Chloroflexus aurantiacus acetyl-CoA carboxylase evolves fused biotin carboxylase and biotin carboxyl carrier protein to complete carboxylation activity.

Acetyl-CoA carboxylases (ACCs) convert acetyl-CoA to malonyl-CoA, a key step in fatty acid biosynthesis and autotrophic carbon fixation pathways. Three functionally distinct components, biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and carboxyltransferase (CT), are either separated or partially fused in different combinations, forming heteromeric ACCs. However, an ACC with fused BC-BCCP and separate CT has not been identified, leaving its catalytic mechanism unclear. Here, we identify two BC isoforms (BC1 and BC2) from Chloroflexus aurantiacus, a filamentous anoxygenic phototroph that employs 3-hydroxypropionate (3-HP) bi-cycle rather than Calvin cycle for autotrophic carbon fixation. We reveal that BC1 possesses fused BC and BCCP domains, where BCCP could be biotinylated by E. coli or C. aurantiacus BirA on Lys553 residue. Crystal structures of BC1 and BC2 at 3.2 Å and 3.0 Å resolutions, respectively, further reveal a tetramer of two BC1-BC homodimers, and a BC2 homodimer, all exhibiting similar BC architectures. The two BC1-BC homodimers are connected by an eight-stranded ß-barrel of the partially resolved BCCP domain. Disruption of ß-barrel results in dissociation of the tetramer into dimers in solution and decreased biotin carboxylase activity. Biotinylation of the BCCP domain further promotes BC1 and CTß-CTα interactions to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-HP via co-expression with a recombinant malonyl-CoA reductase in E. coli cells. This study revealed a heteromeric ACC that evolves fused BC-BCCP but separate CTα and CTß to complete ACC activity.IMPORTANCEAcetyl-CoA carboxylase (ACC) catalyzes the rate-limiting step in fatty acid biosynthesis and autotrophic carbon fixation pathways across a wide range of organisms, making them attractive targets for drug discovery against various infections and diseases. Although structural studies on homomeric ACCs, which consist of a single protein with three subunits, have revealed the "swing domain model" where the biotin carboxyl carrier protein (BCCP) domain translocates between biotin carboxylase (BC) and carboxyltransferase (CT) active sites to facilitate the reaction, our understanding of the subunit composition and catalytic mechanism in heteromeric ACCs remains limited. Here, we identify a novel ACC from an ancient anoxygenic photosynthetic bacterium Chloroflexus aurantiacus, it evolves fused BC and BCCP domain, but separate CT components to form an enzymatically active ACC, which converts acetyl-CoA to malonyl-CoA in vitro and produces 3-hydroxypropionate (3-HP) via co-expression with recombinant malonyl-CoA reductase in E. coli cells. These findings expand the diversity and molecular evolution of heteromeric ACCs and provide a structural basis for potential applications in 3-HP biosynthesis.

Lateral lymph node metastasis in papillary thyroid microcarcinoma: a study of 5241 follow-up patients.

PURPOSE: To investigate the impact of lateral lymph node metastasis in papillary thyroid microcarcinoma (PTMC). METHODS: 5241 PTMC patients with follow-up information were enrolled in the current study. These patients underwent primary surgery in our situation from January 1997 to December 2016. Additionally, a validation cohort consisting of 274 PTMC patients who underwent primary surgery between January 2020 and December 2021 was also included. Univariable and multivariate logistic analyses were conducted to identify the association between clinicopathologic features and lateral lymph node metastasis (LLNM). Kaplan-Meier survival curve analysis was used to calculate the disease-free survival (DFS) rate. The fitting curve was generated to identify the quantitative relationship between central lymph node metastases (CLNM) and LLNM. RESULTS: Of 5241 PTMC patients, cervical lymph node metastasis was detected in 1494 (28.5%) cases, including 1364 (26.0%) with CLNM only and 130 (2.5%) with LLNM. With a median follow-up time of 60 months (interquartile range [IQR], 44-81), recurrence was detected in 114 patients (2.2%). Multivariate Cox regression analyses showed that LNM was the only independent risk factor for recurrence, with HR values of 3.03 in CLNM and 11.14 in LLNM, respectively. Tumor diameter >0.5 cm (hazard ratio [HR]:1.80), multifocality (HR:2.59), bilaterality (HR:2.13), extrathyroidal invasion (HR:2.13), and CLNM (HR:5.11) were independent risk factors for LLNM. The prevalence of LLNM escalated significantly with increasing number of lymph node involvement in CLNM when stratified by the number of metastatic lymph nodes and trend was observed similarly in the validation cohort. The fitting curve showed that the incidence of LLNM could be as high as 20.7% when the number of CLNM ≥ 5. CONCLUSIONS: By analyzing a large database with follow-up information, our study provides evidence that LLNM is significantly correlated with tumor recurrence in patients with PTMC. Tumor size (>0.5 cm), multifocality, bilaterality, extrathyroidal extension (ETE) and CLNM are independent risk factors for LLNM.

