Nanozymes, novel engineered nanomaterial-based artificial enzymes, have been developed to overcome intrinsic drawbacks exist in natural enzymes including high-cost storage, structural instability, and chemical sensitivity. More recently, carbon dots (CDs) have received significant attention due to their biocompatibility, high catalytic activity, and simple surface functionalization, thus emerging as possible alternatives for biomedical and environmental applications. In this review, we analyze methods and precursors used to synthesize CDs with enzyme-mimicking behaviors. In addition, approaches such as doping or constructing hybrid nanozymes are included as possible strategies to enhance the catalytic performance of CDs. Recent studies have reported CDs that mimic different oxidoreductases, exhibiting peroxidase-, catalase-, oxidase/laccase-, and superoxide dismutase-like activities. Therefore, this review presents a detailed discussion of the mechanism, recent advances, and application for each oxidoreductase-like activity reported on nanozymes based on CDs nanomaterials. Finally, current challenges faced in the successful translation of CDs to potential applications are addressed to suggest research directions.

Glutaredoxins (GRXs) are disulfide oxidoreductases that are involved in various biological processes. However, little information on the role of GRXs in the regulation of fruit ripening and the response to stress is available. In this study, we isolated 64 GRX genes from banana genome. Their encoded GRX proteins could be classified into four classes: CC, CGFS, CPYC and GRL types. The distribution and synteny of these GRXs on chromosomes, the gene structures, the promoter sequences, and the possible protein subcellular localizations were characterized. Molecular interaction network analysis suggested that MaGRX might interact with glutathione reductase (GR), sulfiredoxin, peroxiredoxin (Prx), and NADPH-dependent thioredoxin reductase C (NTRC), contributing to the antioxidative defense of banana fruit. MicroRNA prediction showed that MaGRX genes might be targeted by different miRNAs. Transcriptome analysis characterized the expression profiles of different MaGRX genes during banana fruit ripening, and in response to different storage stresses. The results suggested that CC-type, CPYC-type and GRL-type MaGRXs might be more active than CGFS-type MaGRXs during banana fruit ripening and the response to stress. Moreover, MaGRX6/7/9/11/17/23/28 and MaGRL3/16/19 might play important roles in regulating fruit ripening or in response to low and high temperature, or Fusarium proliferatum infection.

Aldehyde dehydrogenases (ALDHs) are NAD(P)+-dependent oxidoreductases that catalyze the oxidation of a variety of aldehydes to their acid forms. In this study, we determined the crystal structures of ALDH from Bacillus cereus (BcALDH), alone, and in complex with NAD+ and NADP+. This enzyme can oxidize all-trans-retinal to all-trans-retinoic acid using either NAD+ or NADP+ with equal efficiency, and atypically, as a minor activity, can reduce all-trans-retinal to all-trans-retinol using NADPH. BcALDH accommodated the additional 2'-phosphate of NADP+ by expanding the cofactor-binding pocket and upshifting the AMP moiety in NADP+. The nicotinamide moiety in NAD+ and NADP+ had direct interactions with the conserved catalytic residues (Cys300 and Glu266) and caused concerted conformational changes. We superimposed the structure of retinoic acid bound to human ALDH1A3 onto the BcALDH structure and speculated a model of the substrate all-trans-retinal bound to BcALDH. We also proposed a plausible mechanism for the minor reducing activity of BcALDH. These BcALDH structures will be useful in understanding cofactor specificity and the catalytic mechanism of an atypical bacterial BcALDH and should help the development of a new biocatalyst to produce retinoic acid and related high-end products.

Laccases or benzenediol oxygen oxidoreductases (EC 1.10.3.2) are polyphenol multicopper oxidases that are known for their structural and functional diversity in various life forms. In the present study, the molecular and physico-chemical properties (redox-potential and secondary structures) of fungal laccase isozymes (FLIs) isolated from a medicinal mushroom Ganoderma lucidum were analyzed and compared with those of the recombinant bacterial laccases (rLac) obtained from different Yersinia enterocolitica strains. It was revealed that the FLIs contained His-Cys-His as the most conserved residue in its domain I Cu site, while the fourth and fifth residues were variable (Ile, Leu, or Phe). Evidently, the cyclic voltammetric measurements of Glac L2 at Type 1 Cu site revealed greater E° for ABTS/ABTS+ (0.312 V) and ABTS+/ABTS2+ (0.773 V) compared to the E° of rLac. Furthermore, circular dichroism-based conformational analysis revealed structural stability of the FLIs at acidic pH (3.0) and low temperature (<30 °C), while the isozymes were destabilized at neutral pH (7.0) and high-temperature conditions (>70 °C). The zymographic studies further confirmed the functional inactivation of FLIs at high temperatures (≥70 °C), predominantly due to domain unfolding. These findings provide novel insight into the evolution of the catalytic efficiency and redox properties of the FLIs, contributing to the existing knowledge regarding stress responses, metabolite production, and the biotechnological utilization of metabolites.

Glutaredoxins (GRXs) are small, ubiquitous, multifunctional, heat-stable and glutathione-dependent thiol-disulphide oxidoreductases, classified under thioredoxin-fold superfamily. In the green lineage, GRXs constitute a complex family of proteins. Based on their active site, GRXs are classified into two subfamilies: dithiol and monothiol. Monothiol GRXs contain 'CGFS' as a redox active motif and assist in maintaining redox state and iron homeostasis within the cell. Using RACE strategy, a full length cDNA of chickpea (Cicer arietinum) glutaredoxin 3 (CarGRX3) was cloned and sequenced. The cDNA contains open reading frame of 537 bp encoding 178 amino acids and exhibits features of other known 'CGFS' type GRXs. Based on the multiple sequence alignment among CarGRX3 and monothiol GRXs of other photosynthetic organisms, the characteristic motif (KGX4PXCGFSX([29/30/32])KX4WPTXPQX4GX3GGXDI) with 18 invariant residues was observed. The proposed structure of CarGRX3 was compared with structurally resolved monothiol GRXs of other organisms. The CarGRX3 and nearest Arabidopsis homolog (AtGRXcp) shares 76% sequence identity which was reflected by their 3D-structure conservation. The structure of chickpea monothiol GRX (CarGRX3) coordinates glutathione ligated [2Fe-2S] cluster in a homodimeric form, highlighting the structural basis for iron-sulfur cluster (ISC) assembly and delivery to acceptor proteins. The present study on CarGRX3 model highlighted the utility of the theoretical approaches to understand complex biological phenomena such as glutathione docking and incorporation of GSH-ligated [2Fe-2S] cluster.