High-Resolution Views and Transport Mechanisms of the NKCC1 and KCC Transporters

Cation-chloride cotransporters (CCCs) are responsible for the coupled co-transport of Cl^- with K⁺ and/or Na⁺ in an electroneutral manner. They play important roles in myriad fundamental physiological processes––from cell volume regulation to transepithelial solute transport and intracellular ion homeostasis––and are targeted by medicines commonly prescribed to treat hypertension and edema. After several decades of studies into the functions and pharmacology of these transporters, there have been several breakthroughs in the structural determination of CCC transporters. The insights provided by these new structures for the Na⁺/K⁺/Cl^- cotransporter NKCC1 and the K⁺/Cl^- cotransporters KCC1, KCC2, KCC3 and KCC4 have deepened our understanding of their molecular basis and transport function. This focused review discusses recent advances in the structural and mechanistic understanding of CCC transporters, including architecture, dimerization, functional roles of regulatory domains, ion binding sites, and coupled ion transport.     

A Conserved Motif in Intracellular Loop 1 Stabilizes the Outward-Facing Conformation of TmrAB

The ATP binding cassette (ABC) family of transporters moves small molecules (lipids, sugars, peptides, drugs, nutrients) across membranes in nearly all organisms. Transport activity requires conformational switching between inward-facing and outward-facing states driven by ATP-dependent dimerization of two nucleotide binding domains (NBDs). The mechanism that connects ATP binding and hydrolysis in the NBDs to conformational changes in a substrate binding site in the transmembrane domains (TMDs) is currently an outstanding question. Here we use sequence coevolution analyses together with biochemical characterization to investigate the role of a highly conserved region in intracellular loop 1 we define as the GRD motif in coordinating domain rearrangements in the heterodimeric peptide exporter from Thermus thermophilus, TmrAB. Mutations in the GRD motif alter ATPase activity as well as transport. Disulfide crosslinking, evolutionary trace, and evolutionary coupling analysis reveal that these effects are likely due to the destabilization of a network in which the GRD motif in TmrA bridges residues of the Q-loop, X-loop, and ABC motif in the NBDs to residues in the TmrAB peptide substrate binding site, thus providing an avenue for conformational coupling. We further find that disruption of this network in TmrA versus TmrB has different functional consequences, hinting at an intrinsic asymmetry in heterodimeric ABC transporters extending beyond that of the NBDs. These results support a mechanism in which the GRD motifs help coordinate a transition to an outward open conformation, and each half of the transporter likely plays a different role in the conformational cycle of TmrAB.     

Triggering Closure of a Sialic Acid TRAP Transporter Substrate Binding Protein through Binding of Natural or Artificial Substrates

The pathogens Vibrio cholerae and Haemophilus influenzae use tripartite ATP-independent periplasmic transporters (TRAPs) to scavenge sialic acid from host tissues. They use it as a nutrient or to evade the innate immune system by sialylating surface lipopolysaccharides. An essential component of TRAP transporters is a periplasmic substrate binding protein (SBP). Without substrate, the SBP has been proposed to rest in an open-state, which is not recognised by the transporter. Substrate binding induces a conformational change of the SBP and it is thought that this closed state is recognised by the transporter, triggering substrate translocation. Here we use real time single molecule FRET experiments and crystallography to investigate the open- to closed-state transition of VcSiaP, the SBP of the sialic acid TRAP transporter from V. cholerae. We show that the conformational switching of VcSiaP is strictly substrate induced, confirming an important aspect of the proposed transport mechanism. Two new crystal structures of VcSiaP provide insights into the closing mechanism. While the first structure contains the natural ligand, sialic acid, the second structure contains an artificial peptide in the sialic acid binding site. Together, the two structures suggest that the ligand itself stabilises the closed state and that SBP closure is triggered by physically bridging the gap between the two lobes of the SBP. Finally, we demonstrate that the affinity for the artificial peptide substrate can be substantially increased by varying its amino acid sequence and by this, serve as a starting point for the development of peptide-based inhibitors of TRAP transporters.     

