Crystal structure of dihydroneopterin aldolase from Mycobacterium tuberculosis associated with 8-mercaptoguanine, and development of novel S8-functionalized analogues as inhibitors: Synthesis, enzyme inhibition, in vitro toxicity and antitubercular activity.

The crystallographic structure of the FolB enzyme from Mycobacterium tuberculosis (MtFolB), complexed with its inhibitor 8-mercaptoguanine (8-MG), was elucidated at a resolution of 1.95 Å. A novel series of S8-functionalized 8-MG derivatives were synthesised and evaluated as in vitro inhibitors of dihydroneopterin aldolase (DHNA, EC 4.1.2.25) activity of MtFolB. These compounds exhibited IC50 values in the submicromolar range. Evaluation of the activity for five compounds indicated their inhibition mode and inhibition constants. Molecular docking analyses were performed to determine the enzyme-inhibitor intermolecular interactions and ligand conformations upon complex formation. The inhibitory activities of all compounds against the M. tuberculosis H37Rv strain were evaluated. Compound 3e exhibited a minimum inhibitory concentration in the micromolar range. Finally, Compound 3e showed no apparent toxicity in both HepG2 and Vero cells. The findings presented herein will advance the quest for novel, specific inhibitors targeting MtFolB, an attractive molecular target for TB drug development.

Diverse roles of aldolase enzymes in cancer development, drug resistance and therapeutic approaches as moonlighting enzymes.

Aldolase enzymes, particularly ALDOA, ALDOB, and ALDOC, play a crucial role in the development and progression of cancer. While the aldolase family is mainly known for its involvement in the glycolysis pathway, these enzymes also have various pathological and physiological functions through distinct signaling pathways such as Wnt/ß-catenin, EGFR/MAPK, Akt, and HIF-1α. This has garnered increased attention in recent years and shed light on other sides of this enzyme. Potential therapeutic strategies targeting aldolases include using siRNA, inhibitors like naphthol AS-E phosphate and TX-2098, and natural compounds such as HDPS-4II and L-carnosine. Additionally, anticancer peptides derived from ALDOA, like P04, can potentially increase cancer cells' sensitivity to chemotherapy. Aldolases also affect cancer drug resistance by different approaches, making them good therapeutic targets. In this review, we extensively explore the role of aldolase enzymes in various types of cancers in proliferation, invasion, migration, and drug resistance; we also significantly explore the possible treatment considering aldolase function.

Hepatic glucokinase regulatory protein and carbohydrate response element binding protein attenuation reduce de novo lipogenesis but do not mitigate intrahepatic triglyceride accumulation in Aldob deficiency.

OBJECTIVE: Stable isotope studies have shown that hepatic de novo lipogenesis (DNL) plays an important role in the pathogenesis of intrahepatic lipid (IHL) deposition. Furthermore, previous research has demonstrated that fructose 1-phosphate (F1P) not only serves as a substrate for DNL, but also acts as a signalling metabolite that stimulates DNL from glucose. The aim of this study was to elucidate the mediators of F1P-stimulated DNL, with special focus on two key regulators of intrahepatic glucose metabolism, i.e., glucokinase regulatory protein (GKRP) and carbohydrate response element binding protein (ChREBP). METHODS: Aldolase B deficient mice (Aldob-/-), characterized by hepatocellular F1P accumulation, enhanced DNL, and hepatic steatosis, were either crossed with GKRP deficient mice (Gckr-/-) or treated with short hairpin RNAs directed against hepatic ChREBP. RESULTS: Aldob-/- mice showed higher rates of de novo palmitate synthesis from glucose when compared to wildtype mice (p < 0.001). Gckr knockout reduced de novo palmitate synthesis in Aldob-/- mice (p = 0.017), without affecting the hepatic mRNA expression of enzymes involved in DNL. In contrast, hepatic ChREBP knockdown normalized the hepatic mRNA expression levels of enzymes involved in DNL and reduced fractional DNL in Aldob-/- mice (p < 0.05). Of interest, despite downregulation of DNL in response to Gckr and ChREBP attenuation, no reduction in intrahepatic triglyceride levels was observed. CONCLUSIONS: Both GKRP and ChREBP mediate F1P-stimulated DNL in aldolase B deficient mice. Further studies are needed to unravel the role of GKRP and hepatic ChREBP in regulating IHL accumulation in aldolase B deficiency.

