Role of second-sphere arginine residues in metal binding and metallocentre assembly in nitrile hydratases.

Two conserved second-sphere ßArg (R) residues in nitrile hydratases (NHase), that form hydrogen bonds with the catalytically essential sulfenic and sulfinic acid ligands, were mutated to Lys and Ala residues in the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) and the Fe-type NHase from Rhodococcus equi TG328-2 (ReNHase). Only five of the eight mutants (PtNHase ßR52A, ßR52K, ßR157A, ßR157K and ReNHase ßR61A) were successfully expressed and purified. Apart from the PtNHase ßR52A mutant that exhibited no detectable activity, the kcat values obtained for the PtNHase and ReNHase ßR mutant enzymes were between 1.8 and 12.4 s-1 amounting to <1% of the kcat values observed for WT enzymes. The metal content of each mutant was also significantly decreased with occupancies ranging from ∼10 to ∼40%. UV-Vis spectra coupled with EPR data obtained on the ReNHase mutant enzyme, suggest a decrease in the Lewis acidity of the active site metal ion. X-ray crystal structures of the four PtNHase ßR mutant enzymes confirmed the mutation and the low active site metal content, while also providing insight into the active site hydrogen bonding network. Finally, DFT calculations suggest that the equatorial sulfenic acid ligand, which has been shown to be the catalytic nucleophile, is protonated in the mutant enzyme. Taken together, these data confirm the necessity of the conserved second-sphere ßR residues in the proposed subunit swapping process and post-translational modification of the α-subunit in the α activator complex, along with stabilizing the catalytic sulfenic acid in its anionic form.

Design, Synthesis, and Mechanistic Studies of (R)-3-Amino-5,5-difluorocyclohex-1-ene-1-carboxylic Acid as an Inactivator of Human Ornithine Aminotransferase.

Human ornithine aminotransferase (hOAT), a pyridoxal 5'-phosphate (PLP)-dependent enzyme, has been shown to play an essential role in the metabolic reprogramming and progression of hepatocellular carcinoma (HCC). HCC accounts for approximately 75% of primary liver cancers and is within the top three causes of cancer death worldwide. As a result of treatment limitations, the overall 5-year survival rate for all patients with HCC is under 20%. The prevalence of HCC necessitates continued development of novel and effective treatment methods. In recent years, the therapeutic potential of selective inactivation of hOAT has been demonstrated for the treatment of HCC. Inspired by previous increased selectivity for hOAT by the expansion of the cyclopentene ring scaffold to a cyclohexene, we designed, synthesized, and evaluated a series of novel fluorinated cyclohexene analogues and identified (R)-3-amino-5,5-difluorocyclohex-1-ene-1-carboxylic acid as a time-dependent inhibitor of hOAT. Structural and mechanistic studies have elucidated the mechanism of inactivation of hOAT by 5, resulting in a PLP-inactivator adduct tightly bound to the active site of the enzyme. Intact protein mass spectrometry, 19F NMR spectroscopy, transient state kinetic studies, and X-ray crystallography were used to determine the structure of the final adduct and elucidate the mechanisms of inactivation. Interestingly, despite the highly electrophilic intermediate species conferred by fluorine and structural evidence of solvent accessibility in the hOAT active site, Lys292 and water did not participate in nucleophilic addition during the inactivation mechanism of hOAT by 5. Instead, rapid aromatization to yield the final adduct was favored.

High-Performance Blue Perovskite Light-Emitting Diodes Enabled by Synergistic Effect of Additives.

While quasi-two-dimensional (quasi-2D) perovskites have good properties of cascade energy transfer, high exciton binding energy, and high quantum efficiency, which will benefit high-efficiency blue PeLEDs, inefficient domain distribution management and unbalanced carrier transport impede device performance improvement. Herein, (2-(9H-carbazol-9-yl)ethyl)phosphonic acid (2PACz) and methyl 2-aminopyridine-4-carboxylate (MAC) were simultaneously introduced to a blue quasi-2D perovskite film. Relying on the synergistic effect of 2PACz and MAC, it not only modulates the phase distribution inhibiting the n = 2 phase but also greatly improves the electrical property of the quasi-2D perovskite film. As a result, the as-modified blue quasi-2D PeLED demonstrated an external quantum efficiency (EQE) of 17.08% and a luminance of 10142 cd m-2. This study exemplifies the synergistic effect among dual additives and offers a new effective additive strategy modulating phase distribution and building balanced carrier transport, which paves the way for the fabrication of highly efficient blue PeLEDs.

