Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for biocatalytic applications

Pils, Sabine; Schnabl, Kordula; Wallner, Silvia; Kljajic, Marko; Kupresanin, Nina; Breinbauer, Rolf; Fuchs, Michael; Rocha, Rosilene Fernandes da; Schrittwieser, Joerg H.; Kroutil, Wolfgang; Daniel, Bastian; Macheroux, Peter

doi:10.1016/j.molcatb.2016.10.018

Cited by 6 publications

(6 citation statements)

References 19 publications

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“…In the case of Pp BBE1, V175 is found in the pertinent ‘gate keeper’ position, and thus, the reduced enzyme is expected to rapidly react with dioxygen, as confirmed by the high rate of reoxidation, that is, 0.5 × 10 5 m ‐1 s ‐1 . In accordance with our ‘gate keeper’ model, replacement of this valine by leucine diminished the rate of reoxidation to 25 m −1 ·s −1 , thus basically suppressing the reaction of the reduced flavin with dioxygen as previously found for other members of this enzyme family .…”

Section: Resultssupporting

confidence: 89%

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase

et al. 2018

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The berberine bridge enzyme from the California poppy Eschscholzia californica (Ec BBE) catalyzes the oxidative cyclization of (S)‐reticuline to (S)‐scoulerine, that is, the formation of the berberine bridge in the biosynthesis of benzylisoquinoline alkaloids. Interestingly, a large number of BBE‐like genes have been identified in plants that lack alkaloid biosynthesis. This finding raised the question of the primordial role of BBE in the plant kingdom, which prompted us to investigate the closest relative of Ec BBE in Physcomitrella patens (Pp BBE1), the most basal plant harboring a BBE‐like gene. Here, we report the biochemical, structural, and in vivo characterization of Pp BBE1. Our studies revealed that Pp BBE1 is structurally and biochemically very similar to Ec BBE. In contrast to Ec BBE, we found that Pp BBE1 catalyzes the oxidation of the disaccharide cellobiose to the corresponding lactone, that is, Pp BBE1 is a cellobiose oxidase. The enzymatic reaction mechanism was characterized by a structure‐guided mutagenesis approach that enabled us to assign a catalytic role to amino acid residues in the active site of Pp BBE1. In vivo experiments revealed the highest level of PpBBE1 expression in chloronema, the earliest stage of the plant's life cycle, where carbon metabolism is strongly upregulated. It was also shown that the enzyme is secreted to the extracellular space, where it may be involved in later steps of cellulose degradation, thereby allowing the moss to make use of cellulose for energy production. Overall, our results suggest that the primordial role of BBE‐like enzymes in plants revolved around primary metabolic reactions in carbohydrate utilization.DatabaseStructural data are available in the PDB under the accession numbers 6EO4 and 6EO5.

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Section: Resultssupporting

confidence: 89%

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase

et al. 2018

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“…Although alcohol oxidase activity has previously been demon- ChemBioChem strated for cyclohexanol, 2-cyclohexen-1-ol and 1-phenyl ethanol, conversions or selectivity were poor. [18,22,29] In contrast to our poor E value for 2-pentanol 16, the alcohol oxidase from Phanerochaete chrysosporium gives E > 200. [22] Our reactions all proceeded with (S)-selectivity suggesting this might be an inherent property of cholesterol oxidases, [24] and indeed, many other alcohol oxidases also display (S)-selectivity.…”

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confidence: 81%

“…Thus we were interested in exploring other alcohol oxidases for the oxidation of secondary alcohols. Previous studies of secondary alcohol oxidation by alcohol oxidases have largely focussed on activated secondary allylic or benzylic alcohols [1,7,17,18] . There are some limited reports of oxidation of aliphatic secondary alcohols, in some cases alongside their enantioselectivity, although the substrate scope is narrow and it is not clear how these biocatalysts might perform more generally for synthetic applications [19–22] …”

Section: Figurementioning

confidence: 99%

“…Previous studies of secondary alcohol oxidation by alcohol oxidases have largely focussed on activated secondary allylic or benzylic alcohols. [1,7,17,18] There are some limited reports of oxidation of aliphatic secondary alcohols, in some cases alongside their enantioselectivity, although the substrate scope is narrow and it is not clear how these biocatalysts might perform more generally for synthetic applications. [19][20][21][22] We targeted cholesterol oxidase (EC 1.1.3.6) in order to develop a broad spectrum biocatalyst for the oxidation of secondary alcohols.…”

mentioning

confidence: 99%

“…[22] Our reactions all proceeded with (S)-selectivity suggesting this might be an inherent property of cholesterol oxidases, [24] and indeed, many other alcohol oxidases also display (S)-selectivity. [4,7,17,18,22,30] Conversely, an engineered galactose oxidase variant displayed (R)-selectivity [1] and has complementary selectivity for substrates 6, 7 and 8.…”

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confidence: 99%

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An Engineered Cholesterol Oxidase Catalyses Enantioselective Oxidation of Non‐steroidal Secondary Alcohols

2022

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The enantioselective oxidation of 2°alcohols to ketones is an important reaction in synthetic chemistry, especially if it can be achieved using O 2 -driven alcohol oxidases under mild reaction conditions. However to date, oxidation of secondary alcohols using alcohol oxidases has focused on activated benzylic or allylic substrates, with unactivated secondary alcohols showing poor activity. Here we show that cholesterol oxidase (EC 1.1.3.6) could be engineered for activity towards a range of aliphatic, cyclic, acyclic, allylic and benzylic secondary alcohols. Additionally, since the variants demonstrated high (S)-selectivity, deracemisation reactions were performed in the presence of ammonia borane to obtain enantiopure (R)-alcohols.

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Biocatalytic Enantioselective Oxidation of Sec‐Allylic Alcohols with Flavin‐Dependent Oxidases

Gandomkar

Jost

et al. 2019

Adv Synth Catal

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The oxidation of allylic alcohols is challenging to perform in a chemo‐ as well as stereo‐selective fashion at the expense of molecular oxygen using conventional chemical protocols. Here, we report the identification of a library of flavin‐dependent oxidases including variants of the berberine bridge enzyme (BBE) analogue from Arabidopsis thaliana (AtBBE15) and the 5‐(hydroxymethyl)furfural oxidase (HMFO) and its variants (V465T, V465S, V465T/W466H and V367R/W466F) for the enantioselective oxidation of sec‐allylic alcohols. While primary and benzylic alcohols as well as certain sugars are well known to be transformed by flavin‐dependent oxidases, sec‐allylic alcohols have not been studied yet except in a single report. The model substrates investigated were oxidized enantioselectively in a kinetic resolution with an E‐value of up to >200. For instance HMFO V465S/T oxidized the (S)‐enantiomer of (E)‐oct‐3‐en‐2‐ol (1 a) and (E)‐4‐phenylbut‐3‐en‐2‐ol with E>200 giving the remaining (R)‐alcohol with ee>99% at 50% conversion. The enantioselectivity could be decreased if required by medium engineering by the addition of cosolvents (e. g. dimethyl sulfoxide).

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Characterization of the monolignol oxidoreductase AtBBE-like protein 15 L182V for biocatalytic applications

Cited by 6 publications

References 19 publications

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase

The single berberine bridge enzyme homolog of Physcomitrella patens is a cellobiose oxidase

An Engineered Cholesterol Oxidase Catalyses Enantioselective Oxidation of Non‐steroidal Secondary Alcohols

Biocatalytic Enantioselective Oxidation of Sec‐Allylic Alcohols with Flavin‐Dependent Oxidases

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