Analysis of Risk Factors of Pelvic Organ Prolapse in Postmenopausal Women and Construction of Prediction Model.

Background: The incidence of Pelvic organ prolapse (POP) was as high as 50% in women, with the main symptoms of vaginal tissue prolapse, accompanied by urination, defecation, and sexual dysfunction, which affected patients' quality of life. POP is more prominent in postmenopausal women due to various factors. By constructing a model, we predict POP and expect to reduce the incidence of POP. Objective: To explore the risk factors for POP in postmenopausal women and develop a predictive model that can identify high-risk individuals early so that targeted preventive measures can be taken to reduce the burden of POP. Methods: Using retrospective studies, 290 menopausal women treated in the Department of Gynecology of the Ninth People's Hospital of Suzhou from January 2019 to December 2022 were selected as the study subjects. Women with menopause were divided into the POP group (62 cases) and a non-POP group (228 cases) according to whether or not POP occurred. Single factor analysis was performed on the two data groups. The risk factors of POP in menopausal women were screened by multivariate logistic regression analysis. Based on the screening results, a graph prediction model expressed as a nomogram is constructed. The model's effectiveness was analyzed by the goodness of fit test and receiver operating characteristic curve (ROC) curve. The decision curve was used to analyze the clinical effectiveness of the model. Results: Multifactor logistic regression analysis showed that Older age (OR = 2.309, P = .007), more childbirth frequency (OR = 3.121, P = .002), low expression of estradiol (E2) (OR = 1.499, P = .023), low expression of serum 25-hydroxyvitamin D3[25-(OH)D3] (OR = 2.073, P = .011), and lower blood calcium (OR = 21.677, P = .014) were all risk factors for POP in menopausal women. Based on the above indicators, a risk prediction model is constructed. The model has been proved to have good recognition ability, areas under curve (AUC) = 0.887 (95%CI: 0.845-0.926), The best cutoff value is 0.37, The sensitivity and specificity were 0.885 and 0.840, respectively; The goodness of fit test showed that the predicted value of the model had no statistical significance with the actual value. The threshold probability is in the range of 1%~99%. The net benefit of menopausal women is higher than the other two extreme curves. It shows that the model is clinically effective. Conclusion: Age, times of delivery, E2, 25-(OH)D3, and blood calcium are related to POP in menopausal women. A nomogram model based on these 5 indicators can effectively assess the risk of POP in postmenopausal women. The clinician can use this column chart to calculate the risk of POP occurrence for each patient and make clinical recommendations accordingly.

Multi-scale failure mechanisms of hydraulic engineering exposed to seasonally frozen salinization environment: Integrating SBAS-InSAR and mechanical experiments.

