Improvement of ST0452 N -Acetylglucosamine-1-Phosphate Uridyltransferase Activity by the Cooperative Effect of Two Single Mutations Identified through Structure-Based Protein Engineering

Abstract

We showed previously that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4 times higher N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase (UTase) activity, compared with that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this increased activity. The overall structure was almost identical to that of the wild-type ST0452 protein (PDB ID 2GGO), with residue 97 (Asn) interacting with the O-5 atom of N-acetylglucosamine (GlcNAc) in the complex without metal ions. The same interaction was observed for Escherichia coli GlmU in the absence of metal ions. These observations indicated that the three-dimensional structure of the Y97N protein was not changed by this substitution but the interactions with the substrate were slightly modified, which might cause the activity to increase. The crystal structure of the Y97N protein also showed that positions 146 (Glu) and 80 (Thr) formed interactions with GlcNAc, and an engineering strategy was applied to these residues to increase activity. All proteins substituted at position 146 had drastically decreased activities, whereas several proteins substituted at position 80 showed higher GlcNAc-1-P UTase activity, compared to that of the wild-type protein. The substituted amino acids at positions 80 and 97 might result in optimized interactions with the substrate; therefore, we predicted that the combination of these two substitutions might cooperatively increase GlcNAc-1-P UTase activity. Of the four double mutant ST0452 proteins generated, T80S/Y97N showed 6.5-times-higher activity, compared to that of the wild-type ST0452 protein, revealing that these two substituted residues functioned cooperatively to increase GlcNAc-1-P UTase activity. IMPORTANCE We demonstrated that the enzymatic activity of a thermostable protein was over 4 times higher than that of the wild-type protein following substitution of a single amino acid, without affecting its thermostability. The three-dimensional structure of the improved mutant protein complexed with substrate was determined. The same overall structure and interaction between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of Tyr at position 97 by Asn might slightly change the interaction. This subtle change in the interaction might potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in the structure of a natural thermostable enzyme is not necessary to increase its activity; a subtle change in the interaction with the substrate might be sufficient. Cooperative effects were observed in the appropriate double mutant protein. This work provides useful information for the future engineering of natural enzymes.

Characterization of a glycolipid glycosyltransferase with broad substrate specificity from the marine bacteriumCandidatusPelagibacter sp. HTCC7211

“…When the residue at position 257 (Thr) in GT cp was substituted with Ser (T257S), the T257S mutant exhibited approximately 1.6-times higher activity than that of wild-type GT cp . Compared with the parental residue Thr, Ser has one fewer methyl group; therefore, there is more free space between the Ser residue and the substrate (45). The generated space might allow more room for the appropriate interaction between the Ser residue and the sugar donor.…”

Architecture, Function, Regulation, and Evolution of α-Glucans Metabolic Enzymes in Prokaryotes

“…In contrast, the archeal glucose/glucosamine-1-phosphate uridylyltransferase from Sulfolobus tokodaii displays a C-terminal LβH by which the enzyme trimerizes similarly to GlmU. 423,424 The catalytic site, including the G-rich loop and key catalytic residues, is essentially preserved among UGPases and AGPases, supporting a common mechanism. Major differences are observed in other loops, including the RL2 loop, whose equivalent in bacterial UGPases is shorter, reflecting its participation/ specialization in the allosteric regulation mechanism of AGPases.…”

“…394 It is worth noting that the UDP-N-acetylglucosamine 3-O-acyltransferase LpxA from E. coli (EC: 2.3.1.129) 395 also comprises an LβH domain involved in catalysis, highlighting the participation of such domains in nucleotide-sugar 404 On the right, the two views of the tetrameric architecture of EcAGPase show the other three protomers in grey, with the C-terminal domain in dark grey. (C) The archaeal structure of the Sulfurisphaera tokodaii glucose 1phosphate thymidylyltransferase (PDB 5Z09) 424 in complex with UTP and two views of the trimeric arrangement. Note the longer C-terminal LβH compared to the AGPase, and the difference in the participation in the oligomeric arrangement.…”

“…On the right, the two views of the tetrameric architecture of Ec AGPase show the other three protomers in grey, with the C-terminal domain in dark grey. (C) The archaeal structure of the Sulfurisphaera tokodaii glucose 1-phosphate thymidylyl­transferase (PDB 5Z09) in complex with UTP and two views of the trimeric arrangement. Note the longer C-terminal LβH compared to the AGPase, and the difference in the participation in the oligomeric arrangement.…”

A Glycolipid Glycosyltransferase with Broad Substrate Specificity from the Marine Bacterium “ Candidatus Pelagibacter sp.” Strain HTCC7211

“…When the residue at position 257 (Thr) in GT cp was substituted with Ser (T257S), the T257S mutant exhibited approximately 1.6-times higher activity than that of wild-type GT cp . Compared with the parental residue Thr, Ser has one fewer methyl group; therefore, there is more free space between the Ser residue and the substrate (45).…”