ATP, or CTP), using VldB (Yang et al, 2011), whereas UDP- and dTDP-valienol were prepared from valienol 1-phosphate and UTP and dTTP, respectively, using RmlA-L89T, an engineered broad spectrum nucleotidyltransferase (Moretti et al, 2011), kindly provided by Prof. Jon Thorson at the University of Kentucky (Figures S2A and S2B). Although RmlA-L89T has been known to have relaxed substrate specificity towards various sugar phosphates (Moretti et al, 2011), the present study showed that RmlA-L89T is also able to process an unsaturated carbasugar phosphate.…”

Increasing the Thermostable Sugar-1-Phosphate Nucleotidylyltransferase Activities of the Archaeal ST0452 Protein through Site Saturation Mutagenesis of the 97th Amino Acid Position

“…Substitution with amino acid residues smaller than the original 97th (103rd in EcGlmU) Tyr residue of the ST0452 protein increased the GlcNAc-1-P UTase activities of both ST0452 and EcGlmU proteins. Similarly, in the bacterial sugar-1-P TTase enzyme, substitution with smaller amino acid residues expands the nucleoside triphosphate or sugar-1-P substrate specificity (12,13,14,15). Structure-based substitution mutants have been designed for expansion of the substrate specificity, primarily due to improvement in K m values (13).…”

Kinetic evaluation of glucose 1-phosphate analogues with a thymidylyltransferase using a continuous coupled enzyme assay

“…A prevalent, pathogenic organism, S. pneumoniae has shown widespread clinical resistance to penicillin and chloramphenicol, as well as to synergistic treatments involving β-lactams and aminoglycosides. 5,6 Furthermore, they have been used to prepare sugar nucleotide analogues for enzymatic glycodiversification studies, [7][8][9] and used to prepare phosphonate 10 and carbacyclic 11 sugar nucleotide analogues that have been put forward as putative glycosyltransferase inhibitors. Cps2L (EC 2.7.7.24) is a bacterial thymidylyltransferase (nucleotidylyltransferase) cloned from S. pneumoniae that catalyses the first step in the biosynthesis of L-rhamnose (Scheme 1), 3 an essential constituent of the cell wall in many bacterial species.…”