De novo biosynthesis of a nonnatural cobalt porphyrin cofactor in E. coli and incorporation into hemoproteins

Enzymes that bear a nonnative or artificially introduced metal center can engender novel reactivity and enable new spectroscopic and structural studies. In the case of metal-organic cofactors, such as metalloporphyrins, no general methods exist to build and incorporate new-to-nature cofactor analogs in vivo. We report here that a common laboratory strain, Escherichia coli BL21(DE3), biosynthesizes cobalt protoporphyrin IX (CoPPIX) under iron-limited, cobalt-rich growth conditions. In supplemented minimal media containing CoCl2, the metabolically produced CoPPIX is directly incorporated into multiple hemoproteins in place of native heme b (FePPIX). Five cobalt-substituted proteins were successfully expressed with this new-to-nature cobalt porphyrin cofactor: myoglobin H64V V68A, dye decolorizing peroxidase, aldoxime dehydratase, cytochrome P450 119, and catalase. We show conclusively that these proteins incorporate CoPPIX, with the CoPPIX making up at least 95% of the total porphyrin content. In cases in which the native metal ligand is a sulfur or nitrogen, spectroscopic parameters are consistent with retention of native metal ligands. This method is an improvement on previous approaches with respect to both yield and ease-of-implementation. Significantly, this method overcomes a long-standing challenge to incorporate nonnatural cofactors through de novo biosynthesis. By utilizing a ubiquitous laboratory strain, this process will facilitate spectroscopic studies and the development of enzymes for CoPPIX-mediated biocatalysis.

Brazilian native metal and the experience of transculturation

Politics and identity go together in Brazilian heavy metal. Headbangers often experience accusations regarding their less-than-Latin-American identity for enjoying a foreign musical style more than their own native styles. Even though this is partially true, Brazilian heavy metal engages national and musical identity in at least two different ways. The first is through the denial of any connection to Brazilian culture and its roots by accepting this anglophone genre. The second is through the transformation of the musical genre itself, thanks to the influence of Brazilian folk music. Based on these changes, we intend to describe how Sepultura laid down the roots that eventually flourished in the music of Arandu Arakuaa, a band associated with a movement known as the ‘Insurgency of Native Metal’, which describes itself as a union of Brazilian metal bands that write and perform songs about their country. We will also delve into how Sepultura, a highly regarded group often added as part of the ‘big five’ of thrash metal, used this influence to pave the way for other Brazilian bands, specifically Arandu Arakuaa, encouraging them to explore further possibilities regarding transculturation. Thus, in this article we intend to contemplate transculturation as a theoretical concept and as a tool to understand Brazilian heavy metal within its contradictions and core beliefs.

The Evolution of Platinum Jewellery Alloys: From the 1920s to the 2020s

Platinum has only been known to Europe since the 16th century. This was impure platinum, found as grains of native metal in alluvial deposits and comprising mainly platinum alloyed with the other 5 platinum group metals. They were exploited by pre-Colombian Indians of Ecuador and Colombia. In more recent times, the use of platinum in jewellery dates from the late 19th/early 20th century, often as a basis for diamond (and other precious gemstone) jewellery. Early jewellery alloys tended to be based on the existing industrial alloys and comparatively little development of specific jewellery alloys was carried out. Its acceptance as a hallmarkable jewellery metal came in 1975 when, with a wider availability of the metal, platinum was promoted as a high value jewellery metal and platinum jewellery started to grow in popularity, mainly at 950 and 900 fineness qualities. Since that time there has been alloy development specifically for jewellery application and tailored to the requirements of different manufacturing technologies. This review examines the evolution of platinum jewellery alloys over the last century against the challenges presented in developing improved alloys for jewellery application. There has been a substantial increase in alloy development over the last 30 years, particularly focused on improved investment (lost wax) casting alloys as well as better mechanical properties.

Elucidating the role of metal ions in carbonic anhydrase catalysis

Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn2+, tetrahedral to octahedral conversion for Co2+, octahedral for Ni2+, and trigonal bipyramidal for Cu2+) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.

Native Electrospray-based Metabolomics Enables the Detection of Metal-binding Compounds

Metals are essential for the molecular machineries of life, and microbes have evolved a variety of small molecules to acquire, compete for, and utilize metals. Systematic methods for the discovery of metal-small molecule complexes from biological samples are limited. Here we describe a two-step native electrospray ionization mass spectrometry method, in which double-barrel post-column metal-infusion and pH adjustment is combined with ion identity molecular networking, a rule-based informatics workflow. This method can be used to identify metal-binding compounds in complex samples based on defined mass (m/z) offsets of ion features with the same chromatographic profiles. As this native metal metabolomics approach can be easily implemented on any liquid chromatography-based mass spectrometry system, this method has the potential to become a key strategy for elucidating and understanding the role of metal-binding molecules in biology.

Comparative analysis of inducible promoters in cyanobacteria

Research in the field of synthetic biology highly depends on efficient, well-characterized promoters. While great progress has been made with other model organisms such as Escherichia coli, photosynthetic cyanobacteria still lag behind. Commonly used promoters that have been tested in cyanobacteria show weaker dynamic range or no regulation at all. Alternatives such as native metal-inducible promoters pose the problem of inducer toxicity.Here, we evaluate four different inducible promoters, both previously published and new, using the modular plasmid pSHDY, in the model cyanobacterium Synechocystis sp. PCC 6803 - namely the vanillate-inducible promoter PvanCC, the rhamnose-inducible Prha, and the aTc-inducible PL03, and the Co2+-inducible PcoaT. We estimate individual advantages and disadvantages, as well as dynamic range and strength of each promoter in comparison with well-established constitutive systems. We observed a delicate balance between transcription factor toxicity and sufficient expression to obtain a dose-dependent response to the inducer. In summary, we expand the current understanding and employability of inducible promoters in order to facilitate the construction of more complex regulatory synthetic networks, as well as more complicated biotechnological pathways for cyanobacteria.