Enhancing tumor-specific recognition of programmable synthetic bacterial consortium for precision therapy of colorectal cancer.

Probiotics hold promise as a potential therapy for colorectal cancer (CRC), but encounter obstacles related to tumor specificity, drug penetration, and dosage adjustability. In this study, genetic circuits based on the E. coli Nissle 1917 (EcN) chassis were developed to sense indicators of tumor microenvironment and control the expression of therapeutic payloads. Integration of XOR gate amplify gene switch into EcN biosensors resulted in a 1.8-2.3-fold increase in signal output, as confirmed by mathematical model fitting. Co-culturing programmable EcNs with CRC cells demonstrated a significant reduction in cellular viability ranging from 30% to 50%. This approach was further validated in a mouse subcutaneous tumor model, revealing 47%-52% inhibition of tumor growth upon administration of therapeutic strains. Additionally, in a mouse tumorigenesis model induced by AOM and DSS, the use of synthetic bacterial consortium (SynCon) equipped with multiple sensing modules led to approximately 1.2-fold increased colon length and 2.4-fold decreased polyp count. Gut microbiota analysis suggested that SynCon maintained the abundance of butyrate-producing bacteria Lactobacillaceae NK4A136, whereas reducing the level of gut inflammation-related bacteria Bacteroides. Taken together, engineered EcNs confer the advantage of specific recognition of CRC, while SynCon serves to augment the synergistic effect of this approach.

A Novel Underwater Acoustic Target Recognition Method Based on MFCC and RACNN.

In ocean remote sensing missions, recognizing an underwater acoustic target is a crucial technology for conducting marine biological surveys, ocean explorations, and other scientific activities that take place in water. The complex acoustic propagation characteristics present significant challenges for the recognition of underwater acoustic targets (UATR). Methods such as extracting the DEMON spectrum of a signal and inputting it into an artificial neural network for recognition, and fusing the multidimensional features of a signal for recognition, have been proposed. However, there is still room for improvement in terms of noise immunity, improved computational performance, and reduced reliance on specialized knowledge. In this article, we propose the Residual Attentional Convolutional Neural Network (RACNN), a convolutional neural network that quickly and accurately recognize the type of ship-radiated noise. This network is capable of extracting internal features of Mel Frequency Cepstral Coefficients (MFCC) of the underwater ship-radiated noise. Experimental results demonstrate that the proposed model achieves an overall accuracy of 99.34% on the ShipsEar dataset, surpassing conventional recognition methods and other deep learning models.

A critical inter-subunit interaction for the transmission of the allosteric signal in the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

ADP-glucose pyrophosphorylase is a key regulatory enzyme involved in starch and glycogen synthesis in plants and bacteria, respectively. It has been hypothesized that inter-subunit communications are important for the allosteric effect in this enzyme. However, no specific interactions have been identified as part of the regulatory signal. The enzyme from Agrobacterium tumefaciens is a homotetramer allosterically regulated by fructose 6-phosphate and pyruvate. Three pairs of distinct subunit-subunit interfaces are present. Here we focus on an interface that features two symmetrical interactions between Arg11 and Asp141 from one subunit with residues Asp141 and Arg11 of the neighbor subunit, respectively. Previously, scanning mutagenesis showed that a mutation at the Arg11 position disrupted the activation of the enzyme. Considering the distance of these residues from the allosteric and catalytic sites, we hypothesized that the interaction between Arg11 and Asp141 is critical for allosteric signaling rather than effector binding. To prove our hypothesis, we mutated those two sites (D141A, D141E, D141N, D141R, R11D, and R11K) and performed kinetic and binding analysis. Mutations that altered the charge affected the regulation the most. To prove that the interaction per se (rather than the presence of specific residues) is critical, we partially rescued the R11D protein by introducing a second mutation (R11D/D141R). This could not restore the activator effect on kcat , but it did rescue the effect on substrate affinity. Our results indicate the critical functional role of Arg11 and Asp141 to relay the allosteric signal in this subunit interface.

Genome-wide association study of drought tolerance traits in sugar beet germplasms at the seedling stage.

