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Heme proteins perform a variety of biological functions and also play significant roles in the field of bio-catalysis. The β-lactamase activity of heme proteins has rarely been reported. Herein, we found, for the first time, that myoglobin (Mb), an O2 carrier, also exhibits novel β-lactamase activity by catalyzing the hydrolysis of ampicillin. The catalytic proficiency ((kcat/KM)/kuncat) was determined to be 6.25 × 1010, which is much higher than the proficiency reported for designed metalloenzymes, although it is lower than that of natural β-lactamases. Moreover, we found that this activity could be regulated by an engineered disulfide bond, such as Cys46-Cys61 in F46C/L61C Mb or by the addition of imidazole to directly coordinate to the heme center. These results indicate that the heme active site is responsible for the β-lactamase activity of Mb. Therefore, the study suggests the potential of heme proteins acting as β-lactamases, which broadens the diversity of their catalytic functions. [ABSTRACT FROM AUTHOR]

Protein design provides a rigorous test of our knowledge about proteins and allows the creation of novel enzymes for biotechnological applications. Whereas progress has been made in designing proteins that mimic native proteins structurally (1-3), it is more difficult to design functional proteins (4-8). In comparison to recent successes in designing non-metalloproteins (4,6,7,9,10), it is even more challenging to rationally design metalloproteins that reproduce both the structure and function of native metalloenzymess (5,8,11-20). This is because protein metal-binding sites are much more varied than non-metal-containing sites, in terms of different metal ion oxidation states, preferred geometry and metal ion ligand donor sets. Because of their variability, it has been difficult to predict metal-binding site properties in silico, as many of the parameters, such as force fields, are ill-defined. Therefore, the successful design of a structural and functional metalloprotein would greatly advance the field of protein design and our understanding of enzymes. Here we report a successful, rational design of a structural and functional model of a metalloprotein, nitric oxide reductase (NOR), by introducing three histidines and one glutamate, predicted as ligands in the active site of NOR, into the distal pocket of myoglobin. A crystal structure of the designed protein confirms that the minimized computer model contains a haem/non-haem [Fe.sub.B] centre that is remarkably similar to that in the crystal structure. This designed protein also exhibits NO reduction activity, and so models both the structure and function of NOR, offering insight that the active site glutamate is required for both iron binding and activity. These results show that structural and functional metalloproteins can be rationally designed in silico., NOR is a metalloenzyme in the denitrification pathway of anaerobic bacteria that is responsible for the two-electron reduction of NO to [N.sub.2]O (ref. 21). It is a key enzyme in [...]

Imbalance in the cellular redox system is thought to be associated with the induction and progression of breast cancers, and heme proteins may regulate the redox balance. Cytochrome b5 (Cyt b5) is a small mitochondrial heme protein. Its function and regulating mechanism in breast cancer remain unknown. In this study, we elucidated the level of endogenous oxidative stress in breast cancer cells, MCF-7 cells (hormone receptor-positive cells) and MDA-MB-231 cells (triple-negative cells), and investigated the difference in Cyt b5 content. Based on the low content of Cyt b5 in MDA-MB-231 cells, the overexpression of Cyt b5 was found to regulate the oxidative stress and apoptosis cascades, including ERK1/2 and Akt signaling pathways. The overexpressed Cyt b5 MDA-MB-231 cells were shown to exhibit decreased oxidative stress, less phosphorylation of ERK1/2 and Akt, and less cleavage of caspases 3 and 9 upon treatment with H2O2, as compared to those of normal MDA-MB-231 cells. Moreover, the overexpressed Cyt b5 most likely functioned by interacting with its protein partner, Cyt c, as suggested by co-immunoprecipitation studies. These results indicated that Cyt b5 has different effects on breast cancer cells of different phenotypes, which provides useful information for understanding the multiple roles of Cyt b5 and provides clues for clinical treatment. [ABSTRACT FROM AUTHOR]

Design of metal‐selective hydrogels is attractive due to potential applications in materials and biological sciences. Although much progress has been made, assembly of both l‐ and d‐amino acid derivatives was less explored for design of metallohydrogels. In this study, we synthesized a facile and small tryptophan derivative containing an imidazole ligand with both l‐ and d‐ configurations (denoted as l/d‐ImW). Intriguingly, the assembly of (l+d)‐ImW gelators was found to selectively form a Ni2+‐hydrogel in aqueous medium at room temperature, which shows a rare purple color and exhibits excellent multi‐responsiveness. In addition to insights into the gelation mechanism, this study provides a novel approach to the design of metallohydrogels, by the assembly of (l+d)‐amino acid derivatives containing both aromatic rings and multiple metal coordination sites. [ABSTRACT FROM AUTHOR]