4-hydroxy-2-oxoglutarate metabolism in a mouse model of Primary Hyperoxaluria Type 3.

Primary Hyperoxaluria Type 3 (PH3) results from 4-hydroxy-2-oxoglutarate (HOG) aldolase (HOGA) deficiency, which causes an increase in endogenous oxalate synthesis leading to calcium oxalate kidney stone disease. The mechanisms underlying HOG metabolism and increased oxalate synthesis in PH3 are not well understood. We used a Hoga1 knock-out mouse model of PH3 to investigate two aspects of HOG metabolism: reduction to dihydroxyglutarate (DHG), a pathway that may limit oxalate synthesis in PH3, and metabolism to glyoxylate, which is a direct precursor to oxalate. The metabolism of HOG to DHG was highest in liver and kidney cortical tissue, enhanced in the cytosolic compartment of the liver, and preferred NADPH as a cofactor. In the absence of HOGA, HOG to glyoxylate aldolase activity was highest in liver mitoplasts, with no activity present in brain tissue lysates. These findings will assist in the identification of enzymes responsible for the metabolism of HOG to DHG and glyoxylate, which may lead to novel therapeutic approaches to limit oxalate synthesis in those afflicted with PH3.

Genome-Wide Characterization of Fructose 1,6-Bisphosphate Aldolase Genes and Expression Profile Reveals Their Regulatory Role in Abiotic Stress in Cucumber.

The fructose-1,6-bisphosphate aldolase (FBA) gene family exists in higher plants, with the genes of this family playing significant roles in plant growth and development, as well as response to abiotic stresses. However, systematic reports on the FBA gene family and its functions in cucumber are lacking. In this study, we identified five cucumber FBA genes, named CsFBA1-5, that are distributed randomly across chromosomes. Phylogenetic analyses involving these cucumber FBAs, alongside eight Arabidopsis FBA proteins and eight tomato FBA proteins, were conducted to assess their homology. The CsFBAs were grouped into two clades. We also analyzed the physicochemical properties, motif composition, and gene structure of the cucumber FBAs. This analysis highlighted differences in the physicochemical properties and revealed highly conserved domains within the CsFBA family. Additionally, to explore the evolutionary relationships of the CsFBA family further, we constructed comparative syntenic maps with Arabidopsis and tomato, which showed high homology but only one segmental duplication event within the cucumber genome. Expression profiles indicated that the CsFBA gene family is responsive to various abiotic stresses, including low temperature, heat, and salt. Taken together, the results of this study provide a theoretical foundation for understanding the evolution of and future research into the functional characterization of cucumber FBA genes during plant growth and development.

Structure of the iminium reaction intermediate in an engineered aldolase explains the carboligation activity toward arylated ketones and aldehydes.

Two structures of fructose 6-phosphate aldolase, the wild-type and an engineered variant containing five active-site mutations, have been solved by cryoelectron microscopy (cryo-EM). The engineered variant affords production of aldols from aryl substituted ketones and aldehydes. This structure was solved to a resolution of 3.1 Å and contains the critical iminium reaction intermediate trapped in the active site. This provides new information that rationalizes the acquired substrate scope and aids in formulating hypotheses of the chemical mechanism. A Tyr residue (Y131) is positioned for a role as catalytic acid/base during the aldol reaction and the different structures demonstrate mobility of this amino acid residue. Further engineering of this fructose 6-phosphate aldolase (FSA) variant, guided by this new structure, identified additional FSA variants that display improved carboligation activities with 2-hydroxyacetophenone and phenylacetaldehyde.

An aldolase-dependent phloroglucinol degradation pathway in Collinsella sp. zg1085.