Introduction: Sugar beets are an important crop for global sugar production. Intense drought and the increasing lack of water resources pose a great threat to sugar beet cultivation. It is a priority to investigate favourable germplasms and functional genes to improve the breeding of drought tolerant plants. Methods: Thus, in this study, 328 sugar beet germplasms were used in a genome-wide association study (GWAS) to identify single nucleotide polymorphism (SNP) markers and candidate genes associated with drought tolerance. Results: The results showed that under drought stress (9% PEG-6000), there were 11 significantly associated loci on chromosomes 2, 3, 5, 7, and 9 from the 108946 SNPs filtered using a mixed linear model (MLM). Genome-wide association analysis combined with qRT-PCR identified 13 genes that were significantly differentially expressed in drought-tolerant extreme materials. Discussion: These candidate genes mainly exhibited functions such as regulating sugar metabolism, maintaining internal environmental stability and participating in photosystem repair. This study provides valuable information for exploring the molecular mechanisms of drought tolerance and improvement in sugar beet.

Genetic diversity and genome-wide association study of 13 agronomic traits in 977 Beta vulgaris L. germplasms.

BACKGROUND: Sugar beet (Beta vulgaris L.) is an economically essential sugar crop worldwide. Its agronomic traits are highly diverse and phenotypically plastic, influencing taproot yield and quality. The National Beet Medium-term Gene Bank in China maintains more than 1700 beet germplasms with diverse countries of origin. However, it lacks detailed genetic background associated with morphological variability and diversity. RESULTS: Here, a comprehensive genome-wide association study (GWAS) of 13 agronomic traits was conducted in a panel of 977 sugar beet accessions. Almost all phenotypic traits exhibited wide genetic diversity and high coefficient of variation (CV). A total of 170,750 high-quality single-nucleotide polymorphisms (SNPs) were obtained using the genotyping-by-sequencing (GBS). Neighbour-joining phylogenetic analysis, principal component analysis, population structure and kinship showed no obvious relationships among these genotypes based on subgroups or regional sources. GWAS was carried out using a mixed linear model, and 159 significant associations were detected for these traits. Within the 25 kb linkage disequilibrium decay of the associated markers, NRT1/PTR FAMILY 6.3 (BVRB_5g097760); nudix hydrolase 15 (BVRB_8g182070) and TRANSPORT INHIBITOR RESPONSE 1 (BVRB_8g181550); transcription factor MYB77 (BVRB_2g023500); and ethylene-responsive transcription factor ERF014 (BVRB_1g000090) were predicted to be strongly associated with the taproot traits of root groove depth (RGD); root shape (RS); crown size (CS); and flesh colour (FC), respectively. For the aboveground traits, UDP-glycosyltransferase 79B6 (BVRB_9g223780) and NAC domain-containing protein 7 (BVRB_5g097990); F-box protein At1g10780 (BVRB_6g140760); phosphate transporter PHO1 (BVRB_3g048660); F-box protein CPR1 (BVRB_8g181140); and transcription factor MYB77 (BVRB_2g023500) and alcohol acyltransferase 9 (BVRB_2g023460) might be associated with the hypocotyl colour (HC); plant type (PT); petiole length (PL); cotyledon size (C); and fascicled leaf type (FLT) of sugar beet, respectively. AP-2 complex subunit mu (BVRB_5g106130), trihelix transcription factor ASIL2 (BVRB_2g041790) and late embryogenesis abundant protein 18 (BVRB_5g106150) might be involved in pollen quantity (PQ) variation. The candidate genes extensively participated in hormone response, nitrogen and phosphorus transportation, secondary metabolism, fertilization and embryo maturation. CONCLUSIONS: The genetic basis of agronomical traits is complicated in heterozygous diploid sugar beet. The putative valuable genes found in this study will help further elucidate the molecular mechanism of each phenotypic trait for beet breeding.

Low nitrogen stress-induced transcriptome changes revealed the molecular response and tolerance characteristics in maintaining the C/N balance of sugar beet (Beta vulgaris L.).

