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Metalloproteins are ubiquitous in all kingdoms of life. Their role and function are tightly related to the local structure of the metal-binding site. In this regard, the MetalPDB database is an invaluable tool since it stores the 3D structure of metal-binding sites and of their corresponding apo forms. In this work, we exploited MetalPDB to compute extensive statistics over >3000 clusters of mononuclear sites about the rearrangements occurring upon change in metalation state. For each cluster, we matched the holo and apo sites so that it was possible to average the distances between all possible pairs of Cα and donor atoms and thus quantitatively assess structural variations by computing the Δ values (mean apo distance – mean holo distance). For most of the structures the backbone is rigid with little to no rearrangement, while donor atoms experience significant changes of their relative position when the metal is removed. Sodium and potassium sites are an exception to this general observation. This is most likely caused by their preference for coordination by the main-chain oxygen atoms, making the rearrangement of donor atoms superimposable to that of the backbone. Magnesium and calcium show a different behavior, despite their chemical similarity: calcium sites undergo a larger reorganization upon metalation although both metals have similar percentage of backbone oxygen as donor atoms. We ascribe this observation to the structural and energetic factors regulating the selectivity for calcium over magnesium. We investigated the structural rearrangements occurring at mononuclear metal-binding sites of metalloproteins using information available from the MetalPDB database. In particular, we focused on the variation of backbone-backbone and donor atom-donor atom distances. The rearrangements were modest for sodium, potassium and magnesium sites and more sizable for all other metals. [Display omitted] • We analyzed >3092 clusters containing the 3D structures of metal-binding sites in their holo and apo forms • The Wilcoxon signed-rank test indicated significant differences between the metal-induced rearrangements of backbone and donor atoms for most physiological metals • Typically, the backbone shows little to no rearrangement between the apo and holo forms, whereas donor atoms feature extensive changes in relative position after metal removal • Calcium and magnesium have different behavior in their metal-induced rearrangements, despite their chemical similarity [ABSTRACT FROM AUTHOR]

Background Insufficient incorporation of heme is considered a central impeding cause in the recombinant production of active heme proteins. Currently, two approaches are commonly taken to overcome this bottleneck; metabolic engineering of the heme biosynthesis pathway in the host organism to enhance intracellular heme production, and supplementation of the growth medium with the desired cofactor or precursors thereof to allow saturation of recombinantly produced apo-forms of the target protein. In this study, we investigated the effect of both, pathway engineering and medium supplementation, to optimize the recombinant production of the heme protein horseradish peroxidase in the yeast Pichia pastoris. Results In contrast to studies with other hosts, co-overexpression of genes of the endogenous heme biosynthesis pathway did not improve the recombinant production of active heme protein. However, medium supplementation with hemin proved to be an efficient strategy to increase the yield of active enzyme, whereas supplementation with the commonly used precursor 5- aminolevulinic acid did not affect target protein yield. Conclusions The yield of active recombinant heme peroxidase from P. pastoris can be easily enhanced by supplementation of the cultivation medium with hemin. Thereby, secreted apo-species of the target protein are effectively saturated with cofactor, maximizing the yield of target enzyme activity. [ABSTRACT FROM AUTHOR]

Abstract: Thiocyanate hydrolase (SCNase) is a cobalt-containing enzyme with a post-translationally modified cysteine ligand, γCys131-SO2H. When the SCNase α, β and γ subunits were expressed in Escherichia coli, the subunits assembled to form a hetero-dodecamer, (αβγ)4, like native SCNase but exhibited no catalytic activity. Metal analysis indicated that SCNase was expressed as an apo-form irrespective of the presence of cobalt in the medium. On the contrary, SCNase co-expressed with P15K, encoded just downstream of SCNase genes, in cobalt-enriched medium under the optimized condition (SCNase(+P15K)) possessed 0.86 Co atom/αβγ trimer and exhibited 78% of the activity of native SCNase. SCNase(+P15K) showed a UV–Vis absorption peak characteristic of the SCNase cobalt center. About 70% of SCNase(+P15K) had the γCys131-SO2H modification. These results indicate that SCNase(+P15K) is the active holo-SCNase. P15K is likely to promote the functional expression of SCNase probably by assisting the incorporation of cobalt ion. [Copyright &y& Elsevier]