Photoswitching of peroxidase activity by position‐specific incorporation of a photoisomerizable non‐natural amino acid into horseradish peroxidase

Abstract

Horseradish peroxidase mutants containing L-p-phenylazophenylalanine (azoAla) at various positions were synthesized by using an Escherichia coli in vitro translation system. Among the 15 mutants examined, four mutants containing a single azoAla unit at the 6th, 68th, 142nd, and 179th positions, respectively, retained the peroxidase activity. The activity of the Phe68azoAla mutant was higher when the azobenzene group was in the cis form than in the trans form. On the contrary, the activity of the Phe179azoAla mutant disappeared when the azobenzene group was photoisomerized to the cis form, but recovered in the trans form. In the latter mutant, therefore, an on/off photoswitching of the peroxidase activity was attained. ß

Synthesis of 3,3′-bis(sulfonato)-4,4′-bis(chloroacetamido)azobenzene and cysteine cross-linking for photo-control of protein conformation and activity

“…Examples of biomolecular systems into which azobenzene has been incorporated for photo-regulation are peptides [1][2][3] , enzymes [4][5][6][7] , oligonucleotides 8,9 and ion channels 10,11 . Methods for incorporating azobenzene chromophores into biomolecules include solid-phase peptide or oligonucleotide synthesis 7,12 , nonsense suppression via azobenzenecharged suppressor tRNAs 4 , and both non-selective 13 and targeted chemical modification of protein side chains 14 .…”

Controlling the enzymatic activity of a restriction enzyme by light

“…Azobenzene can be reversibly isomerized between the extended trans-and the more compact cis-configuration by illumination with UV (trans → cis) or blue-light (cis → trans) as well as by thermal relaxation (cis → trans) (2-4). Four generally applicable approaches have been used to introduce azobenzene groups into peptides or proteins: (i) incorporation during peptide synthesis (5)(6)(7)(8), (ii) incorporation during in vitro translation (9,10), (iii) incorporation in vivo by using an orthogonal tRNA/aminoacyl tRNA synthetase pair specific for phenylalanine-4′-azobenzene (11), and (iv) chemical modification of peptides and proteins (3,12,13). Another more specific approach is to use azobenzenemodified ligands (e.g., inhibitors) for proteins (14,15).…”

“…Monofunctional azobenzene derivatives attached to proteins have also been used to regulate protein function by illumination in vitro (27)(28)(29)(30) and in vivo (31)(32)(33)(34)(35). Whereas few examples are known in which enzymes have been modified with monofunctional azobenzene derivatives or with an azobenzenebearing amino acid such that their catalytic activity could be modulated by light (9,10,36,37), none so far, to our knowledge, have been controlled by bifunctional azobenzene crosslinkers.…”

Molecular scissors under light control

“…The reversible changes in enzymatic activity in response to light have been demonstrated previously for various proteins (4,5). So why is the photoswitchable site-specific PvuII endonuclease so exciting?…”