Minimizing cadmium (Cd) contamination in rice grains is crucial for ensuring food security and promoting sustainable agriculture. Utilizing genetic modification to generate rice varieties with low Cd accumulation is a promising strategy due to its cost-effectiveness and operational simplicity. Our study demonstrated that the CRISPR-Cas9-mediated quadruple mutation of the multicopper oxidase genes OsLPR1/3/4/5 in the japonica rice cultivar Tongjing 981 had little effect on yields. However, a notable increase was observed in the cell wall functional groups that bind with Cd. As a result, the quadruple mutation of OsLPR1/3/4/5 enhanced Cd sequestration within the cell wall while reducing Cd concentrations in both xylem and phloem sap, thereby inhibiting Cd transport from roots to shoots. Consequently, Cd concentrations in brown rice and husk in oslpr1/3/4/5 quadruple mutants (qm) decreased by 52% and 55%, respectively, compared to the wild-type. These findings illustrate that the quadruple mutation of OsLPR1/3/4/5 is an effective method for minimizing Cd contamination in rice grains without compromising yields. Therefore, the quadruple mutation of OsLPR1/3/4/5 via biotechnological pathways may represent a valuable strategy for the generation of new rice varieties with low Cd accumulation.

This work presents the development of an enzyme fuel cell, termed \"BioBattery\", that utilizes multicopper oxidases as the anodic enzyme in a non-diffusion limited system. We evaluated various enzyme variants as the anode, including multicopper oxidase from Pyrobaculum aerophilum, laccase from Trametes versicolor, and bilirubin oxidase from Myrothecium verrucaria. Several combinations of cathodes were also examined, focusing on the reduction of oxygen as the primary electron acceptor. The optimal pairing used multicopper oxidase from Pyrobaculum aerophilum as the anode and amine reactive phenazine ethosulfate modified bovine serum albumin as the cathode. BioBattery was integrated with our previously developed BioCapacitor, proving capable of consistently powering a 470 μF capacitor, positioning it as a modular power source for wearable and implantable systems. This research work addresses and overcomes some of the fundamental limitations seen in enzyme fuel cells, where power and current are often limited by substrate accessibility to the active electrode surface. (152 words).

In order to explore the structural changes and products of histamine degradation by multicopper oxidase (MCO) in Lactiplantibacillus plantarum LPZN19, a 1500 bp MCO gene in L. plantarum LPZN19 was cloned, and the recombinant MCO was expressed in E. coli BL21 (DE3). After purification by Ni2+-NTA affinity chromatography, the obtained MCO has a molecular weight of 58 kDa, and it also has the highest enzyme activity at 50 °C and pH 3.5, with a relative enzyme activity of 100%, and it maintains 57.71% of the relative enzyme activity at 5% salt concentration. The secondary structure of MCO was determined by circular dichroism, in which the proportions of the α-helix, β-sheet, β-turn and random coil were 2.9%, 39.7%, 21.2% and 36.1%, respectively. The 6xj0.1.A with a credibility of 68.21% was selected as the template to predict the tertiary structure of MCO in L. plantarum LPZN19, and the results indicated that the main components of the tertiary structure of MCO were formed by the further coiling and folding of a random coil and β-sheet. Histamine could change the spatial structure of MCO by increasing the content of the α-helix and β-sheet. Finally, the LC-MS/MS identification results suggest that the histamine was degraded into imidazole acetaldehyde, hydrogen peroxide and ammonia.

One way to effectively reduce the number of biogenic amines (BAs) in food is through enzymatic reduction using bacteria, such as lactic acid bacteria. This study focuses on the ability of the bacterial strain Lacticaseibacillus casei CCDM 198 to reduce the number of three important BAs (histamine, putrescine and cadaverine) over time, depending on different conditions (temperature and pH) in vitro and for the real dairy product - skimmed milk. The obtained results show that the studied strain significantly (P < 0.05) affects the number of individual amines, and the content of all amines has a decreasing character compared to the initial relative content of BAs at time zero. Furthermore, a statistical dependence (P < 0.05) of the rate of amine degradation on the combination of investigated factors was demonstrated. The presence and the activity of multicopper oxidase enzyme was also detected in this bacterial strain. This is the first known publication demonstrating multicopper oxidase activity in Lacticaseibacillus casei CCDM 198. Moreover, the studied strain is able to reduce the tested BAs in skimmed milk and would be a good candidate for degrading these toxic compounds in other dairy products, such as cheese. These findings could significantly enhance the food safety of dairy products.

