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

The ST0452 protein is a bifunctional protein exhibiting sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) and amino-sugar-1-phosphate acetyltransferase activities and was isolated from the thermophilic archaeon Sulfolobus tokodaii. Based on the previous observation that five single mutations increased ST0452 sugar-1-P NTase activity, nine double-mutant ST0452 proteins were generated with the intent of obtaining enzymes exhibiting a further increase in catalysis, but all showed less than 15% of the wild-type N-acetyl-d-glucosamine-1-phosphate uridyltransferase (GlcNAc-1-P UTase) activity. The Y97A mutant exhibited the highest activity of the single-mutant proteins, and thus site saturation mutagenesis of the 97th position (Tyr) was conducted. Six mutants showed both increased GlcNAc-1-P UTase and glucose-1-phosphate uridyltransferase activities, eight mutants showed only enhanced GlcNAc-1-P UTase activity, and six exhibited higher GlcNAc-1-P UTase activity than that of the Y97A mutant. Kinetic analyses of three typical mutants indicated that the increase in sugar-1-P NTase activity was mainly due to an increase in the apparent k(cat) value. We hypothesized that changing the 97th position (Tyr) to a smaller amino acid with similar electronic properties would increase activity, and thus the Tyr at the corresponding 103rd position of the Escherichia coli GlmU (EcGlmU) enzyme was replaced with the same residues. The Y103N mutant EcGlmU showed increased GlcNAc-1-P UTase activity, revealing that the Tyr at the 97th position of the ST0452 protein (103rd position in EcGlmU) plays an important role in catalysis. The present results provide useful information regarding how to improve the activity of natural enzymes and how to generate powerful enzymes for the industrial production of sugar nucleotides. IMPORTANCE It is typically difficult to increase enzymatic activity by introducing substitutions into a natural enzyme. However, it was previously found that the ST0452 protein, a thermostable enzyme from the thermophilic archaeon Sulfolobus tokodaii, exhibited increased activity following single amino acid substitutions of Ala. In this study, ST0452 proteins exhibiting a further increase in activity were created using a site saturation mutagenesis strategy at the 97th position. Kinetic analyses showed that the increased activities of the mutant proteins were principally due to increased apparent k(cat) values. These mutant proteins might suggest clues regarding the mechanism underlying the reaction process and provide very important information for the design of synthetic improved enzymes, and they can be used as powerful biocatalysts for the production of sugar nucleotide molecules. Moreover, this work generated useful proteins for three-dimensional structural analysis clarifying the processes underlying the regulation and mechanism of enzymatic activity

The association between objectively-measured physical activity during pregnancy and the risk of cesarean delivery: a prospective study

Objectives: To evaluate the association between physical activity (PA) and risk of cesarean delivery. Material and methods: 197 singleton pregnant women recruited in this study. Participants were divided into vaginal and cesarean delivery group. PA based objectively monitoring between the two groups was compared. Logistic regression was used to analyze the association between PA and cesarean delivery. Results: Moderate PA (MPA) of cesarean delivery group was less in the first (21.5 vs 27.5 min/day; p = 0.006) and second trimester (19.4 vs 26.8 min/day; p = 0.001). Light PA of cesarean delivery group was less (195.9 vs 217.3 min/day; p = 0.006) with more sedentary time (551.7 vs 529.1 min/day; p = 0.041) in the third trimester. Increased risk of cesarean delivery was noted in cases with MPA < 37.8 min/day compared to MPA ≥ 37.8 min/day (aOR 2.62; 95% CI 1.09 to 6.32; p = 0.031) in the first trimester. MPA < 17.9 min/day in the second trimester increased the risk of cesarean delivery (aOR 3.01; 95% CI 1.57 to 5.75; p = 0.001) compared to MPA ≥ 17.9 min/day. Conclusions: MPA in the first two trimesters were associated with the risk of cesarean section. Women should increase MPA from early pregnancy

Comfort Prediction of Office Chair Surface Material Based on the ISSA-LSSVM

This study serves the purpose of assisting users in selecting a comfortable seat surface material for office chairs and enhancing users’ comfort while using office chairs. To address the issue that the selection of traditional seat surface material is too subjective and that the prediction effect is poor, an improved sparrow search algorithm (ISSA) optimized least squares support vector machine (LSSVM) method for office chair seat surface material comfort prediction has been proposed. Sparrow Search Algorithm (SSA) was optimized with Sobol sequences, nonlinear inertial weights, and a crisscross optimization algorithm to produce the Improved Sparrow Search Algorithm (ISSA), and then the relevant parameters of the LSSVM algorithm were optimized with the modified algorithm to improve its prediction performance. The prediction accuracy of the ISSA-LSSVM model is as high as 95.75% by combining the body pressure distribution experiments; the root mean square error (RMSE) is 0.29; the goodness of fit (R2) is 0.92; the mean absolute error (MAE) is 0.24; the standard deviation (RSD) is 5.99%. The ISSA-LSSVM model predicts seat surface material comfort more accurately and reliably. This strategy can assist consumers to narrow down their seat surface material choices and even suggest an optimal selection. In this way, it can boost users’ pleasure with office chairs, which has great potential for wide application