Towards Molecular Understanding of the pH Dependence Characterizing NhaA of Which Structural Fold is Shared by Other Transporters

Na⁺/H⁺ antiporters comprise a super-family (CPA) of membrane proteins that are found in all kingdoms of life and are essential in cellular homeostasis of pH, Na⁺ and volume. Their activity is strictly dependent on pH, a property that underpins their role in pH homeostasis. While several human homologues have long been drug targets, NhaA of Escherichia coli has become the paradigm for this class of secondary active transporters as NhaA crystal structure provided insight into the architecture of this molecular machine. However, the mechanism of the strict pH dependence of NhaA is missing. Here, as a follow up of a recent evolutionary analysis that identified a ‘CPA motif’, we rationally designed three E. coli NhaA mutants: D133S, I134T, and the double mutant D133S-I134T. Exploring growth phenotype, transport activity and Li⁺-binding of the mutants, we revealed that Asp133 does not participate directly in proton binding, nor does it directly dictate the pH-dependent transport of NhaA. Strikingly, the variant I134T lost some of the pH control, and the D133S-Il134T double mutant retained Li⁺ binding in a pH independent fashion. Concurrent to loss of pH control, these mutants bound Li⁺ more strongly than the WT. Both positions are in close vicinity to the ion-binding site of the antiporter, attributing the results to electrostatic interaction between these residues and Asp164 of the ion-binding site. This is consistent with pH sensing resulting from direct coupling between cation binding and deprotonation in Asp164, which applies also to other CPA antiporters that are involved in human diseases.     

Unmasking the Conformational Stability and Inhibitor Binding to SARS-CoV-2 Main Protease Active Site Mutants and Miniprecursor

We recently demonstrated that inhibitor binding reorganizes the oxyanion loop of a monomeric catalytic domain of SARS CoV-2 main protease (MPro) from an unwound (E) to a wound (active, E*) conformation, independent of dimerization. Here we assess the effect of the flanking N-terminal residues, to imitate the MPro precursor prior to its autoprocessing, on conformational equilibria rendering stability and inhibitor binding. Thermal denaturation (T_m) of C145A mutant, unlike H41A, increases by 6.8 °C, relative to wild-type mature dimer. An inactivating H41A mutation to maintain a miniprecursor containing TSAVL[Q or E] of the flanking nsp4 sequence in an intact form [^(-6)MPro^H41A and ^(-6*)MPro^H41A, respectively], and its corresponding mature MPro^H41A were systematically examined. While the H41A mutation exerts negligible effect on T_m and dimer dissociation constant (K_dimer) of MPro^H41A, relative to the wild type MPro, both miniprecursors show a 4–5 °C decrease in T_m and > 85-fold increase in K_dimer as compared to MPro^H41A. The K_d for the binding of the covalent inhibitor GC373 to ^(-6*)MPro^H41A increases ～12-fold, relative to MPro^H41A, concomitant with its dimerization. While the inhibitor-free dimer exhibits a state in transit from E to E* with a conformational asymmetry of the protomers’ oxyanion loops and helical domains, inhibitor binding restores the asymmetry to mature-like oxyanion loop conformations (E*) but not of the helical domains. Disorder of the terminal residues 1–2 and 302–306 observed in both structures suggest that N-terminal autoprocessing is tightly coupled to the E-E* equilibrium and stable dimer formation.     

Conformational analysis by NMR and molecular dynamics of adamantane-doxorubicin prodrugs and their assemblies with β-cyclodextrin: A focus on the design of platforms for controlled drug delivery

