Mechanism-based inhibition of an aldolase at high concentrations of its natural substrate acetaldehyde: structural insights and protective strategies

Abstract

Understanding the deactivation mechanism of 2-deoxy-d-ribose-5-phosphate aldolase by its natural substrate leads to a single mutant showing complete acetaldehyde resistance.

Identification of Mechanism-Based Inactivation in P450-Catalyzed Cyclopropanation Facilitates Engineering of Improved Enzymes

“…Tracing the catalyst’s fate during the reaction enables the identification of such inactivation “hotspots”, and allows for the discovery of beneficial mutations at more distant residues. Echoing a recent report on mitigation of substrate inhibition in a DERA enzyme, 7c our results demonstrate the merits of mechanistic analyses in improving the overall activity of enzymes for biotechnological applications, especially ones that are employed in non-native environments. Given the growing interest in the development of biocatalytic carbene transfer reactions, we expect that the inactivation profiling and rational engineering approach we outline here will be generally useful for creating “carbene transferases” with improved lifetimes and tolerance to reaction conditions for synthetic applications.…”

“…However, introduction of xenobiotic reagents to natural enzymes can lead to undesired repercussions such as inactivation. 7 We have provided clear evidence for mechanism-based inactivation of a His-ligated P450-BM3 variant by ethyl diazoacetate that results in the modification of the heme prosthetic group and several protein side chains, which degrades enzyme performance and causes irreversible activity loss. Site-directed mutagenesis of some of the affected residues to amino acids that are less easily modified created superior variants with improved cyclopropanation activity as both purified enzymes and whole-cell catalysts.…”

“…Because understanding inactivation pathways can facilitate development of more stable and efficient enzymes, 7 we investigated whether the P450-derived ‘carbene transferase’ is inactivated by mechanisms similar to the suicide inactivation induced by iron carbenoid formation in native P450s. Covalent modification of the heme cofactor or protein side chains by the reactive carbenoid intermediate (Figure 1B) would compete with product formation and ultimately destroy the catalyst.…”

DERA in Flow: Synthesis of a Statin Side Chain Precursor in Continuous Flow Employing Deoxyribose-5-Phosphate Aldolase Immobilized in Alginate-Luffa Matrix

“…The temperature ranged between 28 and 36 • C in steps of 4.5 • C, while the pH was set to a value between 6.0 and 8.0 in steps of 0.5. Each reaction was carried out with 10 mg of freeze-dried cells hosting DERA suspended in 500 µL of buffer with 1.5 M of acetaldehyde as substrate and 7 µL of DMSO [24]. The mixture was stirred with 200 rpm at the respective temperature.…”

“…The active site of the wildtype DERA (DERA WT ) is irreversibly inhibited by the covalent binding of the side-product, crotonaldehyde. In 2016, the group of Pietruszka at the Research Center Jülich GmbH tackled this issue and developed a mutant (C47M), which is resistant to acetaldehyde to a high degree [24]. This mutant showed outstanding catalytic activity in tests using acetaldehyde as the donor molecule.…”