Constructing hydraulic engineering ensures agricultural development and improves salinization environments. However, in seasonally frozen salinization regions, hydraulic engineering is prone to deformation failure. Leakage from canal raises the regional groundwater level, triggering secondary salinization environmental issues. Exploring the instability mechanisms is thus necessary for hydraulic engineering. Traditional deformation monitoring techniques and soil experiments are constrained by observation scale and timeliness. In this study, Sentinel-1B data from November 2017 to August 2019 were acquired. The small baseline subset (SBAS) InSAR approach was employed to interpret the seasonal deformation characteristics in both the vertical and slope directions of a damaged canal segment in Songyuan, Northeast China. The mechanical properties of saline-alkali soil under varying water contents were quantified by integrating unconfined compression experiment (UCE). In May, as the soil thawed downward, a frozen lenses with poor permeability formed at a depth of approximately 100 cm, causing the accumulation of meltwater and infiltrated precipitation between the frozen layer and the melting layer in the canal. The soil water content at a depth of 80 to 140 cm exceeded 22 %, reaching a threshold for rapid reduction in unconfined compression strength (UCS). Consequently, in spring, the low soil strength between the frozen layer and the melting layer resulted in interface sliding, with a displacement of -133.88 mm in the canal slope direction. Furthermore, the differential projection of freeze-thaw deformation in the slope direction caused continuous creep of the canal towards the free face, with a value of -23.27 mm, exacerbating the formation of the late spring landslide. Integrating InSAR and engineering geological analysis is beneficial for addressing deformation issues in hydraulic engineering. Ensuring the sustainable operation of hydraulic engineering holds important implications for mitigating the salinization process.

Carotenoid assembly regulates quinone diffusion and the Roseiflexus castenholzii reaction center-light harvesting complex architecture.

Carotenoid (Car) pigments perform central roles in photosynthesis-related light harvesting (LH), photoprotection, and assembly of functional pigment-protein complexes. However, the relationships between Car depletion in the LH, assembly of the prokaryotic reaction center (RC)-LH complex, and quinone exchange are not fully understood. Here, we analyzed native RC-LH (nRC-LH) and Car-depleted RC-LH (dRC-LH) complexes in Roseiflexus castenholzii, a chlorosome-less filamentous anoxygenic phototroph that forms the deepest branch of photosynthetic bacteria. Newly identified exterior Cars functioned with the bacteriochlorophyll B800 to block the proposed quinone channel between LHαß subunits in the nRC-LH, forming a sealed LH ring that was disrupted by transmembrane helices from cytochrome c and subunit X to allow quinone shuttling. dRC-LH lacked subunit X, leading to an exposed LH ring with a larger opening, which together accelerated the quinone exchange rate. We also assigned amino acid sequences of subunit X and two hypothetical proteins Y and Z that functioned in forming the quinone channel and stabilizing the RC-LH interactions. This study reveals the structural basis by which Cars assembly regulates the architecture and quinone exchange of bacterial RC-LH complexes. These findings mark an important step forward in understanding the evolution and diversity of prokaryotic photosynthetic apparatus.

Photosynthesis is a biological process that converts energy from sunlight into a form of chemical energy that supports almost all life on Earth. Over the course of evolution, photosynthesis has gone from being only performed by bacteria to appearing in algae and green plants. While this has given rise to a range of different machineries for photosynthesis, the process always begins the same way: with a structure called the reaction center-light harvesting (RC-LH) complex. Two pigments in the light-harvesting (LH) region ­ known as chlorophyll and carotenoids ­ absorb light energy and transfer it to another part of the complex known as the quinone-type reaction center (RC). This results in the release of electrons that interact with a molecule called quinone converting it to hydroquinone. The electron-bound hydroquinone then shuttles to other locations in the cell where it initiates further steps that ultimately synthesize forms of chemical energy that can power essential cellular processes. In photosynthetic bacteria, hydroquinone must first pass through a ring structure in the light harvesting region in order to leave the reaction center. Previous studies suggest that carotenoids influence the architecture of this ring, but it remains unclear how this may affect the ability of hydroquinone to move out of the RC-LH complex. To investigate, Xin, Shi, Zhang et al. used a technique called cryo-electron microscopy to study the three-dimensional structure of RC-LH complexes in one of the first bacterial species to employ photosynthesis, Roseiflexus castenholzii. The experiments found that fully assembled complexes bind two groups of carotenoids: one nestled in the interior of the LH ring and the other on the exterior. The exterior carotenoids work together with bacteriochlorophyll molecules to form a closed ring that blocks hydroquinone from leaving the RC-LH complex. To allow hydroquinone to leave, two groups of regulatory proteins, including a cytochrome and subunit X, then disrupt the structure of the ring to 'open' it up. These findings broaden our knowledge of the molecules involved in photosynthesis. A better understanding of this process may aid the development of solar panels and other devices that use RC-LH complexes rather than silicon or other inorganic materials to convert energy from sunlight into electricity.

