Distorted octahedral coordination of tungstate in a subfamily of specific binding proteins

Bacteria and archaea import molybdenum and tungsten from the environment in the form of the oxyanions molybdate (MoO4 2−) and tungstate (WO4 2−). These substrates are captured by an external, high-affinity binding protein, and delivered to ATP binding cassette transporters, which move them across the cell membrane. We have recently reported a crystal structure of the molybdate/tungstate binding protein ModA/WtpA from Archaeoglobus fulgidus, which revealed an octahedrally coordinated central metal atom. By contrast, the previously determined structures of three bacterial homologs showed tetracoordinate molybdenum and tungsten atoms in their binding pockets. Until then, coordination numbers above four had only been found for molybdenum/tungsten in metalloenzymes where these metal atoms are part of the catalytic cofactors and coordinated by mostly non-oxygen ligands. We now report a high-resolution structure of A. fulgidus ModA/WtpA, as well as crystal structures of four additional homologs, all bound to tungstate. These crystal structures match X-ray absorption spectroscopy measurements from soluble, tungstate-bound protein, and reveal the details of the distorted octahedral coordination. Our results demonstrate that the distorted octahedral geometry is not an exclusive feature of the A. fulgidus protein, and suggest distinct binding modes of the binding proteins from archaea and bacteri

Synthetic Activity of Recombinant Whole Cell Biocatalysts Containing 2-Deoxy-D-ribose-5-phosphate Aldolase from Pectobacterium atrosepticum

In nature 2-deoxy-D-ribose-5-phosphate aldolase (DERA) catalyses the reversible formation of 2-deoxyribose 5-phosphate from D-glyceraldehyde 3-phosphate and acetaldehyde. In addition, this enzyme can use acetaldehyde as the sole substrate, resulting in a tandem aldol reaction, yielding 2,4,6-trideoxy-D-erythro-hexapyranose, which spontaneously cyclizes. This reaction is very useful for the synthesis of the side chain of statin-type drugs used to decrease cholesterol levels in blood. One of the main challenges in the use of DERA in industrial processes, where high substrate loads are needed to achieve the desired productivity, is its inactivation by high acetaldehyde concentration. In this work, the utility of different variants of Pectobacterium atrosepticum DERA (PaDERA) as whole cell biocatalysts to synthesize 2-deoxyribose 5-phosphate and 2,4,6-trideoxy-D-erythro-hexapyranose was analysed. Under optimized conditions, E. coli BL21 (PaDERA C-His AA C49M) whole cells yields 99 % of both products. Furthermore, this enzyme is able to tolerate 500 mM acetaldehyde in a whole-cell experiment which makes it suitable for industrial applications.Fil: Fernández Varela, Romina Noelia. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres". Grupo Vinculado al INGEBI- Laboratorio de Biocatálisis y Biotransformaciones - LBB - UNQUI; ArgentinaFil: Valino, Ana Laura. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología. Área Química. Laboratorio de Biotransformaciones; ArgentinaFil: Abdelraheem, Eman. Delft University of Technology; Países BajosFil: Médici, Rosario. Delft University of Technology; Países BajosFil: Martínez Sayé, Melisa Soledad. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Pereira, Claudio Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Investigaciones Médicas. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Investigaciones Médicas; ArgentinaFil: Hagedoorn, Peter-Leon. Delft University of Technology; Países BajosFil: Hanefeld, Ulf. Delft University of Technology; Países BajosFil: Iribarren, Adolfo Marcelo. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres". Grupo Vinculado al INGEBI- Laboratorio de Biocatálisis y Biotransformaciones - LBB - UNQUI; ArgentinaFil: Lewkowicz, Elizabeth Sandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigación en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres". Grupo Vinculado al INGEBI- Laboratorio de Biocatálisis y Biotransformaciones - LBB - UNQUI; Argentin

Substrate Induced Movement of the Metal Cofactor between Active and Resting State

Regulation of enzyme activity is vital for living organisms. In metalloenzymes, far-reaching rearrangements of the protein scaffold are generally required to tune the metal cofactor's properties by allosteric regulation. Here structural analysis of hydroxyketoacid aldolase from Sphingomonas wittichii RW1 (SwHKA) revealed a dynamic movement of the metal cofactor between two coordination spheres without protein scaffold rearrangements. In its resting state configuration (M$^{2+}$$_R$), the metal constitutes an integral part of the dimer interface within the overall hexameric assembly, but sterical constraints do not allow for substrate binding. Conversely, a second coordination sphere constitutes the catalytically active state (M$^{2+}$$_A$) at 2.4 Å distance. Bidentate coordination of a ketoacid substrate to M$^{2+}$$_A$ affords the overall lowest energy complex, which drives the transition from M$^{2+}$$_R$ to M$^{2+}$$_A$. While not described earlier, this type of regulation may be widespread and largely overlooked due to low occupancy of some of its states in protein crystal structures

Methyltransferases: Functions and Applications

In this review the current state of the art of S-adenosylmethionine (SAM)-dependent methyltransferases and SAM are evaluated. Their structural classification and diversity is introduced and key mechanistic aspects presented which are then detailed further. Then, catalytic SAM as a target for drugs and approaches to utilise SAM as a cofactor in synthesis is introduced with different recycling approaches evaluated. The use of SAM analogues are also described. Finally O-, N-, C- and S-MTs, their synthetic applications and potential for compound diversification is given

