Flexible NAD+ Binding in Deoxyhypusine Synthase Reflects the Dynamic Hypusine Modification of Translation Factor IF5A.

The eukaryotic and archaeal translation factor IF5A requires a post-translational hypusine modification, which is catalyzed by deoxyhypusine synthase (DHS) at a single lysine residue of IF5A with NAD+ and spermidine as cofactors, followed by hydroxylation to form hypusine. While human DHS catalyzed reactions have been well characterized, the mechanism of the hypusination of archaeal IF5A by DHS is not clear. Here we report a DHS structure from Pyrococcus horikoshii OT3 (PhoDHS) at 2.2 Å resolution. The structure reveals two states in a single functional unit (tetramer): two NAD+-bound monomers with the NAD+ and spermidine binding sites observed in multi-conformations (closed and open), and two NAD+-free monomers. The dynamic loop region V288-P299, in the vicinity of the active site, adopts different positions in the closed and open conformations and is disordered when NAD+ is absent. Combined with NAD+ binding analysis, it is clear that PhoDHS can exist in three states: apo, PhoDHS-2 equiv NAD+, and PhoDHS-4 equiv NAD+, which are affected by the NAD+ concentration. Our results demonstrate the dynamic structure of PhoDHS at the NAD+ and spermidine binding site, with conformational changes that may be the response to the local NAD+ concentration, and thus fine-tune the regulation of the translation process via the hypusine modification of IF5A.

Crystallization and preliminary X-ray crystallographic study of a 3.8-MDa respiratory supermolecule hemocyanin.

Many molluscs transport oxygen using a very large cylindrical multimeric copper-containing protein named hemocyanin. The molluscan hemocyanin forms a decamer (cephalopods) or multidecamer (gastropods) of approximately 330-450kDa subunits, resulting in a molecular mass >3.3MDa. Therefore, molluscan hemocyanin is one of the largest proteins. The reason why these organisms use such a large supermolecule for oxygen transport remains unclear. Atomic-resolution X-ray crystallographic analysis is necessary to unveil the detailed molecular structure of this mysterious large molecule. However, its propensity to dissociate in solution has hampered the crystallization of its intact form. In the present study, we successfully obtained the first crystals of an intact decameric molluscan hemocyanin. The diffraction dataset at 3.0-Å resolution was collected by merging the datasets of two isomorphic crystals. Electron microscopy analysis of the dissolved crystals revealed cylindrical particles. Furthermore, self-rotation function analysis clearly showed the presence of a fivefold symmetry with several twofold symmetries perpendicular to the fivefold axis. The absorption spectrum of the crystals showed an absorption peak around 345nm. These results indicated that the crystals contain intact hemocyanin decamers in the oxygen-bound form.

Structural analysis of the α-glucosidase HaG provides new insights into substrate specificity and catalytic mechanism.

α-Glucosidases, which catalyze the hydrolysis of the α-glucosidic linkage at the nonreducing end of the substrate, are important for the metabolism of α-glucosides. Halomonas sp. H11 α-glucosidase (HaG), belonging to glycoside hydrolase family 13 (GH13), only has high hydrolytic activity towards the α-(1 → 4)-linked disaccharide maltose among naturally occurring substrates. Although several three-dimensional structures of GH13 members have been solved, the disaccharide specificity and α-(1 → 4) recognition mechanism of α-glucosidase are unclear owing to a lack of corresponding substrate-bound structures. In this study, four crystal structures of HaG were solved: the apo form, the glucosyl-enzyme intermediate complex, the E271Q mutant in complex with its natural substrate maltose and a complex of the D202N mutant with D-glucose and glycerol. These structures explicitly provide insights into the substrate specificity and catalytic mechanism of HaG. A peculiar long ß â†’ α loop 4 which exists in α-glucosidase is responsible for the strict recognition of disaccharides owing to steric hindrance. Two residues, Thr203 and Phe297, assisted with Gly228, were found to determine the glycosidic linkage specificity of the substrate at subsite +1. Furthermore, an explanation of the α-glucosidase reaction mechanism is proposed based on the glucosyl-enzyme intermediate structure.

Structures of AzrA and of AzrC complexed with substrate or inhibitor: insight into substrate specificity and catalytic mechanism.

