S T R U C T U R A L B I O L O G Y

S C I E N T I F I C H I G H L I G H T S

4 2 H I G H L I G H T S 2 0 2 1 I

every subsequent mutation improving activity of the enzyme in the cellular context. Therefore, the newly designed enzyme is an important step towards creating a microbe that can efficiently turn formate or methanol into valuable products, a process that is essential to realising

a circular C1 economy. In a broader context, this work also showcases the potential of protein engineering and synthetic biology in addressing today s key challenge of carbon emissions.

Fig. 28: Enzyme engineering by structure-based directed evolution. After a basal initial activity is detected in the enzyme candidate, its structure is evaluated and targets for mutagenesis are identified based on their proximity to the active site. Variant libraries are created and evaluated in a high-throughput fashion. The best performer is used as template for another round of mutagenesis and screening. Once no further improvement is achieved, the final variant is taken forward to in vivo implementation.

PRINCIPAL PUBLICATION AND AUTHORS

Engineering a Highly Efficient Carboligase for Synthetic One-Carbon Metabolism, M. Nattermann (a), S. Burgener (a), P. Pfister (a), A. Chou (b), L. Schulz (a), S.H. Lee (b), N. Nicole (a), J. Zarzycki (a), R. Gonzalez (b), T.J. Erb (a,c), ACS Catal. 11, 5396-5404 (2021); https:/doi.org/10.1021/acscatal.1c01237 (a) Max-Planck-Institute for terrestrial Microbiology, Marburg (Germany) (b) University of South Florida, Tampa (USA) (c) LOEWE Center for Synthetic Microbiology, Marburg (Germany)

REFERENCES

[1] G.A. Olah, Angew. Chem. Int. Ed. Engl. 44, 2636-2639 (2005). [2] O. Yishai et al., Curr. Opin. Chem. Biol. 35, 1-9 (2016). [3] C.L. Berthod et al., Structure 15, 853-861 (2007).

Shining light (and X-rays) on a critical biological switch

Akt is an essential protein kinase at the heart of cell growth, proliferation and survival. Two membrane- based phospholipids generated in response to growth factors are sensed by Akt and transduced into the appropriate cellular response. This work demonstrates that transmission of these signals by Akt is strictly dependent on physical engagement of Akt with one of its lipid activators.

Discovered almost 30 years ago as a viral oncogene, the protein kinase Akt has been the subject of more than 100 000 scientific articles. An effector of the phosphoinositide 3-kinase (PI3K) pathway, Akt is hyperactivated in the majority of human cancers, while mutations in Akt itself are causative of rare overgrowth disorders such as Proteus Syndrome [1].

Akt is activated by two phospholipids: phosphatidylinositol- 3,4,5-trisphosphate (PIP3), synthesised at the plasma membrane by class I PI3K, and phosphatidylinositol-3,4- bisphosphate (PI(3,4)P2), the product of SHIP-mediated dephosphorylation of PIP3 and found throughout the endolyosomal membrane system. Binding of Akt to PIP3 or PI(3,4)P2 is accomplished by its N-terminal pleckstrin homology (PH) domain, while its C-terminal kinase domain is responsible for signal transduction. Early work established that Akt requires phosphorylation of two regulatory amino acids in its kinase domain for activity, events mediated by phosphoinositide-dependent kinase 1 (PDK1) and mammalian target of rapamycin complex 2 (mTORC2). Binding of Akt to PIP3 or PI(3,4)P2 promotes phosphorylation by inducing a conformational change that exposes the respective motifs.

Many substrates of Akt, however, are localised in subcellular compartments distal to the plasma membrane, raising the