Tolrestat acts atypically as a competitive inhibitor of the thermostable aldo-keto reductase Tm1743 from Thermotoga maritima.

Tolrestat and epalrestat have been characterized as noncompetitive inhibitors of aldo-ketone reductase 1B1 (AKR1B1), a leading drug target for the treatment of type 2 diabetes complications. However, clinical applications are limited for most AKR1B1 inhibitors due to adverse effects of cross-inhibition with other AKRs. Here, we report an atypical competitive binding and inhibitory effect of tolrestat on the thermostable AKR Tm1743 from Thermotoga maritima. Analysis of the Tm1743 crystal structure in complex with tolrestat alone and epalrestat-NADP+ shows that tolrestat, but not epalrestat, binding triggers dramatic conformational changes in the anionic site and cofactor binding pocket that prevents accommodation of NADP+ . Enzymatic and molecular dynamics simulation analyses further confirm tolrestat as a competitive inhibitor of Tm1743.

The C-terminal domain conformational switch revealed by the crystal structure of malyl-CoA lyase from Roseiflexus castenholzii.

Malyl-coenzyme A lyase (MCL) is a carbon-carbon bond lyase that catalyzes the reversible cleavage of coenzyme A (CoA) thioesters in multiple carbon metabolic pathways. This enzyme contains a CitE-like TIM barrel and an additional C-terminal domain that undergoes conformational changes upon substrate binding. However, the structural basis underlying these conformational changes is elusive. Here, we report the crystal structure of MCL from the thermophilic photosynthetic bacterium Roseiflexus castenholzii (RfxMCL) in the apo- and oxalate-bound forms at resolutions of 2.50 and 2.65 Å, respectively. Molecular dynamics simulations and structural comparisons with MCLs from other species reveal the deflection of the C-terminal domain to close the adjacent active site pocket in the trimer and contribute active site residues for CoA coordination. The deflection angles of the C-terminal domain are not only related to the occupation but also the type of bound substrates in the adjacent active site pocket. Our work illustrates that a conformational switch of the C-terminal domain accompanies the substrate-binding of MCLs. The results provide a framework for further investigating the reaction mechanism and multifunctionality of MCLs in different carbon metabolic pathways.

Substrate Specificity of Acyltransferase Domains for Efficient Transfer of Acyl Groups.

Acyltransferase domains (ATs) of polyketide synthases (PKSs) are critical for loading of acyl groups on acyl carrier protein domains (A) via self- and trans-acylation reactions, to produce structurally diverse polyketides. However, the interaction specificity between ATs and unusual acyl units is rarely documented. In Streptomycestsukubaensis YN06, we found that AT4FkbB [an AT in the fourth module of tacrolimus (FK506) PKS] transferred both allylmalonyl (allmal) and emthylmalonyl (ethmal) units to ACPs, which was supposed responsible for the production of both FK506 and its analog FK520, respectively. Mutations of five residues in AT4FkbB (Q119A, L185I-V186D-V187T, and F203L) caused decreased efficiency of allmal transfer, but a higher ratio of ethmal transfer, supposedly due to less nucleophilic attacks between Ser599 in the active site of AT4FkbB and the carbonyl carbon in the allmal unit, as observed from molecular dynamics simulations. Furthermore, reverse mutations of these five residues in ethmal-specific ATs to the corresponding residues of AT4FkbB increased its binding affinity to allmal-CoA. Among these residues, Val187 of AT4FkbB mainly contributed to allmal recognition, and V187K mutant produced less FK520 than wild type. Our findings thus suggested that five critical residues within AT4FkbB were important for AT functionality in polyketide extension and potentially for targeting biosynthesis by generating desirable products and eliminating undesirable analogs.