Nitrogen (N) is an essential macronutrient for plants, acting as a common limiting factor for crop yield. The application of nitrogen fertilizer is related to the sustainable development of both crops and the environment. To further explore the molecular response of sugar beet under low nitrogen (LN) supply, transcriptome analysis was performed on the LN-tolerant germplasm '780016B/12 superior'. In total, 580 differentially expressed genes (DEGs) were identified in leaves, and 1,075 DEGs were identified in roots (log2 |FC| ≥ 1; q value < 0.05). Gene Ontology (GO), protein-protein interaction (PPI), and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses clarified the role and relationship of DEGs under LN stress. Most of the downregulated DEGs were closely related to "photosynthesis" and the metabolism of "photosynthesis-antenna proteins", "carbon", "nitrogen", and "glutathione", while the upregulated DEGs were involved in flavonoid and phenylalanine biosynthesis. For example, GLUDB (glutamate dehydrogenase B) was identified as a key downregulated gene, linking carbon, nitrogen, and glutamate metabolism. Thus, low nitrogen-tolerant sugar beet reduced energy expenditure mainly by reducing the synthesis of energy-consuming amino acids, which in turn improved tolerance to low nitrogen stress. The glutathione metabolism biosynthesis pathway was promoted to quench reactive oxygen species (ROS) and protect cells from oxidative damage. The expression levels of nitrogen assimilation and amino acid transport genes, such as NRT2.5 (high-affinity nitrate transporter), NR (nitrate reductase [NADH]), NIR (ferredoxin-nitrite reductase), GS (glutamine synthetase leaf isozyme), GLUDB, GST (glutathione transferase) and GGT3 (glutathione hydrolase 3) at low nitrogen levels play a decisive role in nitrogen utilization and may affect the conversion of the carbon skeleton. DFRA (dihydroflavonol 4-reductase) in roots was negatively correlated with NIR in leaves (coefficient = -0.98, p < 0.05), suggesting that there may be corresponding remote regulation between "flavonoid biosynthesis" and "nitrogen metabolism" in roots and leaves. FBP (fructose 1,6-bisphosphatase) and PGK (phosphoglycerate kinase) were significantly positively correlated (p < 0.001) with Ci (intercellular CO2 concentration). The reliability and reproducibility of the RNA-seq data were further confirmed by real-time fluorescence quantitative PCR (qRT-PCR) validation of 22 genes (R2 = 0.98). This study reveals possible pivotal genes and metabolic pathways for sugar beet adaptation to nitrogen-deficient environments.

Cercospora leaf spot disease of sugar beet.

Leaf spot disease caused by Cercospora beticola Sacc. is the most damaging foliar disease threatening sugar beet production worldwide. The wide spread of disease incurs a reduction of yield and economic losses. The in-depth knowledge of disease epidemiology and virulence factor of pathogen is crucial and basic for preventing fungal disease. The integrated control strategies are needed for an efficient and sustainable disease management. The rotation of fungicides and crop could reduce the initial inoculum and delay the emergence of resistant pathogens. Spraying fungicides under the guide of forecasting models and molecular detecting techniques may hinder the onset of disease prevalence. The resistant varieties of sugar beet to cercospora leaf spot could be obtained by combining classical and molecular breeding methods. More effective approaches are supposed to develop for prevention and control for fungal disease of sugar beet.

Drought resistance evaluation of sugar beet germplasms by response of phenotypic indicators.

Sugar beet is a main sugar crop worldwide that often faces drought stress. The identification of drought tolerance of sugar beet germplasms is beneficial for breeding, but the research about it has been rarely reported. In this study, the drought tolerance of germplasms 92005-1, 94002-2 and 92021-1-1 was tested under simulated conditions. Seven days and 9% PEG treatment were the optimal conditions for evaluation, under which more phenotypic indicators showed significant difference in drought tolerance coefficient. The objective weighting and membership function method were established for evaluating the drought tolerance of different sugar beet germplasms. Drought stress decreased the biomass of leaves and roots of sugar beet germplasms. The drought-sensitive germplasm responded faster for leaf weight, root weight, plant height and root length. These indicators declined more significantly under long-term and severe stress. Increasing the root-shoot ratio and proline content were universal strategies of sugar beet germplasms to overcome drought stress. The drought-tolerant germplasms held higher peroxidase activity and better ability to scavenge reactive oxygen for preventing the damage.

Structural and Mechanistic Basis for the Inactivation of Human Ornithine Aminotransferase by (3S,4S)-3-Amino-4-fluorocyclopentenecarboxylic Acid.