Azo dyes are major synthetic dyestuffs with one or more azo bonds and are widely used for various industrial purposes. The biodegradation of residual azo dyes via azoreductase-catalyzed cleavage is very efficient as the initial step of wastewater treatment. The structures of the complexes of azoreductases with various substrates are therefore indispensable to understand their substrate specificity and catalytic mechanism. In this study, the crystal structures of AzrA and of AzrC complexed with Cibacron Blue (CB) and the azo dyes Acid Red 88 (AR88) and Orange I (OI) were determined. As an inhibitor/analogue of NAD(P)H, CB was located on top of flavin mononucleotide (FMN), suggesting a similar binding manner as NAD(P)H for direct hydride transfer to FMN. The structures of the AzrC-AR88 and AzrC-OI complexes showed two manners of binding for substrates possessing a hydroxy group at the ortho or the para position of the azo bond, respectively, while AR88 and OI were estimated to have a similar binding affinity to AzrC from ITC experiments. Although the two substrates were bound in different orientations, the hydroxy groups were located in similar positions, resulting in an arrangement of electrophilic C atoms binding with a proton/electron-donor distance of ∼3.5 Što N5 of FMN. Catalytic mechanisms for different substrates are proposed based on the crystal structures and on site-directed mutagenesis analysis.

Enantioselectivity regulation of antibody against chiral herbicide metolachlor based on interaction at chiral center.

Enantioselective antibodies have emerged as great potential biomaterials in the fields of immunoassays and chiral separation. However, cross-reactivity of antibodies to the distomer may severely restrict the application. Comprehending the interaction mechanism between antibodies and enantiomers could be beneficial to produce superior enantioselective antibodies. In this study, a pair of recombinant antibodies (RAbs) against metolachlor enantiomers at chiral carbon (αSS-MET and αSR-MET) were generated and characterized. The αSS-MET-RAb and αSR-MET-RAb showed comparable sensitivity and specificity to the parental monoclonal antibodies by icELISA, with IC50 values of 3.45 and 223.77 ng/mL, respectively. Moreover, the complex structures of RAbs and corresponding eutomer were constructed and analyzed, and site-specific mutagenesis was utilized to verify the reliability of the enantioselective mechanism elucidated. It demonstrated that the strength of the interaction between the chiral center region of eutomer and the antibody was the key factor for the enantioselectivity of antibody. Increasing this interaction could limit the conformational adjustment of the distomer in a specific chiral recognition cavity, thus decreasing the affinity of the antibody to the distomer. This work provided the in-depth analysis of enantioselective mechanism for two RAbs and paved the way to regulate antibody enantioselective performance for immunoassays of chiral compounds.

Crystal structure analysis, overexpression and refolding behaviour of a DING protein with single mutation.

After crystallization of a certain protein-RNA complex, well diffracting crystals were obtained. However, the asymmetric unit of the crystal was too small to locate any components. Mass spectrometry and X-ray crystal structure analysis showed that it was a member of the DING protein family (HPBP). Surprisingly, the structure of HPBP reported previously was also determined accidentally as a contaminant, suggesting that HPBP has a strong tendency to crystallize. Furthermore, DING proteins were reported to relate in disease. These observations suggest that DING has potential for application in a wide range of research fields. To enable further analyses, a system for preparation of HPBP was constructed. As HPBP was expressed in insoluble form in Escherichia coli, it was unfolded chemically and refolded. Finally, a very high yield preparation method was constructed, in which 43 mg of HPBP was obtained from 1 L of culture. Furthermore, to evaluate the validity of refolding, its crystal structure was determined at 1.03 Å resolution. The determined structure was identical to the native structure, in which two disulfide bonds were recovered correctly and a phosphate ion was captured. Based on these results, it was concluded that the refolded HPBP recovers its structure correctly.

A Rapid RPA-CRISPR/Cas12a Detection Method for Adulteration of Goat Milk Powder.

Because of the serious adulteration of goat milk, the rapid on-site detection of goat milk powder adulteration is needed. In this study, the CRISPR/Cas12a detection system combined with recombinase polymerase amplification (RPA) was employed to qualitatively detect the adulteration of goat milk powder with cattle-derived components. Specific primers and crRNA were designed and screened. After the optimization of RPA and the Cas system, the RPA-CRISPR/Cas12a detection method was established. The detection can complete the rapid identification of cattle-derived components in 45 min, without the assistant of large equipment. The absolute detectability of the RPA-CRISPR/Cas12a assay could reach 10-2 ng/µL for cattle genomic DNA, and 1% (w/w) for cattle milk powder, which is suitable to meet the testing requirements for on-site detection. In total, 55 commercial goat milk powder products were collected for blind testing. The results showed that 27.3% of the samples were adulterated with cattle ingredients, revealing a serious adulteration situation in goat milk powder market. The RPA-CRISPR/Cas12a assay established in this research exhibited its potential for practical use of on-site detection to detect cow milk powder in goat milk powder and can provide reliable technical reference for combating food fraud of adulteration of goat milk products.

