Structural and functional characterization of a mycobacterial methylenetetrahydrofolate reductase utilizing NADH as the exclusive cofactor.

5,10-Methylenetetraydrofolate reductase (MTHFR) is a key enzyme in folate metabolism. MSMEG_6649, a non-canonical MTHFR from Mycobacterium smegmatis, was previously reported as a monomeric protein lacking the flavin coenzyme. However, the structural basis for its unique flavin-independent catalytic mechanism remains poorly understood. Here, we determined the crystal structures of apo MTHFR MSMEG_6649 and its complex with NADH from M. smegmatis. Structural analysis revealed that the groove formed by the loops 4 and 5 of non-canonical MSMEG_6649 interacting with FAD was significantly larger than that of canonical MTHFR. Meanwhile, the NADH-binding site in MSMEG_6649 is highly similar to the FAD binding site in canonical MTHFR, suggesting that NADH plays the same role (immediate hydride donor for methylenetetraydrofolate) as FAD in the catalytic reaction. Using biochemical analysis, molecular modeling, and site-directed mutagenesis, the critical residues participating in the binding of NADH and the substrate 5,10-methylenetetrahydrofolate as well as the product 5-methyltetrahydrofolate were identified and validated. Taken together, this work not only provides a good starting point for understanding the potential catalytic mechanism for MSMEG_6649, but also identifies an exploitable target for the development of anti-mycobacterial drugs.

Identification and Structural Modeling of the RNA Polymerase Omega Subunits in Chlamydiae and Other Obligate Intracellular Bacteria.

Gene transcription in bacteria is carried out by the multisubunit RNA polymerase (RNAP), which is composed of a catalytic core enzyme and a promoter-recognizing σ factor. The core enzyme comprises two α subunits, one ß subunit, one ß' subunit, and one ω subunit. The ω subunit plays critical roles in the assembly of the core enzyme and other cellular functions, including the regulation of bacterial growth, the stress response, and biofilm formation. However, the identity of an ω subunit for the obligate intracellular bacterium Chlamydia has not previously been determined. Here, we report the identification of the hypothetical protein CTL0286 as the probable chlamydial ω subunit based on sequence, synteny, and AlphaFold and AlphaFold-Multimer three-dimensional-structure predictions. Our findings indicate that CTL0286 functions as the missing ω subunit of chlamydial RNAP. Our extended analysis also indicates that all obligate intracellular bacteria have ω orthologs. IMPORTANCE Chlamydiae are obligate intracellular bacteria that replicate only inside eukaryotic cells. Previously, it has not been possible to identify a candidate gene encoding the chlamydial RNA polymerase ω subunit, and it has been hypothesized that the chlamydial RNA polymerase ω subunit was lost in the evolutionary process through which Chlamydiae reduced their genome size and proteome sizes to adapt to an obligate intracellular lifestyle. Here, we report the identification of the chlamydial RNA polymerase ω subunit, based on conserved sequence, conserved synteny, AlphaFold-predicted conserved three-dimensional structure, and AlfaFold-Multimer-predicted conserved interactions. Our identification of the previously elusive chlamydial RNA polymerase ω subunit sets the stage for investigation of its roles in regulation of gene expression during chlamydial growth, development, and stress responses, and sets the stage for preparation and study of the intact chlamydial RNA polymerase and its interactions with inhibitors.

Functional Prediction of trans-Prenyltransferases Reveals the Distribution of GFPPSs in Species beyond the Brassicaceae Clade.

Terpenoids are the most diverse class of plant primary and specialized metabolites, and trans-prenyltransferases (trans-PTs) are the first branch point to synthesize precursors of various chain lengths for further metabolism. Whereas the catalytic mechanism of the enzyme is known, there is no reliable method for precisely predicting the functions of trans-PTs. With the exponentially increasing number of available trans-PTs genes in public databases, an in silico functional prediction method for this gene family is urgently needed. Here, we present PTS-Pre, a web tool developed on the basis of the "three floors" model, which shows an overall 86% prediction accuracy for 141 experimentally determined trans-PTs. The method was further validated by in vitro enzyme assays for randomly selected trans-PTs. In addition, using this method, we identified nine new GFPPSs from different plants which are beyond the previously reported Brassicaceae clade, suggesting these genes may have occurred via convergent evolution and are more likely lineage-specific. The high accuracy of our blind prediction validated by enzymatic assays suggests that PTS-Pre provides a convenient and reliable method for genome-wide functional prediction of trans-PTs enzymes and will surely benefit the elucidation and metabolic engineering of terpenoid biosynthetic pathways.