Ornithine aminotransferase (OAT) is overexpressed in hepatocellular carcinoma (HCC), and we previously showed that inactivation of OAT inhibits the growth of HCC. Recently, we found that (3S,4S)-3-amino-4-fluorocyclopentenecarboxylic acid (5) was a potent inactivator of γ-aminobutyric acid aminotransferase (GABA-AT), proceeding by an enamine mechanism. Here we describe our investigations into the activity and mechanism of 5 as an inactivator of human OAT. We have found that 5 exhibits 10-fold less inactivation efficiency (kinact/KI) against hOAT than GABA-AT. A comprehensive mechanistic study was carried out to understand its inactivation mechanism with hOAT. pKa and electrostatic potential calculations were performed to further support the notion that the α,ß-unsaturated alkene of 5 is critical for enhancing acidity and nucleophilicity of the corresponding intermediates and ultimately responsible for the improved inactivation efficiency of 5 over the corresponding saturated analogue (4). Intact protein mass spectrometry and the crystal structure complex with hOAT provide evidence to conclude that 5 mainly inactivates hOAT through noncovalent interactions, and that, unlike with GABA-AT, covalent binding with hOAT is a minor component of the total inhibition which is unique relative to other monofluoro-substituted derivatives. Furthermore, based on the results of transient-state measurements and free energy calculations, it is suggested that the α,ß-unsaturated carboxylate group of PLP-bound 5 may be directly involved in the inactivation cascade by forming an enolate intermediate. Overall, compound 5 exhibits unusual structural conversions which are catalyzed by specific residues within hOAT, ultimately leading to an enamine mechanism-based inactivation of hOAT through noncovalent interactions and covalent modification.

Interfacial Engineering of Au@Nb2CTx-MXene Modulates the Growth Strain, Suppresses the Auger Recombination, and Enables an Open-Circuit Voltage of over 1.2 V in Perovskite Solar Cells.

Defects at the interface of charge transport layers can cause severe charge accumulation and poor charge transferability, which greatly affect the efficiency and stability of stannic oxide (SnO2)-based perovskite solar cells (PSCs). Herein, a new type of MXene (Nb2CTx-MXene) is applied to the interface of SnO2 layers to passivate the interfacial defects and promote charge transport. Nb2CTx-MXene in PSCs realizes the role of boosting the conductivity, reducing the tin vacancies in the interstitial void of the SnO2 layer, decreasing the defect density, and aligning the bandgap. Afterward, Nb2CTx-MXene is decorated with gold nanospheres, which has the ability to modulate the tensile strain of perovskites and suppress the Auger recombination. Eventually, the Au@Nb2CTx-MXene-modified device yields an excellent power conversion efficiency (PCE) of 23.78% with a relatively high open-circuit voltage of 1.215 V (Eg ∼ 1.60 eV). The unencapsulated devices maintain 90% of their initial PCE values after storage in the air with a relative humidity of 40% for 1000 h and remain above 80% of their initial efficiency after operation at the maximum power point for 500 h under 1 sun illumination. Our work provides an avenue to fabricate high-efficiency and stable PSCs with MXene adapting to commercial development.

Mammalian dihydropyrimidine dehydrogenase: Added mechanistic details from transient-state analysis of charge transfer complexes.

Dihydropyrimidine dehydrogenase (DPD) is a flavin dependent enzyme that catalyzes the reduction of the 5,6-vinylic bond of pyrimidines uracil and thymine with electrons from NADPH. DPD has two active sites that are separated by ∼60 Å. At one site NADPH binds adjacent to an FAD cofactor and at the other pyrimidine binds proximal to an FMN. Four Fe4S4 centers span the distance between these active sites. It has recently been established that the enzyme undergoes reductive activation prior to reducing the pyrimidine. In this initial process NADPH is oxidized at the FAD site and electrons are transmitted to the FMN via the Fe4S4 centers to yield the active state with a cofactor set of FAD•4(Fe4S4)•FMNH2. The catalytic chemistry of DPD can be studied in transient-state by observation of either NADPH consumption or charge transfer absorption associated with complexation of NADPH adjacent to the FAD. Here we have utilized both sets of absorption transitions to find evidence for specific additional aspects of the DPD mechanism. Competition for binding with NADP+ indicates that the two charge transfer species observed in activation/single turnover reactions arise from NADPH populating the FAD site before and after reductive activation. An additional charge transfer species is observed to accumulate at longer times when high NADPH concentrations are mixed with the enzyme•pyrimidine complex and this data can be modelled based on asymmetry in the homodimer. It was also shown that, like pyrimidines, dihydropyrimidines induce rapid reductive activation indicating that the reduced pyrimidine formed in turnover can stimulate the reinstatement of the active state of the enzyme. Investigation of the reverse reaction revealed that dihydropyrimidines alone can reductively activate the enzyme, albeit inefficiently. In the presence of dihydropyrimidine and NADP+ DPD will form NADPH but apparently without measurable reductive activation. Pyrimidines that have 5-substituent halogens were utilized to probe both reductive activation and turnover. The linearity of the Hammett plot based on the rate of hydride transfer to the pyrimidine establishes that, at least to the radius of an iodo-group, the 5-substituent volume does not have influence on the observed kinetics of pyrimidine reduction.