Biogenic amines (BAs) are produced by microbial decarboxylation in various foods. Histamine and tyramine are recognized as the most toxic of all BAs. Applying degrading amine enzymes such as multicopper oxidase (MCO) is considered an effective method to reduce BAs in food systems. This study analyzed the characterization of heterologously expressed MCO from L. sakei LS. Towards the typical substrate 2,2\'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), the optimal temperature and pH for recombinant MCO (rMCO) were 25 °C and 3.0, respectively, with the specific enzyme activity of 1.27 U/mg. Then, the effect of different environmental factors on the degrading activity of MCO towards two kinds of BAs was investigated. The degradation activity of rMCO is independent of exogenous copper and mediators. Additionally, the oxidation ability of rMCO was improved for histamine and tyramine with an increased NaCl concentration. Several food matrices could influence the amine-oxidizing activity of rMCO. Although the histamine-degrading activities of rMCO were affected, this enzyme reached a degradation rate of 28.1% in the presence of surimi. Grape juice improved the tyramine degradation activity of rMCO by up to 31.18%. These characteristics of rMCO indicate that this enzyme would be a good candidate for degrading toxic biogenic amines in food systems.

Geobacillus sp. ID17 is a gram-positive thermophilic bacterium isolated from Deception Island, Antarctica, which has shown to exhibit remarkable laccase activity in crude extract at high temperatures. A bioinformatic search using local databases led to the identification of three putative multicopper oxidase sequences in the genome of this microorganism. Sequence analysis revealed that one of those sequences contains the four-essential copper-binding sites present in other well characterized laccases. The gene encoding this sequence was cloned and overexpressed in Escherichia coli, partially purified and preliminary biochemically characterized. The resulting recombinant enzyme was recovered in active and soluble form, exhibiting optimum copper-dependent laccase activity at 55 °C, pH 6.5 with syringaldazine substrate, retaining over 60% of its activity after 1 h at 55 and 60 °C. In addition, this thermophilic enzyme is not affected by common inhibitors SDS, NaCl and L-cysteine. Furthermore, biodecolorization assays revealed that this laccase is capable of degrading 60% of malachite green, 54% of Congo red, and 52% of Remazol Brilliant Blue R, after 6 h at 55 °C with aid of ABTS as redox mediator. The observed properties of this enzyme and the relatively straightforward overexpression and partial purification of it could be of great interest for future biotechnology applications.

Sporopollenin is one of the most structurally sophisticated and chemically recalcitrant biopolymers. In higher plants, sporopollenin is the dominant component of exine, the outer wall of pollen grains, and contains covalently linked phenolics that protect the male gametes from harsh environments. Although much has been learned about the biosynthesis of sporopollenin precursors in the tapetum, the nutritive cell layer surrounding developing microspores, little is known about how the biopolymer is assembled on the microspore surface. We identified SCULP1 (SKS clade universal in pollen) as a seed plant conserved clade of the multicopper oxidase family. We showed that SCULP1 in common wheat (Triticum aestivum) is specifically expressed in the microspore when sporopollenin assembly takes place, localized to the developing exine, and binds p-coumaric acid in vitro. Through genetic, biochemical, and 3D reconstruction analyses, we demonstrated that SCULP1 is required for p-coumaroylation of sporopollenin, exine integrity, and pollen viability. Moreover, we found that SCULP1 accumulation is compromised in thermosensitive genic male sterile wheat lines and its expression partially restored exine integrity and male fertility. These findings identified a key microspore protein in autonomous sporopollenin polymer assembly, thereby laying the foundation for elucidating and engineering sporopollenin biosynthesis.

The chemistry of metal ions with human pathogens is essential for their survival, energy generation, redox signaling, and niche dominance. To regulate and manipulate the metal ions, various enzymes and metal chelators are present in pathogenic bacteria. Metalloenzymes incorporate transition metal such as iron, zinc, cobalt, and copper in their reaction centers to perform essential metabolic functions; however, iron and copper have gained more importance. Multicopper oxidases have the ability to perform redox reaction on phenolic substrates with the help of copper ions. They have been reported from Enterobacteriaceae, namely Salmonella enterica, Escherichia coli, and Yersinia enterocolitica, but their role in virulence is still poorly understood. Similarly, superoxide dismutases participate in reducing oxidative stress and allow the survival of pathogens. Their role in virulence and survival is well established in Salmonella typhimurium and Mycobacterium tuberculosis. Further, to ensure survival against stress, like metal starvation or metal toxicity, redox metalloenzymes and metal transportation systems of pathogens actively participate in metal homeostasis. Recently, the omics and protein structure biology studies have helped to predict new targets for regulation the colonization potential of the pathogenic strains. The current review is focused on the major roles of redox metalloenzymes, especially MCOs and SODs of human pathogenic bacteria.

