Chemical Modification of 1-Aminocyclopropane Carboxylic Acid (ACC) Oxidase: Cysteine Mutational Analysis, Characterization, and Bioconjugation with a Nitroxide Spin Label

Abstract

1-AminoCyclopropane Carboxylic acid Oxidase (ACCO) catalyzes the last step of ethylene biosynthesis in plants. Although some sets of structures have been described, there are remaining questions on the active conformation of ACCO and in particular, on the conformation and potential flexibility of the C-terminal part of the enzyme. Several techniques based on the introduction of a probe through chemical modification of amino acid residues have been developed for determining the conformation and dynamics of proteins. Cysteine residues are recognized as convenient targets for selective chemical modification of proteins, thanks to their relatively low abundance in protein sequences and to their well-mastered chemical reactivity. ACCO have generally 3 or 4 cysteine residues in their sequences. By a combination of approaches including directed mutagenesis, activity screening on cell extracts, biophysical and biochemical characterization of purified enzymes, we evaluated the effect of native cysteine replacement and that of insertion of cysteines on the C-terminal part in tomato ACCO.

The Hunt for the Closed Conformation of the Fruit‐Ripening Enzyme 1‐Aminocyclopropane‐1‐carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study

“…ACCO from tomato contains four cysteines in the main core: C28, C60, C133 and C165. In a previous study, we showed that only two of the existing native cysteines are accessible to the P label: C60 and C165 . We then performed a complete mutagenesis study to provide proteins displaying unique modification sites in the main core while retaining their primary structure and function.…”

“…We first generated the double mutants C165H/L292C and C165H/M307C. The enzymes were expressed and purified according to previously reported procedures . Labeling experiments were conducted with the P spin label (Figure C) and the labeled enzymes were analyzed by mass spectrometry (MS) to confirm the introduction of the two labels at the desired positions, namely C60*C292* and C60*C307* (see Table S2 in the Supporting Information).…”

“…Hence, we will refer to this crystal structure as the “open” conformation of ACCO (Figure A). It has been shown that the C‐terminal region is essential for the enzymatic activity and C‐terminal truncated enzymes are poorly active . In addition, several residues in this part of the enzyme (e.g., K297 and R300) are important for cofactor binding .…”

“…It is, however, important to note that a C‐terminal truncated form of At ACO2 has been crystallized (303 amino acids compared with 320 at full length). The lack of the extremity of the C‐terminal part may have an influence on both the conformation and the activity of the enzyme . Overall, these structural differences might reflect a flexibility of the C terminus that is possibly important for the activity of ACCO enzymes.…”

“…Tomato ACCO contains four cysteine residues (C28, C60, C133 and C165) in the sequence located in the main core of the protein. In a previous study, we reported on mutagenesis experiments designed to systematically replace native cysteines by other amino acids to reduce the number of cysteines for chemical modification . We have also shown that several positions in the C‐terminal part of the enzyme are suitable for the introduction of cysteines without losing activity (in particular, L292C and M307C mutants were successfully produced and chemically modified).…”