5z0a
ST0452(Y97N)-GlcNAc binding form
Structural highlights
FunctionS1PNA_SULTO Bifunctional enzyme involved in the synthesis of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylgalactosamine (UDP-GalNAc). It has multiple amino-sugar-1-phosphate acetyltransferase activities, including glucosamine-1-phosphate (GlcN-1-P) acetyltransferase and galactosamine-1-phosphate (GalN-1-P) acetyltransferase activities, and multiple sugar-1-phosphate nucleotidylyltransferase activities, including N-acetylglucosamine-1-phosphate (GlcNAc-1-P) uridyltransferase and N-acetylgalactosamine-1-phosphate (GalNAc-1-P) uridyltransferase activities (PubMed:15598657, PubMed:20400541, PubMed:25567746). Also catalyzes the formation of dTDP-glucose from dTTP and glucose-1-phosphate (Glc-1-P), and the reverse reaction, which produces dTTP from dTDP-glucose and diphosphate (PubMed:15598657). Can also catalyze the formation of UDP-glucose from UTP and glucose-1-phosphate (PubMed:27864169, PubMed:30291121).[1] [2] [3] [4] [5] Publication Abstract from PubMedWe previously showed that the Y97N mutant of the ST0452 protein, isolated from Sulfolobus tokodaii, exhibited over 4-times higher N-acetylglucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity than that of the wild-type ST0452 protein. We determined the three-dimensional structure of the Y97N protein to explore the detailed mechanism underlying this activity increasing. Its overall structure was almost identical to that of the wild-type ST0452 protein (2GGO), with the 97th residue (Asn) interacting with the O5 atom of GlcNAc in the complex without metal ions. The same interaction was observed in 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 increasing. The crystal structure of the Y97N protein also showed that the 146th (Glu) and 80th (Thr) positions formed interactions with GlcNAc and to these residues engineering strategy was applied for activity increasing. All 146th-substituted proteins drastically decreased their activities, whereas several proteins mutated at the 80th position showed higher GlcNAc-1-P UTase activity compared to the wild-type. The substituted amino acids at the 80th and 97th positions might result in optimized interactions with the substrate, thus 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 than that of the wild-type ST0452 protein, revealing that these two substituted residues function 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 interactions between the substituted residue and the GlcNAc substrate as observed in the well-characterized bacterial enzyme suggested that the substitution of 97th Tyr to Asn might slightly change the interaction. This subtle changing of interaction may potentially increase the GlcNAc-1-P UTase activity of the mutant protein. These observations indicated that a drastic change in structure of a natural thermostable enzyme is unnecessary to increase its activity: a subtle change in the interaction with substrate might be sufficient. A cooperative effect was observed in the appropriate double-mutant protein. This work provides useful information for the future engineering of natural enzymes. Improvement of ST0452 GlcNAc-1-phosphate uridyltransferase activity by the cooperative effect of two single mutations identified through structure-based protein engineering.,Honda Y, Nakano S, Ito S, Dadashipour M, Zhang Z, Kawarabayasi Y Appl Environ Microbiol. 2018 Oct 5. pii: AEM.02213-18. doi: 10.1128/AEM.02213-18. PMID:30291121[6] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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