Metabolic sensing is a crucial prerequisite for cells to adjust their physiology to rapidly changing environments. In bacteria, the response to intra- and extracellular ligands is primarily controlled by transcriptional regulators, which activate or repress gene expression to ensure metabolic acclimation. Translational control, such as ribosomal stalling, can also contribute to cellular acclimation and has been shown to mediate responses to changing intracellular molecules. In the current study, we demonstrate that the cotranslational export of the Rhodobacter capsulatus protein CutF regulates the translation of the downstream cutO-encoded multicopper oxidase CutO in response to extracellular copper (Cu). Our data show that CutF, acting as a Cu sensor, is cotranslationally exported by the signal recognition particle pathway. The binding of Cu to the periplasmically exposed Cu-binding motif of CutF delays its cotranslational export via its C-terminal ribosome stalling-like motif. This allows for the unfolding of an mRNA stem-loop sequence that shields the ribosome-binding site of cutO, which favors its subsequent translation. Bioinformatic analyses reveal that CutF-like proteins are widely distributed in bacteria and are often located upstream of genes involved in transition metal homeostasis. Our overall findings illustrate a highly conserved control mechanism using the cotranslational export of a protein acting as a sensor to integrate the changing availability of extracellular nutrients into metabolic acclimation. IMPORTANCE Metabolite sensing is a fundamental biological process, and the perception of dynamic changes in the extracellular environment is of paramount importance for the survival of organisms. Bacteria usually adjust their metabolisms to changing environments via transcriptional regulation. Here, using Rhodobacter capsulatus, we describe an alternative translational mechanism that controls the bacterial response to the presence of copper, a toxic micronutrient. This mechanism involves a cotranslationally secreted protein that, in the presence of copper, undergoes a process resembling ribosomal stalling. This allows for the unfolding of a downstream mRNA stem-loop and enables the translation of the adjacent Cu-detoxifying multicopper oxidase. Bioinformatic analyses reveal that such proteins are widespread, suggesting that metabolic sensing using ribosome-arrested nascent secreted proteins acting as sensors may be a common strategy for the integration of environmental signals into metabolic adaptations.

Iron (Fe) is an essential metal ion that plays a major role as a cofactor in many biological processes. The balance between the Fe2+ and Fe3+ forms is central for cellular Fe homeostasis because it regulates its transport, utilization, and storage. Contrary to Fe3+ reduction that is crucial for Fe uptake by roots in deficiency conditions, ferroxidation has been much less studied. In this work, we have focused on the molecular characterization of two members of the MultiCopper Oxidase family (MCO1 and MCO3) that share high identity with the Saccharomyces cerevisiae ferroxidase Fet3. The heterologous expression of MCO1 and MCO3 restored the growth of the yeast fet3fet4 mutant, impaired in high and low affinity Fe uptake and otherwise unable to grow in Fe deficient media, suggesting that MCO1 and MCO3 were functional ferroxidases. The ferroxidase enzymatic activity of MCO3 was further confirmed by the measurement of Fe2+-dependent oxygen consumption, because ferroxidases use oxygen as electron acceptor to generate water molecules. In planta, the expression of MCO1 and MCO3 was induced by increasing Fe concentrations in the medium. Promoter-GUS reporter lines showed that MCO1 and MCO3 were mostly expressed in shoots and histochemical analyses further showed that both promoters were highly active in mesophyll cells. Transient expression of MCO1-RFP and MCO3-RFP in tobacco leaves revealed that both proteins were localized in the apoplast. Moreover, cell plasmolysis experiments showed that MCO1 remained closely associated to the plasma membrane whereas MCO3 filled the entire apoplast compartment. Although the four knock out mutant lines isolated (mco1-1, mco1-2, mco3-1, and mco3-2) did not display any macroscopic phenotype, histochemical staining of Fe with the Perls/DAB procedure revealed that mesophyll cells of all four mutants overaccumulated Fe inside the cells in Fe-rich structures in the chloroplasts, compared with wild-type. These results suggested that the regulation of Fe transport in mesophyll cells had been disturbed in the mutants, in both standard condition and Fe excess. Taken together, our findings strongly suggest that MCO1 and MCO3 participate in the control of Fe transport in the mesophyll cells, most likely by displacing the Fe2+/Fe3+ balance toward Fe3+ in the apoplast and therefore limiting the accumulation of Fe2+, which is more mobile and prone to be transported across the plasma membrane.