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 8 H I G H L I G H T S 2 0 2 2 I

notion that the Michaelase activity of DERA-MA proceeds via an iminium-based catalytic mechanism in which the enzyme-bound substrate is activated as an electrophile.

In contrast, the natural aldolase reaction proceeds via an enamine-based catalytic mechanism in which the enzyme- bound substrate is activated as a nucleophile.

This work demonstrates the power of directed evolution to redesign a natural aldolase into an unnatural Michaelase , and provides a stepping stone for further exploring rare- to-nature carboligation reactions with DERA-derived catalysts.

PRINCIPAL PUBLICATION AND AUTHORS

Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution, A. Kunzendorf (a), G. Xu (a), J.J.H. van der Velde (a), H.J. Rozeboom (b), A.M.W.H. Thunnissen (b), G.J. Poelarends (a), ACS Catal. 11, 13236-13243 (2021); https:/doi.org/10.1021/acscatal.1c03911 (a) Department of Chemical and Pharmaceutical Biology, Groningen Research Institute of Pharmacy, University of Groningen (The Netherlands) (b) Molecular Enzymology Group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen (The Netherlands)

REFERENCES

[1] A. Heine et al., Science 294, 369- 374 (2001). [2] D. Chambre et al., Chem. Commun. 55, 7498-7501 (2019). [3] G. DeSantis et al., Bioorg. Med. Chem. 11, 43-52 (2003). [4] M.A. Huffman et al., Science 366, 1255-1259 (2019).

Visualising breathing motions of protein structures

Proteins are not rigid structures: they move, breathe and adapt to their environment in order to optimise their shapes and functions. Cutting-edge work in nuclear magnetic resonance spectroscopy and X-ray crystallography maps the structural details of breathing motions in the core of a protein.

Nuclear magnetic resonance (NMR) studies carried out in the 1970s surprisingly demonstrated that aromatic amino acids in proteins can undergo so-called ring flipping (i.e., 180° rotations of the aromatic side chain). Paradoxically, these aromatic amino acids are often

located in the tightly packed protein core, where they engage in multiple interactions to maintain the protein fold, and thereby function. At that time, it was proposed that large-scale protein breathing motions would be necessary to accommodate these ring flips; however, until now, the structural details of these motions have remained enigmatic.

This work combined NMR spectroscopy and X-ray crystallography techniques in order to map the structural changes associated with aromatic ring flipping in the core of a protein (Figure 40). Using state-of-the-art macromolecular crystallography beamlines ID30A, ID23-1 and ID23-2, and beamlines at Diamond Light Source (I04 and I04-1), the study shows how specific

Fig. 39: Crystal structure of DERA-MA trapped in an intermediate catalytic state upon reaction with

cinnamaldehyde. a) Overall structure of DERA- MA in cartoon representation with the Lys167-

cinnamaldehyde iminium intermediate (INT1 in Figure 38b) shown as sticks. b) Comparison of

the active sites of DERA-MA (orange) and wildtype DERA (cyan, PDB entry 1JCJ) showing the different

covalent reaction intermediates formed with Lys167 (orange-blue, INT1 intermediate formed

in DERA-MA upon reaction with CIN; cyan-white, second intermediate formed in wildtype DERA upon reaction with acetaldehyde and D-glyceraldehyde- 3-phosphate. Movement of loop1 displaces Leu20 out of the active site, creating a pocket for binding the phenyl ring of cinnamaldehyde. Movements of

loop2 and loop3 disrupt the phosphate binding site of D-glyceraldehyde-3-phosphate. The positions of the mutated residues in the DERA-MA backbone are indicated with spheres and highlighted in magenta.

The mesh represents the 2Fo-Fc electron density map for the Lys167-cinnamaldehyde adduct, calculated

at 1.90 Å resolution and contoured at 1s.