Phloroglucinol (1,3,5-trihydroxybenzene) is a key intermediate in the degradation of polyphenols such as flavonoids and hydrolysable tannins and can be used by certain bacteria as a carbon and energy source for growth. The identification of enzymes that participate in the fermentation of phloroglucinol to acetate and butyrate in Clostridia was recently reported. In this study, we present the discovery and characterization of a novel metabolic pathway for phloroglucinol degradation in the bacterium Collinsella sp. zg1085, from marmot respiratory tract. In both the Clostridial and Collinsella pathways, phloroglucinol is first reduced to dihydrophoroglucinol by the NADPH-dependent phloroglucinol reductase (PGR), followed by ring opening to form (S)-3-hydroxy-5-oxohexanoate by a Mn2+-dependent dihydrophloroglucinol cyclohydrolase (DPGC). In the Collinsella pathway, (S)-3-hydroxy-5-oxohexanoate is then cleaved to form malonate semialdehyde and acetone by a newly identified aldolase (HOHA). Finally, a NADP+-dependent malonate-semialdehyde dehydrogenase converts malonate semialdehyde to CO2 and acetyl-CoA, an intermediate in carbon and energy metabolism. Recombinant expression of the Collinsella PGR, DPGC, and HOHA in E. coli enabled the conversion of phloroglucinol into acetone, providing support for the proposed pathway. Experiments with Olsenella profusa, another bacterium containing the gene cluster of interest, show that the PGR, DPGC, HOHA, and MSDH are induced by phloroglucinol. Our findings add to the variety of metabolic pathways for the degradation of phloroglucinol, a widely distributed phenolic compound, in the anaerobic microbiome.IMPORTANCEPhloroglucinol is an important intermediate in the bacterial degradation of polyphenols, a highly abundant class of plant natural products. Recent research has identified key enzymes of the phloroglucinol degradation pathway in butyrate-producing anaerobic bacteria, which involves cleavage of a linear triketide intermediate by a beta ketoacid cleavage enzyme, requiring acetyl-CoA as a co-substrate. This paper reports a variant of the pathway in the lactic acid bacterium Collinsella sp. zg1085, which involves cleavage of the triketide intermediate by a homolog of deoxyribose-5-phosphate aldolase, highlighting the variety of mechanisms for phloroglucinol degradation by different anaerobic bacterial taxa.

Characterization of Arabidopsis aldolases AtFBA4, AtFBA5, and their inhibition by morin and interaction with calmodulin.

Fructose bisphosphate aldolases (FBAs) catalyze the reversible cleavage of fructose 1,6-bisphosphate into dihydroxyacetone phosphate and glyceraldehyde 3-phosphate. We analyzed two previously uncharacterized cytosolic Arabidopsis FBAs, AtFBA4 and AtFBA5. Based on a recent report, we examined the interaction of AtFBA4 with calmodulin (CaM)-like protein 11 (AtCML11). AtFBA4 did not bind AtCML11; however, we found that CaM bound AtFBA5 in a Ca2+-dependent manner with high specificity and affinity (KD ~ 190 nm) and enhanced its stability. AtFBA4 and AtFBA5 exhibited Michaelis-Menten kinetics with Km and Vmax values of 180 µm and 4.9 U·mg-1 for AtFBA4, and 6.0 µm and 0.30 U·mg-1 for AtFBA5, respectively. The flavonoid morin inhibited both isozymes. Our study suggests that Ca2+ signaling and flavanols may influence plant glycolysis/gluconeogenesis.

The metal cofactor: stationary or mobile?

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.

Identification and characterization of FBA genes in moso bamboo reveals PeFBA8 related to photosynthetic carbon metabolism.