A Novel Exopolysaccharide with Metal Adsorption Capacity Produced by a Marine Bacterium Alteromonas sp. JL2810

Most marine bacteria can produce exopolysaccharides (EPS). However, very few structures of EPS produced by marine bacteria have been determined. The characterization of EPS structure is important for the elucidation of their biological functions and ecological roles. In this study, the structure of EPS produced by a marine bacterium, Alteromonas sp. JL2810, was characterized, and the biosorption of the EPS for heavy metals Cu2+, Ni2+, and Cr6+ was also investigated. Nuclear magnetic resonance (NMR) analysis indicated that the JL2810 EPS have a novel structure consisting of the repeating unit of [-3)-α-Rhap-(1→3)-α-Manp-(1→4)-α-3OAc-GalAp-(1→]. The biosorption of the EPS for heavy metals was affected by a medium pH; the maximum biosorption capacities for Cu2+ and Ni2+ were 140.8 ± 8.2 mg/g and 226.3 ± 3.3 mg/g at pH 5.0; however, for Cr6+ it was 215.2 ± 5.1 mg/g at pH 5.5. Infrared spectrometry analysis demonstrated that the groups of O-H, C=O, and C-O-C were the main function groups for the adsorption of JL2810 EPS with the heavy metals. The adsorption equilibrium of JL2810 EPS for Ni2+ was further analyzed, and the equilibrium data could be better represented by the Langmuir isotherm model. The novel EPS could be potentially used in industrial applications as a novel bio-resource for the removal of heavy metals

Identification of Novel Acetyltransferase Activity on the Thermostable Protein ST0452 from Sulfolobus tokodaii Strain 7▿

A 401-residue-long protein, ST0452, has been identified from a thermophilic archaeon, Sulfolobus tokodaii strain 7, as a glucose-1-phosphate thymidylyltransferase (Glc-1-P TTase) homolog with a 170-residue-long extra C-terminus portion. Functional analyses of the ST0452 protein have confirmed that the protein possessed dual sugar-1-phosphate nucleotidylyltransferase (sugar-1-P NTase) activities. The 24 repeats of a signature motif sequence which has been found in bacterial acetyltransferases, (L/I/V)-(G/A/E/D)-XX-(S/T/A/V)-X, were detected at the C terminus of the ST0452 protein. This observation prompted our group to investigate the acetyltransferase activity of the ST0452 protein. Detection of the release of coenzyme A (CoA) from acetyl-CoA and the production of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc) from glucosamine-1-phosphate (GlcN-1-P) and UTP in the presence of the ST0452 protein revealed that this protein possesses the GlcN-1-P-specific acetyltransferase activity. In addition, analyses of substrate specificity showed that acetyltransferase activity of the ST0452 protein is capable of catalyzing the change of galactosamine-1-phosphate (GalN-1-P) to N-acetyl-d-galactosamine-1-phosphate (GalNAc-1-P) as well as GlcN-1-P and that its sugar-1-P NTase activity is capable of producing UDP-GalNAc from GalNAc-1-P and UTP. This is the first report of a thermostable bifunctional enzyme with GalN-1-P acetyltransferase and GalNAc-1-P uridyltransferase activities. The observation reveals that the bacteria-type UDP-GlcNAc biosynthetic pathway from fructose-6-phospate is utilized in this archaeon and represents a novel biosynthetic pathway for producing UDP-GalNAc from GalN-1-P in this microorganism

Reference

gdstc.gov.cn. The funders had no role in study design, data collection and analysis, decision t

Effects of d-Amino Acids on the EPS Production and Cell Aggregation of Alteromonas macleodii Strain JL2069

National Key Basic Research Program of China [2013CB955700]; NSFC project [91028001]; SOA project [201105021]Exopolysaccharide (EPS) is produced by many marine bacteria and is important for cell aggregation in the ocean. d-amino acids are important components in bacteria and are recently recognized as signal molecules for regulation of bacterial growth. In this study, the effects of d-amino acids on EPS production, cell aggregation, and metabolic activity were investigated using an EPS-producing bacterium Alteromonas macleodii strain JL2069. EPS produced by JL2069 was inhibited by 1 mM of d-Ala and d-Ser, but not by d-Glu. The formation of particulate organic matter (POM) was promoted by the three amino acids. A new technique of microcalorimetry analysis indicated that the metabolic activity of the JL2069 cells was inhibited by these d-amino acids. Our results suggested that d-amino acids may reduce the bacterial metabolism by changing bacterial lifestyle from planktonic to cell aggregation growth which occurs independent of the production of EPS