Effects of cadmium stress on the morphology, physiology, cellular ultrastructure, and BvHIPP24 gene expression of sugar beet (Beta vulgaris L.).

To clarify the mechanism of the response of sugar beet (Beta vulgaris L.) to cadmium (Cd) stress, this study investigated changes in the phenotype, physiological indexes, and subcellular structure of B. vulgaris under Cd treatment and the transcriptional pattern of the BvHIPP24 gene (a heavy metal-associated isoprenylated plant protein involved in heavy metal detoxification). The plant height and shoot and root growth of B. vulgaris seedlings were inhibited to some extent under 0.5 and 1 mM Cd, with gradually wilting and yellowing of leaves and dark brown roots. When the Cd concentration was increased, malondialdehyde content and the activities of peroxidase, superoxide dismutase, and glutathione S-transferase increased differentially. qPCR indicated that the expression of BvHIPP24 was induced by different concentrations of Cd. Although transmission electron microscopy revealed damage to nuclei, mitochondria, and chloroplasts, B. vulgaris exhibited strong adaptability to 0.5 mM Cd according to a comprehensive analysis using the membership function. The results showed that B. vulgaris may reduce cell damage and improve its Cd tolerance by regulating functional gene expression and antioxidant enzymes. This study increases our understanding of the Cd-tolerance mechanism of B. vulgaris and provides insights into the use of B. vulgaris in Cd bioremediation.

Sugar beet is a novel energy crop with superior characteristics for both heavy metal phytoremediation and biomass energy development. This work is the first to investigate both the morphological, physiological, and ultrastructural response of sugar beet to cadmium stress and the induction of a functional metallochaperone gene by cadmium. This study explains the cadmium tolerance mechanism of sugar beet based on a comprehensive evaluation and provides an important theoretical basis for further application of beet in heavy metal bioremediation.

Tolerance and adaptation characteristics of sugar beet (Beta vulgaris L.) to low nitrogen supply.

Nitrogen (N) is an essential element required for sugar beet growth. Sugar beets with low N (LN) tolerance and high N use efficiency are excellent materials for breeding. Here, we comprehensively evaluated the morphological and physiological responses of nine sugar beet genotypes to LN supply. It was found that 0.5 mmol·L-1 N (LN) significantly influenced the performance of leaves and the topology of roots by reducing the bioproduction of chlorophyll a (Chl a) and soluble protein (SP) and the accumulation of N in leaves and roots (LNA and RNA), thus differentially restricting the growth (hypocotyl diameter, HD; root length, RL) and biomass (leaf and root fresh weight; LFW and RFW; leaf dry weight, LDW) of these sugar beets. Principal component and cluster analyses showed that 780016B/12 superior (F) exhibited excellent tolerance to LN; it had higher SOD activity (62.70%) and APX activity (188.92%) and a higher proline content (131.82%) than 92011 (G, LN sensitive). These attributes helped 780016B/12 superior (F) to better endure LN stress, and the morphology and N distribution changed to adapt to N deficiency, such that the root length increased by 112.48%, leaf area increased by 101.23%, and leaf nitrogen accumulation reached a peak of 14.13 g/plant. It seems that LN-tolerant genotypes increased their root length and surface area by reducing the difference in biomass, thereby expanding the contact between roots and soil, which was conducive to the absorption of nutrients (N) by sugar beets and helped distribute more assimilation products to the roots.

Acclimation of sugar beet in morphological, physiological and BvAMT1.2 expression under low and high nitrogen supply.