Fructose 1,6-bisphosphate aldolase (FBA) is a pivotal enzyme, which plays a critical role in fixing CO2 through the process of in the Calvin cycle. In this study, a comprehensive exploration of the FBA family genes in moso bamboo (Phyllostachys edulis) was conducted by the bioinformatics and biological analyses. A total of nine FBA genes (PeFBA1-PeFBA9) were identified in the moso bamboo genome. The expression patterns of PeFBAs across diverse tissues of moso bamboo suggested that they have multifaceted functionality. Notably, PeFBA8 might play an important role in regulating photosynthetic carbon metabolism. Co-expression and cis-element analyses demonstrated that PeFBA8 was regulated by a photosynthetic regulatory transcription factor (PeGLK1), which was confirmed by yeast one-hybrid and dual-luciferase assays. In-planta gene editing analysis revealed that the edited PeFBA8 mutants displayed compromised photosynthetic functionality, characterized by reduced electron transport rate and impaired photosystem I, leading to decreased photosynthesis rate overall, compared to the unedited control. The recombinant protein of PeFBA8 from prokaryotic expression exhibited enzymatic catalytic function. The findings suggest that the expression of PeFBA8 can affect photosynthetic efficiency of moso bamboo leaves, which underlines the potential of leveraging PeFBA8's regulatory mechanism to breed bamboo varieties with enhanced carbon fixation capability.

A Case Study of a Rare Disease (Fructosemia) Diagnosed in a Patient with Abdominal Pain.

Hereditary fructose intolerance is a rare genetic disorder that is inherited in an autosomal recessive manner, with mutations sometimes occurring spontaneously. Consuming fructose triggers biochemical abnormalities, disrupting liver processes like glycogenolysis and gluconeogenesis. Recent studies have revealed elevated intrahepatic fat levels in affected individuals. Symptoms include aversion to fructose-containing foods, hypoglycemia, liver and kidney dysfunction, and growth delays, with severe cases leading to liver enlargement, fatty liver disease, kidney failure, and life-threatening hypoglycemia. In this case study, we present a 20-month-old child with symptoms including difficulty passing stool, abdominal rigidity, abdominal pain with bloating and hypoglycemia. Initial clinical findings revealed elevated liver enzymes, a mildly enlarged hyperechoic liver, hypercholesterolemia, and borderline alpha-fetoprotein values. Diagnostic assessments identified hereditary fructose intolerance (HFI) with pathogenic variants in the ALDOB gene, along with a diagnosis of celiac disease. Genetic testing of the parents revealed carrier status for pathological aldolase B genes. This case underscores the importance of comprehensive clinical evaluation and genetic testing in pediatric patients with complex metabolic presentations.

PPAR-γ agonists reactivate the ALDOC-NR2F1 axis to enhance sensitivity to temozolomide and suppress glioblastoma progression.

Glioblastoma (GBM) is a type of brain cancer categorized as a high-grade glioma. GBM is characterized by limited treatment options, low patient survival rates, and abnormal serotonin metabolism. Previous studies have investigated the tumor suppressor function of aldolase C (ALDOC), a glycolytic enzyme in GBM. However, it is unclear how ALDOC regulates production of serotonin and its associated receptors, HTRs. In this study, we analyzed ALDOC mRNA levels and methylation status using sequencing data and in silico datasets. Furthermore, we investigated pathways, phenotypes, and drug effects using cell and mouse models. Our results suggest that loss of ALDOC function in GBM promotes tumor cell invasion and migration. We observed that hypermethylation, which results in loss of ALDOC expression, is associated with serotonin hypersecretion and the inhibition of PPAR-γ signaling. Using several omics datasets, we present evidence that ALDOC regulates serotonin levels and safeguards PPAR-γ against serotonin metabolism mediated by 5-HT, which leads to a reduction in PPAR-γ expression. PPAR-γ activation inhibits serotonin release by HTR and diminishes GBM tumor growth in our cellular and animal models. Importantly, research has demonstrated that PPAR-γ agonists prolong animal survival rates and increase the efficacy of temozolomide in an orthotopic brain model of GBM. The relationship and function of the ALDOC-PPAR-γ axis could serve as a potential prognostic indicator. Furthermore, PPAR-γ agonists offer a new treatment alternative for glioblastoma multiforme (GBM).

Turn-on Coumarin Precursor: From Hydrazine Sensor to Covalent Inhibition and Fluorescence Detection of Rabbit Muscle Aldolase.

