Function and versatile location of Met-rich inserts in blue oxidases involved in bacterial copper resistance.

Cuproxidases form a subgroup of the blue multicopper oxidase family. They display disordered methionine-rich loops, not observable in most available crystal structures, which have been suggested to bind toxic Cu(I) ions before oxidation into less harmful Cu(II) by the core enzyme. We found that the location of the Met-rich regions is highly variable in bacterial cuproxidases, but always inserted in solvent exposed surface loops, at close proximity of the conserved T1 copper binding site. We took advantage of the large differences in loop length between cold-adapted, mesophilic and thermophilic oxidase homologs to unravel the function of the methionine-rich regions involved in copper detoxification. Using a newly developed anaerobic assay for cuprous ions, it is shown that the number of Cu(I) bound is nearly proportional to the loop lengths in these cuproxidases and to the number of potential Cu(I) ligands in these loops. In order to substantiate this relation, the longest loop in the cold-adapted oxidase was deleted, lowering bound extra Cu(I) from 9 in the wild-type enzyme to 2-3 Cu(I) in deletion mutants. These results demonstrate that methionine-rich loops behave as molecular octopus scavenging toxic cuprous ions in the periplasm and that these regions are essential components of bacterial copper resistance.

Improving control in microbial cell factories: from single-cell to large-scale bioproduction.

Bioprocess deviations are likely to occur at different operating scales, leading in most of the case to substrate deviation from main metabolic routes and impact product synthesis. Correlating qS and qP is of utmost importance for bioprocess observability and control and can be modeled actually by advanced metabolic flux models. However, if most of these models are able to make prediction about metabolic switches, they still do not incorporate deviation due to biological noise, i.e. phenotypic and genotypic heterogeneity. These limitations impair observability and thus the use of fundamental knowledge about biological network for practical application, i.e. metabolic engineering or bioprocess scale-up.

Activity-stability relationships revisited in blue oxidases catalyzing electron transfer at extreme temperatures.

Cuproxidases are a subset of the blue multicopper oxidases that catalyze the oxidation of toxic Cu(I) ions into less harmful Cu(II) in the bacterial periplasm. Cuproxidases from psychrophilic, mesophilic, and thermophilic bacteria display the canonical features of temperature adaptation, such as increases in structural stability and apparent optimal temperature for activity with environmental temperature as well as increases in the binding affinity for catalytic and substrate copper ions. In contrast, the oxidative activities at 25 °C for both the psychrophilic and thermophilic enzymes are similar, suggesting that the nearly temperature-independent electron transfer rate does not require peculiar adjustments. Furthermore, the structural flexibilities of both the psychrophilic and thermophilic enzymes are also similar, indicating that the firm and precise bindings of the four catalytic copper ions are essential for the oxidase function. These results show that the requirements for enzymatic electron transfer, in the absence of the selective pressure of temperature on electron transfer rates, produce a specific adaptive pattern, which is distinct from that observed in enzymes possessing a well-defined active site and relying on conformational changes such as for the induced fit mechanism.