Rapid and Simple Detection of Burkholderia gladioli in Food Matrices Using RPA-CRISPR/Cas12a Method.

Pathogenic variants of Burkholderia gladioli pose a serious threat to human health and food safety, but there is a lack of rapid and sensitive field detection methods for Burkholderia gladioli. In this study, the CRISPR/Cas12a system combined with recombinant enzyme polymerase amplification (RPA) was used to detect Burkholderia gladioli in food. The optimized RPA-CRISPR/Cas12a assay was able to specifically and stably detect Burkholderia gladioli at a constant 37 °C without the assistance of large equipment. The detection limit of the method was evaluated at two aspects, the genomic DNA (gDNA) level and bacterial quantity, of which there were 10-3 ng/µL and 101 CFU/mL, respectively. Three kinds of real food samples were tested. The detection limit for rice noodles, fresh white noodles, and glutinous rice flour samples was 101 CFU/mL, 102 CFU/mL, and 102 CFU/mL, respectively, without any enrichment steps. The whole detection process, including sample pretreatment and DNA extraction, did not exceed one hour. Compared with the qPCR method, the established RPA-CRISPR /Cas12a method was simpler and even more sensitive. Using this method, a visual detection of Burkholderia gladioli that is suitable for field detection can be achieved quickly and easily.

The binding mechanism of eIF2ß with its partner proteins, eIF5 and eIF2Bε.

The eukaryotic translation initiation factor eIF2 delivers Met-tRNAiMet to the ribosomal small subunit in GTP-bound form associated with eIF1, eIF1A, eIF3 and eIF5, and dissociates together with eIF5 as eIF5-eIF2-GDP complex from the ribosomal small subunit after formation of start codon-anticodon base pairing between Met-tRNAiMet and mRNA. The inactive form eIF2-GDP is then exchanged for the active form eIF2-GTP by eIF2B for further initiation cycle. Previous studies showed that the C-terminal domains of eIF5 (eIF5-CTD) and eIF2Bε (eIF2Bε-CTD) have a common eIF2ß-binding site for interacting with an N-terminal region of eIF2ß (eIF2ß-NTD). Here we have reconstructed the complexes of (eIF5-CTD)-(eIF2ß-NTD) and (eIF2Bε-CTD)-(eIF2ß-NTD) in vitro, and investigated binding mechanism by circular dichroism spectroscopy and small angle X-ray scattering in solution. The results showed the conformation of eIF2ß-NTD was changed when bound to partner proteins, whereas the structures of eIF5-CTD and eIF2Bε-CTD were similar in both isolated and complex states. We propose that eIF2ß-NTD works as an intrinsically disordered domain which is disorder in the isolated state, but folds into a definite structure when bound to its partner proteins. Such flexibility of eIF2ß-NTD is expected to be responsible for its binding capability.

Influence of ultrasound pretreatment on the subsequent glycation of dietary proteins.

The influence of ultrasound treatment on the subsequent glycation process of proteins is controversial. Glycation behaviors of bovine serum albumin (BSA), ß-lactoglobulin (ß-Lg) and ß-casein (ß-CN) after ultrasound pretreatment (UP) were compared by both evaluating glycation kinetics and analyzing structural changes of proteins. UP resulted in both unfolding and aggregation behavior in protein samples, which altered the accessibility of the Lys and Arg. Five cycles of UP up-regulated the glycation degree of BSA and ß-Lg, possibly due to the unfolding behavior induced by UP, which exposed additional glycation sites. In contrast, 30 cycles of UP induced a dramatic increase (by 97.9 nm) in particle size of BSA, thus burying portions of glycation sites and suppressing the glycation process. Notably, UP had minimal influence on glycation kinetics of ß-CN, due to its intrinsic disordered structure. Based on proteomics analysis, the preference of Lys and Arg during glycation was found to be changed by UP in BSA and ß-Lg. Four, 3 and 3 unique carboxyethylated lysine residues were identified in glycated BSA after 0, 5 and 30 cycles of UP, respectively. This study suggests that the protein glycation can be affected by UP, depending on the ultrasonication duration and native structure of the protein.