An acyltransferase domain of FK506 polyketide synthase recognizing both an acyl carrier protein and coenzyme A as acyl donors to transfer allylmalonyl and ethylmalonyl units.

UNLABELLED: Acyltransferase (AT) domains of polyketide synthases (PKSs) usually use coenzyme A (CoA) as an acyl donor to transfer common acyl units to acyl carrier protein (ACP) domains, initiating incorporation of acyl units into polyketides. Two clinical immunosuppressive agents, FK506 and FK520, are biosynthesized by the same PKSs in several Streptomyces strains. In this study, characterization of AT4FkbB (the AT domain of the fourth module of FK506 PKS) in transacylation reactions showed that AT4FkbB recognizes both an ACP domain (ACPT csA) and CoA as acyl donors for transfer of a unique allylmalonyl (AM) unit to an acyl acceptor ACP domain (ACP4FkbB), resulting in FK506 production. In addition, AT4FkbB uses CoA as an acyl donor to transfer an unusual ethylmalonyl (EM) unit to ACP4FkbB, resulting in FK520 production, and transfers AM units to non-native ACP acceptors. Characterization of AT4FkbB in self-acylation reactions suggests that AT4FkbB controls acyl unit specificity in transacylation reactions but not in self-acylation reactions. Generally, AT domains of PKSs only recognize one acyl donor; however, we report here that AT4FkbB recognizes two acyl donors for the transfer of different acyl units. DATABASE: Nucleotide sequence data have been submitted to the GenBank database under accession numbers KJ000382 and KJ000383.

Hydrogen sulfide improves drought tolerance in Arabidopsis thaliana by microRNA expressions.

Hydrogen sulfide (H2S) is a gasotransmitter and plays an important role in many physiological processes in mammals. Studies of its functions in plants are attracting ever growing interest, for example, its ability to enhance drought resistance in Arabidopsis. A general role of microRNAs (miRNAs) in plant adaptive responses to drought stress has thereby increased our interest to delve into the possible interplay between H2S and miRNAs. Our results showed that treating wild type (WT) Arabidopsis seedlings with polyethylene glycol 8000 (PEG8000) to simulate drought stress caused an increase in production rate of endogenous H2S; and a significant transcriptional reformation of relevant miRNAs, which were also triggered by exogenous H2S in WT. When lcd mutants (with lower H2S production rate than WT) were treated with PEG8000, they showed lower levels of miRNA expression changes than WT. In addition, we detected significant changes in target gene expression of those miRNAs and the corresponding phenotypes in lcd, including less roots, retardation of leaf growth and development and greater superoxide dismutase (SOD) activity under drought stress. We thereby conclude that H2S can improve drought resistance through regulating drought associated miRNAs in Arabidopsis.

Hydrogen sulfide improves drought resistance in Arabidopsis thaliana.

Hydrogen sulfide (H(2)S) plays a crucial role in human and animal physiology. Its ubiquity and versatile properties have recently caught the attention of plant physiologists and biochemists. Two cysteine desulfhydrases (CDes), L-cysteine desulfhydrase and D-cysteine desulfhydrase, were identified as being mainly responsible for the degradation of cysteine in order to generate H(2)S. This study investigated the expression regulation of these genes and their relationship to drought tolerance in Arabidopsis. First, the expression pattern of CDes in Arabidopsis was investigated. The expression levels of CDes gradually increased in an age-dependent manner. The expression of CDes was significantly higher in stems and cauline leaves than in roots, rosette leaves and flowers. Second, the protective effect of H(2)S against drought was evaluated. The expression pattern of CDes was similar to the drought associated genes induced by dehydration, and H(2)S fumigation was found to stimulate further the expression of drought associated genes. Drought also significantly induced increased H(2)S production, a process that was reversed by re-watering. In addition, seedlings after treatment with NaHS (a H(2)S donor) showed a higher survival rate and displayed a significant reduction in the size of the stomatal aperture compared to the control. These findings provide evidence that H(2)S, as a gasotransmitter, improves drought resistance in Arabidopsis.