Hydrazine, a highly toxic compound, demands sensitive and selective detection methods. Building upon our previous studies with pre-coumarin OFF-ON sensors for fluoride anions, we extended our strategy to hydrazine sensing by adapting phenol protecting groups (propionate, levulinate, and γ-bromobutanoate) to our pre-coumarin scaffold. These probes reacted with hydrazine, yielding a fluorescent signal with low micromolar limits of detection. Mechanistic studies revealed that hydrazine deprotection may be outperformed by a retro-Knoevenagel reaction, where hydrazine acts as a nucleophile and a base yielding a fluorescent diimide compound (6,6'-((1E,1'E)-hydrazine-1,2diylidenebis(methaneylylidene))bis(3(diethylamino)phenol, 7). Additionally, our pre-coumarins unexpectedly reacted with primary amines, generating a fluorescent signal corresponding to phenol deprotection followed by cyclization and coumarin formation. The potential of compound 3 as a theranostic Turn-On coumarin precursor was also explored. We propose that its reaction with ALDOA produced a γ-lactam, blocking the catalytic nucleophilic amine in the enzyme's binding site. The cleavage of the ester group in compound 3 induced the formation of fluorescent coumarin 4. This fluorescent signal was proportional to ALDOA concentration, demonstrating the potential of compound 3 for future theranostic studies in vivo.

Optimization of the Diagnostic Capacity of Traditional Biomarkers in Muscle Damage and Its Use in the Diagnosis of Dermatomyositis and Polymyositis.

BACKGROUND-AIM: Creatine kinase (CK) and aldolase are markers traditionally used in the study of muscle damage (MD). As CK determination is more specific to muscle damage, the demand for both determinations in routine laboratory tests would entail an extra cost. METHODS: Retrospective observational study conducted between 2019-2020. CK and aldolase concentrations from 218 patients were studied.ROC curves were analyzed for CK and aldolase for muscle damage detection. Cut-off values were selected for both strategies. Specifity of CK and aldolase for dermatomyositis or polymyositis diagnosis in our population was studied using the McNemar's test. RESULTS: The area under the ROC curve (AUC) for total CK was 0.716 (95%CI: 0.651-0.775), for CK in males it was 0.703 (95%CI: 0.592-0.799), and for CK in females was 0.719 (95%CI: 0.636-0.793). For aldolase, AUC was 0.505 (95%CI: 0.437-0.573). Optimized cut-off points for each determination were: 112 U/L for CK in men, with a sensitivity of 73.9% (95%CI: 51.6-89.8) and a specificity of 49.2% (95%CI: 35.9-62.5); 88 U/L for CK in women, with a sensitivity of 75.0% (95%CI: 57.8-87.9) and specificity of 50.5% (95%CI: 40.4-60.6); and 5.6 U/L for aldolase, with a sensitivity of 61.0% (95%CI: 53.2-68.8) and a specificity of 38.8% (95%CI: 26.5-52.6).Regarding the individuals diagnosed with dermatomyositis or polymyositis, 66.7% and 44.4% of them were correctly classified as pathological by CK and aldolase results, respectively. McNemar's test did not reveal significant differences. CONCLUSION: The determination of CK offers a better diagnostic performance of MD and, in addition, does not present significant differences regarding the determination of aldolase in cases of polymyositis and dermatomyositis. Therefore, the single determination of CK would be sufficient for MD screening.

Biotransformation of 2-keto-4-hydroxybutyrate via aldol condensation using an efficient and thermostable carboligase from Deinococcus radiodurans.

The bioconversion of 4-hydroxy-2-keto acid derivatives via aldol condensation of formaldehyde and pyruvate has received substantial attention as potential source of chemicals for production of amino acids, hydroxy carboxylic acids, and chiral aldehydes. We developed an environmentally friendly biocatalyst consisting of a novel thermostable class II pyruvate aldolase from Deinococcus radiodurans with maltose-binding protein (MBP-DrADL), which has specific activity of 46.3 µmol min-1 mg-1. Surprisingly, MBP-DrADL maintained over 60% of enzyme activity for 4 days at 50 to 65 °C, we used MBP-DrADL as the best candidate enzyme to produce 2-keto-4-hydroxybutyrate (2-KHB) from formaldehyde and pyruvate via aldol condensation. The optimum reaction conditions for 2-KHB production were 50 °C, pH 8.0, 5 mM Mg2+, 100 mM formaldehyde, and 200 mM pyruvate. Under these optimized conditions, MBP-DrADL produced 76.5 mM (8.94 g L-1) 2-KHB over 60 min with a volumetric productivity of 8.94 g L-1 h-1 and a specific productivity of 357.6 mg mg-enzyme-1 h-1. Furthermore, 2-KHB production was improved by continuous addition of substrates, which produced approximately 124.8 mM (14.6 g L-1) of 2-KHB over 60 min with a volumetric productivity and specific productivity of 14.6 g L-1 h-1 and 583.4 mg mg-enzyme-1 h-1, respectively. MBP-DrADL showed the highest specific productivity for 2-KHB production yet reported. Our study provides a highly efficient biocatalyst for the synthesis of 2-KHB and lays the foundation for large-scale production and application of high-value compounds from formaldehyde.

