Oxidative Alkene Cleavage Catalysed by Manganese‐Dependent Cupin TM1459 from Thermotoga maritima

Abstract

An ovel biocatalytic oxidative alkene cleavagea ctivity was identifiedi np rotein TM1459 from Thermotoga maritima,aso far uncharacterised metalloprotein with ac upinf old, which preferentially binds manganese (over iron and zinc). Va rious styrene derivatives were converted with high chemoselectivity to the corresponding carbonylc ompounds by the manganese-containing protein,u sing organic hydroperoxide and molecular oxygen as oxidant. 4-Chloroacetophenone could be obtainedi n4 0% conversion from 4-chloro-a-methylstyrene (50 mM) in ab iphasic systemu sing ethyl acetate as organic cosolvent (5% v/v), while 76% conversion was obtained at al ower substrate concentration (10 mM). This novel biocatalyst can be easily over-expressed in Escherichia coli in exceptionally high yield andp urified, andt hus mayo ffer av aluablea nd safer alternative in oxidative C=Cb ond cleavage reactions for synthetic applications.

Engineering of TM1459 from Thermotoga maritima for Increased Oxidative Alkene Cleavage Activity

“…Site-saturation libraries of active site amino acids (Arg39, Phe41, Ile49, Trp56, Ile60, Phe94, Phe104, Cys106, and Ile108) were generated using primers with NNK codons. The plasmid pET26b(+)-TM1459 ( Hajnal et al, 2015 ) was used as template. For subsequent double and triple mutants, site-directed mutagenesis was performed using plasmids with TM1459 carrying one or two of the desired mutations as templates.…”

“…The reaction conditions for the bioconversion reactions were derived from Hajnal et al (2015) . The reactions were performed in a biphasic reaction system in 2 mL microreaction tubes.…”

“…Recently, biocatalytic oxidative alkene cleavage activity was also identified in TM1459 from Thermotoga maritima , an up to then uncharacterized metalloprotein with cupin fold, which preferentially binds manganese ( Hajnal et al, 2015 ). TM1459 was found active in the oxidative cleavage of styrene derivatives, working under mild conditions and using organic hydroperoxide and molecular oxygen as oxidant pair.…”

A DyP-Type Peroxidase of Pleurotus sapidus with Alkene Cleaving Activity

“…Different proteins of different enzyme classes, which are heme, non-heme iron, or non-iron metal dependent and have different protein structures as well as different reaction mechanism are known to catalyze alkene cleavage reactions [3]. Specifically, an isoeugnol and trans-anethole oxygenase from Pseudomonas putida and two manganese dependent enzymes from Thermotoga maritima (manganese-dependent Cupin TM1459) and Trametes hirsuta (Mn 3+ -dependent proteinase A homologue) oxidatively cleaved the benzylic double bond of different aryl alkenes, such as isoeugenol and trans-anethole to form the respective aldehydes [6][7][8][9]. In addition, alkene cleavage activity towards aryl alkenes was also detected for several peroxidases.…”

Cupin Variants as Macromolecular Ligand Library for Stereoselective Michael Addition of Nitroalkanes

“…[28] TM1459 is derived from hyperthermophile, whose scaffold is very thermally stable, and has a molecular mass of only 12 977 Da (residues 1-114) with a metal-binding site consisting of a 4-His metal binding motif (Figure 1 A); however, its function is unknown. [29,30] Herein, we have demonstrated that the small cupin variants of TM1459 ( Figure 1 B) can be effectively used to create efficient and robust biocatalysts for the stereoselective Michael addition reaction of nitroalkanes to a,b-unsaturated ketone, 2-azachalcone 1, achieving excellent diastereo-and enantioselectivity as well as high TON.…”