Degradation of Peptide-Bound Maillard Reaction Products in Gastrointestinal Digests of Glyoxal-Glycated Casein by Human Colonic Microbiota.

A large portion of Maillard reaction products (MRPs) cannot be absorbed in the upper gut and therefore may be further decomposed and utilized by colonic microbiota (CM). This work reported the stability of UV-absorbent MRPs, fluorescent MRPs and peptide-bound N(ε)-(carboxymethyl)-lysine (CML) in high molecular weight (HMW, >10 kDa), medium molecular weight (MMW, 1-10 kDa), and low molecular weight (LMW, <1 kDa) gastrointestinal digests of glyoxal-glycated casein in the presence of CM. Fluorescent MRPs showed high stability, whereas UV-absorbent MRPs may be partially decomposed. A higher depletion rate of CML was found in the LMW fraction (38.7%) than in the MMW (21.7%) and HMW (9.6%) fractions. The 16S rRNA sequencing results revealed both beneficial and detrimental changes in CM composition induced by the glycated fractions. Generation of short-chain and branched-chain fatty acids in fermentation solutions with glycated fractions was significantly suppressed compared with that in fermentation solution with unglycated digests. This work revealed the possible interplay between peptide-bound MRPs and CM.

Preparation, characterization and application of rod-like chitin nanocrystal by using p-toluenesulfonic acid/choline chloride deep eutectic solvent as a hydrolytic media.

Chitin nanocrystal (ChiNC) was fabricated based on p-toluenesulfonic acid -choline chloride deep eutectic solvent treatment. The obtained ChiNC was about 12-44 nm in width and 206-399 nm in length. The crystalline structure and the functional groups of ChiNC were maintained during the preparation process. Moreover, porcine pancreas lipase (PPL) was successfully immobilized onto the ChiNC to form the immobilized PPL (PPL@ChiNC). The resulting PPL@ChiNC has enzyme loading and activity recovery of 35.6 mg/g and 82.5%, respectively. The thermal stability, pH and temperature adaptabilities of PPL@ChiNC was improved, comparing with free PPL. The demonstrated DES treatment process was efficient for ChiNC preparation and the as-prepared ChiNC exhibited great potentials in biocatalysis and biomedical field.

Crystal Structure of the 3.8-MDa Respiratory Supermolecule Hemocyanin at 3.0 Å Resolution.

Molluscan hemocyanin, a copper-containing oxygen transporter, is one of the largest known proteins. Although molluscan hemocyanins are currently applied as immunotherapeutic agents, their precise structure has not been determined because of their enormous size. Here, we have determined the first X-ray crystal structure of intact molluscan hemocyanin. The structure unveiled the architecture of the 3.8-MDa supermolecule composed of homologous functional units (FUs), wherein the dimers of FUs hierarchically associated to form the entire cylindrical decamer. Most of the specific inter-FU interactions were localized at narrow regions in the FU dimers, suggesting that rigid FU dimers formed by specific interactions assemble with flexibility. Furthermore, the roles of carbohydrates in assembly and allosteric effect, and conserved sulfur-containing residues in copper incorporation, were revealed. The precise structural information obtained in this study will accelerate our understanding of the molecular basis of hemocyanin and its future applications.

Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG from Halomonas sp. strain H11.

The α-glucosidase HaG from the halophilic bacterium Halomonas sp. strain H11 catalyzes the hydrolysis of the glucosidic linkage at the nonreducing end of α-glucosides, such as maltose and sucrose, to release α-glucose. Based on its amino-acid sequence, this enzyme is classified as a member of glycoside hydrolase family 13. HaG has three unique characteristics: (i) a very narrow substrate specificity, almost exclusively hydrolyzing disaccharides; (ii) activation by monovalent cations, such as K(+), Rb(+), Cs(+) and NH4(+); and (iii) high transfer activity of the glucose moiety to the OH group of low-molecular-weight compounds, including glycerol and 6-gingerol. Crystallographic studies have been performed in order to understand these special features. An expression vector was constructed and recombinant HaG protein was overexpressed, purified and crystallized. A data set to 2.15 Šresolution was collected and processed. The crystal belonged to space group P212121, with unit-cell parameters a = 60.2, b = 119.2, c = 177.2 Å. The structure has been determined by molecular replacement using the isomaltulose synthase PalI as the search model (PDB entry 1m53).