Discovery and directed evolution of C-C bond formation enzymes for the biosynthesis of ß-hydroxy-α-amino acids and derivatives.

ß-Hydroxy-α-amino acids (ß-HAAs) have extensive applications in the pharmaceutical, chemical synthesis, and food industries. The development of synthetic methodologies aimed at producing optically pure ß-HAAs has been driven by practical applications. Among the various synthetic methods, biocatalytic asymmetric synthesis is considered a sustainable approach due to its capacity to generate two stereogenic centers from simple prochiral precursors in a single step. Therefore, extensive efforts have been made in recent years to search for effective enzymes which enable such biotransformation. This review provides an overview on the discovery and engineering of C-C bond formation enzymes for the biocatalytic synthesis of ß-HAAs. We highlight examples where the use of threonine aldolases, threonine transaldolases, serine hydroxymethyltransferases, α-methylserine aldolases, α-methylserine hydroxymethyltransferases, and engineered alanine racemases facilitated the synthesis of ß-HAAs. Additionally, we discuss the potential future advancements and persistent obstacles in the enzymatic synthesis of ß-HAAs.

Overexpression of the FBA and TPI genes promotes high production of HDMF in Zygosaccharomyces rouxii.

4-Hydroxy-2,5-dimethyl-3 (2H)-furanone (HDMF) is widely used in the food industry as a spice and flavoring agent with high market demand. In this study, fructose-1,6-bisphosphate aldolase (FBA) and triose phosphate isomerase (TPI) were overexpressed in Zygosaccharomyces rouxii in the form of single and double genes, respectively, via electroporation. High-yield HDMF-engineered yeast strains were constructed by combining the analysis of gene expression levels obtained by real-time fluorescence quantitative PCR technology and HDMF production measured by HPLC. The results showed that there was a significant positive correlation between the production of HDMF and the expression levels of the FBA and TPI genes in yeast; the expression levels of the FBA and TPI genes were also positively correlated (p < 0.05). Compared with the wild type (WT), the engineered strains F10-D, T17-D, and TF15-A showed marked increases in HDMF production and FBA and TPI gene expression (p < 0.05) and exhibited great genetic stability with no obvious differences in biomass or colony morphology. In addition, the exogenous addition of d-fructose promoted the growth of Z. rouxii. Among the engineered strains, when fermented in YPD media supplemented with d-fructose for 5 days, TF15-A (overexpressing the FBA and TPI genes) generated the highest HDMF production of 13.39 mg/L, which is 1.91 times greater than that of the wild-type strain. The results above indicated that FBA and TPI, which are key enzymes involved in the process of HDMF biosynthesis by Z. rouxii, positively regulate the synthesis of HDMF at the transcriptional level. d-fructose can be used as a precursor for the biosynthesis of HDMF by engineered yeast in industrial production.

Unveiling the Biological Function of Phyllostachys edulis FBA6 (PeFBA6) through the Identification of the Fructose-1,6-Bisphosphate Aldolase Gene.