Differences of Cd uptake and expression of OAS and IRT genes in two varieties of ryegrasses.

Pot experiment was conducted to study the difference of cadmium uptake and OAS and IRT genes' expression between the two ryegrass varieties under cadmium stress. The results showed that with the increase of cadmium levels, the dry weights of roots of the two ryegrass varieties, and the dry weights of shoots and plants of Abbott first increased and then decreased. When exposed to 75 mg kg-1 Cd, the dry weights of shoot and plant of Abbott reached the maximum, which increased by 11.13 and 10.67% compared with the control. At 75 mg kg-1 Cd, cadmium concentrations in shoot of the two ryegrass varieties were higher than the critical value of Cd hyperaccumulator (100 mg kg-1), 111.19 mg kg-1 (Bond), and 133.69 mg kg-1 (Abbott), respectively. The OAS gene expression in the leaves of the two ryegrass varieties showed a unimodal curve, which was up to the highest at the cadmium level of 150 mg kg-1, but fell back at high cadmium levels of 300 and 600 mg kg-1. The OAS gene expression in Bond and Abbott roots showed a bimodal curve. The OAS gene expression in Bond root and Abbott stem mainly showed a unimodal curve. The expression of IRT genes family in the leaves of ryegrass varieties was basically in line with the characteristics of unimodal curve, which was up to the highest at cadmium level of 75 or 150 mg kg-1, respectively. The IRT expression in the ryegrass stems showed characteristics of bimodal and unimodal curves, while that in the roots was mainly unimodal. The expression of OAS and IRT genes was higher in Bond than that in Abbott due to genotype difference between the two varieties. The expression of OAS and IRT was greater in leaves than that in roots and stems. Ryegrass tolerance to cadmium can be increased by increasing the expression of OAS and IRT genes in roots and stems, and transfer of cadmium from roots and stems to the leaves can be enhanced by increasing expression OAS and IRT in leaves.

Differences of Cd uptake and expression of MT family genes and NRAMP2 in two varieties of ryegrasses.

In order to understand the mechanism of the difference of Cd absorption and Cd enrichment in different ryegrass varieties, pot experiment was conducted to study on the response of two varieties of ryegrass (Bond and Abbott) to Cd stress as well as the differences of Cd uptake and expression of MT family genes and NRAMP2. Results showed that root dry weights of two varieties and shoot dry weights of Abbott increased first and then decreased with the increase of Cd level in soil. When exposed to 75 mg kg-1 Cd, shoot dry weight and plant dry weight of Abbott both reached maximum values (10.92 and 12.03 g pot-1), which increased by 11.09 and 10.67% compared with the control, respectively. Shoot dry weight and plant dry weight of Bond decreased with the increase of Cd level in soil. When the Cd level in soil was 75 mg kg-1, shoot Cd concentrations of the two varieties were 111.19 mg kg-1 (Bond) and 133.69 mg kg-1 (Abbott), respectively, both of which exceeded the critical value of Cd hyperaccumulator (100 mg kg-1). The expression of MT gene family and NRAMP2 in the leaf of Bond variety significantly increased at the Cd level of 75 mg kg-1 and reached maximum value (except MT2C) at Cd level of 150 mg kg-1. The expression of MT gene family in the stem of Bond variety showed a double-peak pattern, while the expression of NRAMP2 was a single-peak pattern. The expression of MT gene family and NRAMP2 in Abbott variety was consistent with single-peak pattern. The expression of MT gene family and NRAMP2 in leaf both significantly increased at Cd level of 150 mg kg-1, while that in stem and root significantly increased at Cd level of 75 mg kg-1. For both varieties of ryegrass, the expression amount of MT family genes and Nramp2 in leaf was higher than that in root and stem, indicating the Cd tolerance of ryegrass can be improved by increasing the expression levels of MT family genes and Nramp2 in stem and root. There was significant genotypic difference in the expression of MT gene family and NRAMP2 between the two varieties of ryegrass, and the expression of MT gene family and NRAMP2 in leaves and stems of Bond variety was higher than that in Abbott variety, while the expression of MT gene family and NRAMP2 in roots of Abbott variety was higher than that in Bond variety. The two gene families investigated in this study may be closely related to Cd uptake, but not related to Cd transport from root to leaf and Cd enrichment in shoot.