Understanding the response and tolerance mechanisms of nitrogen (N) stress is essential for the taproot plant of sugar beet. Hence, in this study, low (0.5 and 3 mmol/L; N0.5 and N3), moderate (5 mmol/L; N5; control) and high (10 and 12 mmol/L; N10 and N12) N were imposed to sugar beet to comparatively investigate the growth and physiological changes, and expression pattern of the gene involving ammonia transporting at different seedling stages. The results showed that, different from N5 which could induce maximum biomass of beet seedlings, low N was more likely to inhibit the growth of beet seedlings than high N treatments. Morphological differences and adverse factors increased significantly with extension of stress time, but sugar beet seedlings displayed a variety of physical responses to different N concentrations to adapt to N abnormal. At 14 d, the chlorophyll content, leaf and root surface area, total dry weight and nitrogen content of seedlings treated with N0.5 decreased 15.83%, 53.65%, 73.94%, 78.08% and 24.88% respectively, compared with N12; however, the root shoot ratio increased significantly as well as superoxide dismutase (SOD), peroxidase (POD), glutamine synthetase (GS) activity and malondialdehyde (MDA) and proline content, especially in root. The expression of BvAMT1.2 was also regulated in an N concentration-dependent manner, and was mainly involved in the tolerance of beet leaves to N stress, which significantly positively correlated to GS activity on the basis of its high affinity to N. It can be deduced that the stored nutrients under low N could only maintain relatively stable root growth, and faced difficulty in being transported to the shoots. Sugar beet was relatively resilient to N0.5 stress according to the mean affiliation function analysis. These results provide a theoretical basis for the extensive cultivation of sugar beet in N-stressed soil.

Site-directed mutagenesis of Serine-72 reveals the location of the fructose 6-phosphate regulatory site of the Agrobacterium tumefaciens ADP-glucose pyrophosphorylase.

The allosteric regulation of ADP-glucose pyrophosphorylase is critical for the biosynthesis of glycogen in bacteria and starch in plants. The enzyme from Agrobacterium tumefaciens is activated by fructose 6-phosphate (Fru6P) and pyruvate (Pyr). The Pyr site has been recently found, but the site where Fru6P binds has remained unknown. We hypothesize that a sulfate ion previously found in the crystal structure reveals a part of the regulatory site mimicking the presence of the phosphoryl moiety of the activator Fru6P. Ser72 interacts with this sulfate ion and, if the hypothesis is correct, Ser72 would affect the interaction with Fru6P and activation of the enzyme. Here, we report structural, binding, and kinetic analysis of Ser72 mutants of the A. tumefaciens ADP-glucose pyrophosphorylase. By X-ray crystallography, we found that when Ser72 was replaced by Asp or Glu side chain carboxylates protruded into the sulfate-binding pocket. They would present a strong steric and electrostatic hindrance to the phosphoryl moiety of Fru6P, while being remote from the Pyr site. In agreement, we found that Fru6P could not activate or bind to S72E or S72D mutants, whereas Pyr was still an effective activator. These mutants also blocked the binding of the inhibitor AMP. This could potentially have biotechnological importance in obtaining enzyme forms insensitive to inhibition. Other mutations in this position (Ala, Cys, and Trp) confirmed the importance of Ser72 in regulation. We propose that the ADP-glucose pyrophosphorylase from A. tumefaciens have two distinct sites for Fru6P and Pyr working in tandem to regulate glycogen biosynthesis.

Determination of the pH dependence, substrate specificity, and turnovers of alternative substrates for human ornithine aminotransferase.

Hepatocellular carcinoma (HCC) is the most common primary cancer of the liver and occurs predominantly in patients with underlying chronic liver diseases. Over the past decade, human ornithine aminotransferase (hOAT), which is an enzyme that catalyzes the metabolic conversion of ornithine into an intermediate for proline or glutamate synthesis, has been found to be overexpressed in HCC cells. hOAT has since emerged as a promising target for novel anticancer therapies, especially for the ongoing rational design effort to discover mechanism-based inactivators (MBIs). Despite the significance of hOAT in human metabolism and its clinical potential as a drug target against HCC, there are significant knowledge deficits with regard to its catalytic mechanism and structural characteristics. Ongoing MBI design efforts require in-depth knowledge of the enzyme active site, in particular, pKa values of potential nucleophiles and residues necessary for the molecular recognition of ligands. Here, we conducted a study detailing the fundamental active-site properties of hOAT using stopped-flow spectrophotometry and X-ray crystallography. Our results quantitatively revealed the pH dependence of the multistep reaction mechanism and illuminated the roles of ornithine α-amino and δ-amino groups in substrate recognition and in facilitating catalytic turnover. These findings provided insights of the catalytic mechanism that could benefit the rational design of MBIs against hOAT. In addition, substrate recognition and turnover of several fragment-sized alternative substrates of hOATs, which could serve as structural templates for MBI design, were also elucidated.