Radical-SAM dependent nucleotide dehydratase (SAND), rectification of the names of an ancient iron-sulfur enzyme using NC-IUBMB recommendations

In 1789, the influential French chemist Antoine-Laurent Lavoisier described his view of science and its langague in his book Traité élémentaire de chimie. According to the Robert Kerr’s translation it states (Lavoisier, 1790): “As ideas are preserved and communicated by means of words, it necessarily follows that we cannot improve the language of any science without at the same time improving the science itself; neither can we, on the other hand, improve a science without improving the language or nomenclature which belongs to it.” This view reminds us of Confucius’s earlier doctrine, the rectification of names (Steinkraus, 1980; Lau, 2000). Confucius believed that rectification of names is imperative. He explained (Steinkraus, 1980; Lau, 2000): “If language is incorrect, then what is said does not concord with what was meant, what is to be done cannot be affected. If what is to be done cannot be affected, then rites and music will not flourish. If rites and music do not flourish, then mutilations and lesser punishments will go astray. And if mutilations and lesser punishments go astray, then the people have nowhere to put hand or foot. Therefore the gentleman uses only such language as is proper for speech, and only speaks of what it would be proper to carry into effect. The gentleman in what he says leaves nothing to mere chance.” Inspired by these views, we make the analogy that the progress of science and the language used to describe it are two entangled electrons. This entanglement highlights the importance of introducing systemic names for enzymes using EC classification and the ever-growing problem of protein names (McDonald and Tipton, 2021). Here, we tackle one specific case of iron-sulfur ([FeS]) enzymes. We show that the language used to describe a conserved [FeS] enzyme of the innate immune system, i.e., viperin or RSAD2, is now inadequate and disentangled from its science. We discuss that the enzyme has cellular functions beyond its antiviral activity and that eukaryotic and prokaryotic enzymes catalyse the same chemical reactions. To prevent bias towards antiviral activity while studying various biochemical activities of the enzyme and using scientifically incorrect terms like “prokaryotic viperins,” we rectify the language describing the enzyme. Based on NC-IUBMB recommendations, we introduce the nomenclature S-adenosylmethionine (SAM) dependent Nucleotide Dehydratase (SAND)

Catalysis of iron core formation in Pyrococcus furiosus ferritin

The hollow sphere-shaped 24-meric ferritin can store large amounts of iron as a ferrihydrite-like mineral core. In all subunits of homomeric ferritins and in catalytically active subunits of heteromeric ferritins a diiron binding site is found that is commonly addressed as the ferroxidase center (FC). The FC is involved in the catalytic Fe(II) oxidation by the protein; however, structural differences among different ferritins may be linked to different mechanisms of iron oxidation. Non-heme ferritins are generally believed to operate by the so-called substrate FC model in which the FC cycles by filling with Fe(II), oxidizing the iron, and donating labile Fe(III)–O–Fe(III) units to the cavity. In contrast, the heme-containing bacterial ferritin from Escherichia coli has been proposed to carry a stable FC that indirectly catalyzes Fe(II) oxidation by electron transfer from a core that oxidizes Fe(II). Here, we put forth yet another mechanism for the non-heme archaeal 24-meric ferritin from Pyrococcus furiosus in which a stable iron-containing FC acts as a catalytic center for the oxidation of Fe(II), which is subsequently transferred to a core that is not involved in Fe(II)-oxidation catalysis. The proposal is based on optical spectroscopy and steady-state kinetic measurements of iron oxidation and dioxygen consumption by apoferritin and by ferritin preloaded with different amounts of iron. Oxidation of the first 48 Fe(II) added to apoferritin is spectrally and kinetically different from subsequent iron oxidation and this is interpreted to reflect FC building followed by FC-catalyzed core formation

Inhibition and stimulation of formation of the ferroxidase center and the iron core in Pyrococcus furiosus ferritin

Ferritin is a ubiquitous iron-storage protein that has 24 subunits. Each subunit of ferritins that exhibit high Fe(II) oxidation rates has a diiron binding site, the so-called ferroxidase center (FC). The role of the FC appears to be essential for the iron-oxidation catalysis of ferritins. Studies of the iron oxidation by mammalian, bacterial, and archaeal ferritin have indicated different mechanisms are operative for Fe(II) oxidation, and for inhibition of the Fe(II) oxidation by Zn(II). These differences are presumably related to the variations in the amino acid residues of the FC and/or transport channels. We have used a combination of UV–vis spectroscopy, fluorescence spectroscopy, and isothermal titration calorimetry to study the inhibiting action of Zn(II) ions on the iron-oxidation process by apoferritin and by ferritin aerobically preloaded with 48 Fe(II) per 24-meric protein, and to study a possible role of phosphate in initial iron mineralization by Pyrococcus furiosus ferritin (PfFtn). Although the empty FC can accommodate two zinc ions, binding of one zinc ion to the FC suffices to essentially abolish iron-oxidation activity. Zn(II) no longer binds to the FC nor does it inhibit iron core formation once the FC is filled with two Fe(III). Phosphate and vanadate facilitate iron oxidation only after formation of a stable FC, whereupon they become an integral part of the core. These results corroborate our previous proposal that the FC in PfFtn is a stable prosthetic group, and they suggest that its formation is essential for iron-oxidation catalysis by the protein