Resistance of alfalfa and Indian mustard to Cd and the correlation of plant Cd uptake and soil Cd form.

Phytoremediation of heavy metal-contaminated soil is considered to be one ecological environmental protection way that is effective and economical. The selection of suitable hyperaccumulators is a key issue for phytoremediation of heavy metal-contaminated soil. Pot experiments were conducted to study the effects of different Cd levels (0, 75, 150, 300, and 600 mg kg-1 Cd) on the dry weight, antioxidant enzyme activities, malondialdehyde (MDA) contents, Cd concentration, Cd accumulation, and soil Cd form distribution ratio (FDC) of alfalfa (Medicago sativa L.) and Indian mustard (Brassica juncea L.). The correlations between Cd concentration in shoots and roots of alfalfa and Indian mustard and soil Cd form were also investigated. The results showed that with the increase of soil Cd levels, dry weight of shoot and root of alfalfa and Indian mustard significantly decreased, which decreased by 50.0-71.8% and 29.6-59.3% (alfalfa), 59.6-89.0% and 64.3-74.8% (India mustard), respectively, compared with the control. With the increase of soil Cd level, superoxide dismutase (SOD) activity in shoot of alfalfa significantly increased. Catalase (CAT) activity and malondialdehyde contents in shoots and roots of alfalfa and Indian mustard, as well as superoxide dismutase activity in the roots of alfalfa and Indian mustard increased first and then decreased with the increase of soil Cd level. With increasing Cd stress, Cd concentration in shoots and roots of alfalfa and Indian mustard significantly increased. At soil Cd level of 75 mg kg-1, Cd concentrations in shoots of alfalfa and Indian mustard exceeded the critical value of Cd-hyperaccumulator (100 mg kg-1), which was 356.46 mg kg-1 and 308.74 mg kg-1, respectively. Cadmium concentrations in shoots and roots of plants were in the order of that of alfalfa > Indian mustard; total Cd accumulation in the aboveground tissues and roots of the plants was in the order of that of Indian mustard > alfalfa at the same Cd level. With increasing soil Cd level, Cd concentrations of exchangeable form (EXC-Cd), carbonate-bound form (CAB-Cd), iron-manganese oxide-bound form (FeMn-Cd), organic-bound form (OM-Cd), and residual form (RES-Cd) showed an increasing trend. The form distribution ratio of soil Cd forms in alfalfa's rhizosphere was in the order of that of exchangeable form Cd > carbonate-bound form Cd > iron-manganese oxide-bound form Cd > residual form Cd > organic-bound form Cd. Except for organic-bound form Cd, soil Cd forms were significantly positively correlated with Cd concentration in shoot and root (P < 0.01). Comprehensively considering the biomass and Cd accumulation, Indian mustard is more suitable as remediation material for soil Cd pollution.

Omnidirectional thermal emitter based on plasmonic nanoantenna arrays.

In this paper, the optical properties of a plasmonic nanoantenna array have been investigated. The proposed plasmonic structure presents omnidirectional resonance properties, such as omnidirectional reflection dip and omnidirectional emission peak. In addition, the reflection and emission of the plasmonic nanoantenna array with various metal/insulator/metal cavity thicknesses are theoretically and experimentally investigated. The simulation reveals a fair agreement with the experimental results.