Nanoscale design and construction of affinity-based drug delivery systems (ADDS) is an active research area with enormous potential for the improvement of cancer treatment. For the therapeutic load of these ADDS, a promising strategy is the design of pH-sensitive prodrugs based on the construction of conjugates between adamantane and doxorubicin (Ad-Dox), which stands out as an excellent model system to obtain novel supramolecular materials. Construction of these prodrugs involves a modification of three zones of doxorubicin which in principle does not affect the action mechanism: the carbonyl group C13 (hydrazone linker), the primary alcohol neighboring the carbonyl (ester linker) and the 3′ amino group of daunosamine sugar (amide linker). These modifications are aimed to improve the efficacy and reduce the systemic toxicity of the drug chemotherapy by controlling its release in cancer cells. In this work, we performed 2D NMR experiments and molecular dynamics simulations to characterize the conformational changes of three constructed prodrugs. Our results demonstrated that ring A and the daunsamine sugar of the hydrazone and amide linkers conserve the half-chair state ⁹H₈, while the ester linker disrupts this conformation. Our study also showed that the hydrazone-linked compound (Ad-h-Dox) does not modify the conformation of the original drug and maintains cytotoxic activity. Moreover, the inclusion complex (IC) of Ad-h-Dox with β-cyclodextrin (βCD) generated a highly soluble platform in water, whereas the ester-linked compound (Ad-e-Dox) causes the loss of biological activity. This study proves that Ad-h-Dox prodrug can be an optimum prodrug and act as a building block for a more complex drug transport system.     

Recent Advances in the Structural Biology of Mg2+ Channels and Transporters

Magnesium ions (Mg²⁺) are the most abundant divalent cations in living organisms and are essential for various physiological processes, including ATP utilization and the catalytic activity of numerous enzymes. Therefore, the homeostatic mechanisms associated with cellular Mg²⁺ are crucial for both eukaryotic and prokaryotic organisms and are thus strictly controlled by Mg²⁺ channels and transporters. Technological advances in structural biology, such as the expression screening of membrane proteins, in meso phase crystallization, and recent cryo-EM techniques, have enabled the structure determination of numerous Mg²⁺ channels and transporters. In this review article, we provide an overview of the families of Mg²⁺ channels and transporters (MgtE/SLC41, TRPM6/7, CorA/Mrs2, CorC/CNNM), and discuss the structural biology prospects based on the known structures of MgtE, TRPM7, CorA and CorC.     

Effect of saline irrigation on plant water traits,photosynthesis and ionic balance in durum wheat genotypes

In the era of climate change, decreased precipitation and increased evapo-transpiration hampers the yield of several cereal crops along with the soil salinity and poor ground water resource. Wheat being the moderately tolerant crop face many challenges in the arid and semi-arid regions under irrigated agriculture. In view of this, the study was planned to explore the potential of durum wheat genotypes under salinity on the basis of physiological traits. Experiment was designed as RBD in three replications to evaluate 15 wheat genotypes with moderate saline irrigation (EC_iw – 6 dS m⁻¹) and extreme saline irrigation (EC_iw – 10 dS m⁻¹) along with one set of control (Best available water). Different physiological traits such as water potential (ψ_p), osmotic potential (ψ_s), relative water content (RWC), Na⁺ and K⁺ content were recorded in roots as well as shoots at the reproductive stage whereas photosynthetic rate and chlorophyll content were measured in the flag leaves. A significant variability (p < 0.001) was noted among the genotypes under different stress environments and it was observed that durum genotype HI 8728 and HI 8737 showed less reduction in plant water traits (RWC, ψ_p and ψ_s) than the salinity tolerant checks of bread wheat KRL 99 and KRL 3–4. HD 4728 and HI 8708 maintained higher photosynthetic rate as well as higher chlorophyll content under the extreme salinity level of EC_iw – 10 dSm⁻¹. No significant differences were found in root Na⁺ in genotypes KRL 99 (3.17^g), KRL 3–4 (3.34^g) and HI 8737 (3.41^g) while in shoots, lowest accumulation was seen in KRL 99, MACS 3949 and KRL 3–4 at EC_iw – 10 dSm⁻¹. The mean range of K⁺ content was 7.60–9.74% in roots and 4.21–6.61% in shoots under control environment which decreased to 50.77% in roots and 46.05% in shoots under extreme salinity condition of EC_iw – 10 dSm⁻¹. At EC_iw – 10 dSm⁻¹, KRL 99 maintained highest K^+/Na⁺ in both root and shoot followed by KRL 3–4, HI 8737, MACS 3949, HD 4728 in roots and MACS 3949, KRL 3–4, MACS 4020, HD 4758, MACS 3972 and HI 8713 in shoots. The differential response of durum wheat genotypes under salinity particularly for physiological traits, confer their adaptability towards stress environments and exhibit their potential as genetic sources in breeding programs for improving salt stress tolerance.     