Fructose-1,6-bisphosphate aldolase (FBA) is a pivotal enzyme in various metabolic pathways, including glycolysis, gluconeogenesis, and the Calvin cycle. It plays a critical role in CO2 fixation. Building on previous studies on the FBA gene family in Moso bamboo, our study revealed the biological function of PeFBA6. To identify CSN5 candidate genes, this study conducted a yeast two-hybrid library screening experiment. Subsequently, the interaction between CSN5 and PeFBA6 was verified using yeast two-hybrid and LCI experiments. This investigation uncovered evidence that FBA may undergo deubiquitination to maintain glycolytic stability. To further assess the function of PeFBA6, it was overexpressed in rice. Various parameters were determined, including the light response curve, CO2 response curve, and the levels of glucose, fructose, sucrose, and starch in the leaves of overexpressing rice. The results demonstrated that overexpressed rice exhibited a higher saturation light intensity, net photosynthetic rate, maximum carboxylation rate, respiration rate, and increased levels of glucose, fructose, and starch than wild-type rice. These findings indicated that PeFBA6 not only enhanced the photoprotection ability of rice but also improved the photosynthetic carbon metabolism. Overall, this study enhanced our understanding of the function of FBA and revealed the biological function of PeFBA6, thereby providing a foundation for the development of excellent carbon fixation bamboo varieties through breeding.

Characterization of FBA genes in potato (Solanum tuberosum L.) and expression patterns in response to light spectrum and abiotic stress.

Fructose-1, 6-bisphosphate aldolase (FBA) plays vital roles in plant growth, development, and response to abiotic stress. However, genome-wide identification and structural characterization of the potato (Solanum tuberosum L.) FBA gene family has not been systematically analyzed. In this study, we identified nine StFBA gene members in potato, with six StFBA genes localized in the chloroplast and three in the cytoplasm. The analysis of gene structures, protein structures, and phylogenetic relationships indicated that StFBA genes were divided into Class I and II, which exhibited significant differences in structure and function. Synteny analysis revealed that segmental duplication events promoted the expansion of the StFBA gene family. Promoter analysis showed that most StFBA genes contained cis-regulatory elements associated with light and stress responses. Expression analysis showed that StFBA3, StFBA8, and StFBA9 showing significantly higher expression levels in leaf, stolon, and tuber under blue light, indicating that these genes may improve photosynthesis and play an important function in regulating the induction and expansion of microtubers. Expression levels of the StFBA genes were influenced by drought and salt stress, indicating that they played important roles in abiotic stress. This work offers a theoretical foundation for in-depth understanding of the evolution and function of StFBA genes, as well as providing the basis for the genetic improvement of potatoes.

Protein kinase Cγ negatively regulates the intrinsic excitability in zebrin-negative cerebellar Purkinje cells.

Protein kinase C γ (PKCγ), a neuronal isoform present exclusively in the central nervous system, is most abundantly expressed in cerebellar Purkinje cells (PCs). Targeted deletion of PKCγ causes a climbing fiber synapse elimination in developing PCs and motor deficit. However, physiological roles of PKCγ in adult mouse PCs are little understood. In this study, we aimed to unravel the roles of PKCγ in mature mouse PCs by deleting PKCγ from adult mouse PCs of PKCγfl/fl mice via cerebellar injection of adeno-associated virus (AAV) vectors expressing Cre recombinase under the control of the PC-specific L7-6 promoter. Whole cell patch-clamp recording of PCs showed higher intrinsic excitability in PCs virally lacking PKCγ [PKCγ-conditional knockout (PKCγ-cKO) PCs] than in wild-type (WT) mouse PCs in the zebrin-negative module, but not in the zebrin-positive module. AAV-mediated PKCγ re-expression in PKCγ-deficient mouse PCs in the zebrin-negative module restored the enhanced intrinsic excitability to a level comparable to that of wild-type mouse PCs. In parallel with higher intrinsic excitability, we found larger hyperpolarization-activated cyclic nucleotide-gated (HCN) channel currents in PKCγ-cKO PCs located in the zebrin-negative module, compared with those in WT mouse PCs in the same region. However, pharmacological inhibition of the HCN currents did not restore the enhanced intrinsic excitability in PKCγ-cKO PCs in the zebrin-negative module. These results suggested that PKCγ suppresses the intrinsic excitability in zebrin-negative PCs, which is likely independent of the HCN current inhibition.