Structural Basis of Drug Recognition by the Multidrug Transporter ABCG2

ABCG2 is an ATP-binding cassette (ABC) transporter whose function affects the pharmacokinetics of drugs and contributes to multidrug resistance of cancer cells. While its interaction with the endogenous substrate estrone-3-sulfate (E₁S) has been elucidated at a structural level, the recognition and recruitment of exogenous compounds is not understood at sufficiently high resolution. Here we present three cryo-EM structures of nanodisc-reconstituted, human ABCG2 bound to anticancer drugs tariquidar, topotecan and mitoxantrone. To enable structural insight at high resolution, we used Fab fragments of the ABCG2-specific monoclonal antibody 5D3, which binds to the external side of the transporter but does not interfere with drug-induced stimulation of ATPase activity. We observed that the binding pocket of ABCG2 can accommodate a single tariquidar molecule in a C-shaped conformation, similar to one of the two tariquidar molecules bound to ABCB1, where tariquidar acts as an inhibitor. We also found single copies of topotecan and mitoxantrone bound between key phenylalanine residues. Mutagenesis experiments confirmed the functional importance of two residues in the binding pocket, F439 and N436. Using 3D variability analyses, we found a correlation between substrate binding and reduced dynamics of the nucleotide binding domains (NBDs), suggesting a structural explanation for drug-induced ATPase stimulation. Our findings provide additional insight into how ABCG2 differentiates between inhibitors and substrates and may guide a rational design of new modulators and substrates.     

Halonatronomonas betaini gen. nov., sp. nov., a haloalkaliphilic isolate from soda lake capable of betaine degradation and proposal of Halarsenatibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the order Halanaerobiales

A search for the organisms responsible for anaerobic betaine degradation in soda lakes resulted in isolation of a novel bacterial strain, designated Z-7014^T. The cells were Gram-stain-negative, non-endospore-forming rods. Growth occurred at 8–52 °C (optimum 40–45 °C), pH 7.1–10.1 (optimum pH 8.1–8.8) and 1.0–3.5 M Na⁺ (optimum 1.8 M), i.e. it can be regarded as a haloalkaliphile. The strain utilized a limited range of substrates, mostly peptonaceous but not amino acids, and was able to degrade betaine. Growth on betaine occurred only in the presence of peptonaceous substances which could not be replaced by vitamins. The G + C content of the genomic DNA of strain Z-7014^T was 36.1 mol%. The major cellular fatty acids (>5% of the total) were C_16:0 DMA, C_{18: 0} DMA, C_16:1ω8, C_16:0, C_18:1 DMA, C_16:1 DMA, C_18:1ω9, and C_18:0. Phylogenetic analysis of the 16S rRNA gene sequence revealed that strain Z-7014^T formed a distinct evolutionary lineage in the order Halanaerobiales with the highest similarity to Halarsenitibacter silvermanii SLAS-1^T (83.6%), Halothermothrix orenii H168^T (85.6%), and Halocella cellulosilytica DSM 7362^T (85.6%). AAI and POCP values between strain Z-7014^T and type strains of the order Halanaerobiales were 51.7–57.8%, and 33.8–58.3%, respectively. Based on polyphasic results including phylogenomic data, the novel strain could be distinguished from other genera, which suggests that strain Z-7014^T represents a novel species of a new genus, for which the name Halonatronomonas betaini gen. nov., sp. nov. is proposed. The type strain is Z-7014^T (=KCTC 25237^T = VKM B-3506^T). On the basis of phylogenomic data, it is also proposed to evolve two novel families Halarsenitibacteraceae fam. nov. and Halothermotrichaceae fam. nov. within the current order Halanaerobiales.     

Principles of Alternating Access in Multidrug and Toxin Extrusion (MATE) Transporters

The multidrug and toxin extrusion (MATE) transporters catalyze active efflux of a broad range of chemically- and structurally-diverse compounds including antimicrobials and chemotherapeutics, thus contributing to multidrug resistance in pathogenic bacteria and cancers. Multiple methodological approaches have been taken to investigate the structural basis of energy transduction and substrate translocation in MATE transporters. Crystal structures representing members from all three MATE subfamilies have been interpreted within the context of an alternating access mechanism that postulates occupation of distinct structural intermediates in a conformational cycle powered by electrochemical ion gradients. Here we review the structural biology of MATE transporters, integrating the crystallographic models with biophysical and computational studies to define the molecular determinants that shape the transport energy landscape. This holistic analysis highlights both shared and disparate structural and functional features within the MATE family, which underpin an emerging theme of mechanistic diversity within the framework of a conserved structural scaffold.     

Conformational Dynamics Allows Sampling of an “Active-like” State by Oncogenic K-Ras-GDP

Mutations in K-Ras GTPase replacing Gly12 with either Asp or Val are common in cancer. These mutations decelerate intrinsic and catalyzed GTP hydrolysis, leading to accumulation of K-Ras-GTP in cells. Signaling cascades initiated by K-Ras-GTP promote cell proliferation, survival, and invasion. Despite functional differences between the most frequent G12D mutation and the most aggressive and chemotherapy resistant G12V mutation, their long-suspected distinct structural features remain elusive. Using NMR, X-ray structures, and computational methods, we found that oncogenic mutants of K-Ras4B, the predominant splice variant of K-Ras, exhibit distinct conformational dynamics when GDP-bound, visiting the “active-like” conformational state similar to the one observed in GTP-bound K-Ras. This behavior distinguishes G12V from wild type and G12D K-Ras4B-GDP. The likely reason is interactions between the aliphatic sidechain of V12 and the Switch II region of K-Ras4B^G12V-GDP, which are distinct in K-Ras4B^G12D-GDP. In the X-ray structures, crystal contacts reduce the dynamics of the sidechain at position 12 by stabilizing the Switch I region of the protein. This explains why structural differences between G12V and G12D K-Ras have yet not been reported. Together, our results suggest a previously unknown mechanism of K-Ras activation. This mechanism relies on conformational dynamics caused by specific oncogenic mutations in the GDP-bound state. Our findings also imply that the therapeutic strategies decreasing the level of K-Ras-GTP by interfering with nucleotide exchange or by expediting GTP hydrolysis may work differently in different oncogenic mutants.     

Heteroarylamide smoothened inhibitors: Discovery of N-[2,4-dimethyl-5-(1-methylimidazol-4-yl)phenyl]-4-(2-pyridylmethoxy)benzamide (AZD8542) and N-[5-(1H-imidazol-2-yl)-2,4-dimethyl-phenyl]-4-(2- pyridylmethoxy)benzamide (AZD7254)

Aberrant hedgehog (Hh) pathway signaling is implicated in multiple cancer types and targeting the Smoothened (SMO) receptor, a key protein of the Hh pathway, has proven effective in treating metastasized basal cell carcinoma. Our lead optimization effort focused on a series of heteroarylamides. We observed that a methyl substitution ortho to the heteroaryl groups on an aniline core significantly improved the potency of this series of compounds. These findings predated the availability of SMO crystal structure in 2013. Here we retrospectively applied quantum mechanics calculations to demonstrate the o-Me substitution favors the bioactive conformation by inducing a dihedral twist between the heteroaryl rings and the core aniline. The o-Me also makes favorable hydrophobic interactions with key residue side chains in the binding pocket. From this effort, two compounds (AZD8542 and AZD7254) showed excellent pharmacokinetics across multiple preclinical species and demonstrated in vivo activity in abrogating the Hh paracrine pathway as well as anti- tumor effects.     

Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Subsets of Slow Dynamic Modes Reveal Global Information Sources as Allosteric Sites

Allostery is a key biological control mechanism, and dynamic information flow provides a perspective to describe allosteric interactions in causal relationships. Here, as a novel implementation of the Gaussian Network Model (GNM) based Transfer Entropy (TE) calculations, we show that the dissection of dynamic information into subsets of slow dynamic modes discloses different layers of multi-directional allosteric pathways inherent in a given protein structure. In these subsets of slow modes, the degree of collectivity (Col) in the information transfer of residues with their TE values (TECol score) identifies distinct residues as powerful effectors, global information sources; showing themselves with a high dynamic capacity to collectively disseminate information to others. As exemplified on aspartate transcarbamoylase (ATCase), Na⁺/K⁺-adenosine triphosphatase (Na⁺/K⁺-ATPase), and human transient receptor potential melastatin 2 (TRPM2) along with a dataset of 20 proteins, these specific residues are associated with known active and allosteric sites. These information source residues, which collectively control others and lead allosteric communication pathways, hint at plausible binding sites for structure-based rational drug design.     

Genome-wide analysis revealed novel molecular features and evolution of Anti-codons in cyanobacterial tRNAs

Transfer RNAs (tRNAs) play important roles to decode the genetic information contained in mRNA in the process of translation. The tRNA molecules possess conserved nucleotides at specific position to regulate the unique function. However, several nucleotides at different position of the tRNA undergo modification to maintain proper stability and function. The major modifications include the presence of pseudouridine (Ψ) residue instead of uridine and the presence of m⁵-methylation sites. We found that, Ψ13 is conserved in D-stem, whereas Ψ38 & Ψ39 were conserved in the anti-codon loop (AL) and anti-codon arm (ACA), respectively. Furthermore, Ψ55 found to be conserved in the Ψ loop. Although, fourteen possible methylation sites can be found in the tRNA, cyanobacterial tRNAs were found to possess conserved G9, m³C₃₂, C₃₆, A₃₇, m⁵C₃₈ and U₅₄ methylation sites. The presence of multiple conserved methylation sites might be responsible for providing necessary stability to the tRNA. The evolutionary study revealed, tRNA^Met and tRNA^Ile were evolved earlier than other tRNA isotypes and their evolution is date back to at least 4000 million years ago. The presence of novel pseudouridination and m⁵-methylation sites in the cyanobacterial tRNAs are of particular interest for basic biology. Further experimental study can delineate their functional significance in protein translation.     

Partial substitution of organic nitrogen with synthetic nitrogen enhances rice yield,grain starch metabolism and related genes expression under the dual cropping system

Improving grain filling in the presernt farming systems is crucial where grain filling is a concern due to the extreme use of chemical fertilizers (CF). A field experiment was conducted at the experimental station of Guangxi University, China in 2019 to test the hypothesis that cattle manure (CM) and poultry manure (PM) combined with CF could improve rice grain filling rate, yield, biochemical and qualitative attributes. A total of six treatments, i.e., no fertilizer (T₁), 100% CF (T₂), 60% CM + 40% CF (T₃), 30% CM + 70% CF (T₄), 60% PM + 40% CF (T₅), and 30% PM + 70% CF (T₆) were used in this study. Results showed that the combined treatment T₆increased starch metabolizing enzymes activity (SMEs), such as ADP-glucose phosphorylase (ADGPase) by 8 and 12%, soluble starch synthase (SSS) by 7 and 10%, granule bound starch synthesis (GBSS) by 7 and 9%, and starch branching enzyme (SBE) by 14 and 21% in the early and late seasons, respectively, compared with T₂. Similarly, higher rice grain yield, grain filling rate, starch, and amylose content were also recorded in combined treatments. In terms of seasons, higher activity of SMEs , grain starch, and amylose content was noted in the late-season compared to the early season. The increment in these traits was mainly attributed to a lower temperature in the late season during the grain filling period. Furthermore, our results suggested that an increment in starch accumulation and grain filling rate were mainly associated with the enhanced sink capacity by regulating key enzyme activities involved in Suc-to-starch conversion. In-addition, RT-qPCR analysis showed higher expression levels of AGPS2b, SSS1, GBSS1, and GBSE11b genes, which resultantly increased the activities of SMEs during the grain filling period under combined treatments. Linear regression analysis revealed that the activity of ADGPase, SSS, GBSS, and SBE were highly positively correlated with starch and amylose accumulation. Thus, we concluded that a combination of 30% N from PM or CM with 70% N from CF is a promising option in terms of improving rice grain yield and quality. Our study provides a sustainable fertilizer management strategy to enhance rice grain yield and quality at the lowest environmental cost.