Identification and characterization of a novel “thermophilic-like” Old Yellow Enzyme from the genome of Chryseobacterium sp. CA49

Xu, Mengyu; Pei, Xiaokun; Wu, Zhong‐Liu

doi:10.1016/j.molcatb.2014.07.002

Cited by 16 publications

(12 citation statements)

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“…OYE Ro 2 showed strong and almost identical activity with maleimide substrates ( Table 3 ). The specific activities with maleimides (45–51 U mg -1 at 25°C) count to the highest reported for ERs among LeOPR with N -ethylmaleimide (41.3 U mg -1 ; Straßner et al, 1999 ), TOYE with N -methylmaleimide (40.3 U mg -1 ; Adalbjörnsson et al, 2010 ), Chr -OYE3 with 2-methyl- N -phenylmaleimide (52.9 U mg -1 , Xu et al, 2014 ), and Bac-OYE2 with 2-methylmaleimide (44 U mg -1 ; Zhang et al, 2014 ). OYE Ro 2 was much less active with cyclic enones.…”

Section: Resultsmentioning

confidence: 83%

“…A second group of bacterial OYEs was designated as ‘YqjM-like’ or ‘thermophilic-like’ OYEs ( Toogood et al, 2010 ). Members of this group, e.g., Ts ER (CAP16804, Opperman et al, 2008 ), TOYE (ABY93685, Adalbjörnsson et al, 2010 ), Gk OYE (BAD76617, Schittmayer et al, 2011 ), Geo ER (BAO37313, Tsuji et al, 2014 ), and Chr -OYE3 (AHV90721, Xu et al, 2014 ) are stable at temperatures up to 80°C and range in oligomeric state from dimers to dodecamers.…”

Section: Introductionmentioning

confidence: 99%

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Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

et al. 2015

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Ene-reductases (ERs) are widely applied for the asymmetric synthesis of relevant industrial chemicals. A novel ER OYERo2 was found within a set of 14 putative old yellow enzymes (OYEs) obtained by genome mining of the actinobacterium Rhodococcus opacus 1CP. Multiple sequence alignment suggested that the enzyme belongs to the group of ‘thermophilic-like’ OYEs. OYERo2 was produced in Escherichia coli and biochemically characterized. The enzyme is strongly NADPH dependent and uses non-covalently bound FMNH2 for the reduction of activated α,β-unsaturated alkenes. In the active form OYERo2 is a dimer. Optimal catalysis occurs at pH 7.3 and 37°C. OYERo2 showed highest specific activities (45-50 U mg-1) on maleimides, which are efficiently converted to the corresponding succinimides. The OYERo2-mediated reduction of prochiral alkenes afforded the (R)-products with excellent optical purity (ee > 99%). OYERo2 is not as thermo-resistant as related OYEs. Introduction of a characteristic intermolecular salt bridge by site-specific mutagenesis raised the half-life of enzyme inactivation at 32°C from 28 to 87 min and improved the tolerance toward organic co-solvents. The suitability of OYERo2 for application in industrial biocatalysis is discussed.

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Section: Resultsmentioning

confidence: 83%

Section: Introductionmentioning

confidence: 99%

Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

et al. 2015

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“…The same protein was the basis for the first OYE crystal structure, uncovering a TIM-barrel topology, related to trimethylamine dehydrogenase [83]. Since then, many classical OYEs were identified from proteobacteria (NCR [84], MR [85], PETNR [8]), flavobacteria (Chr-OYE2 [74]), cyanobacteria [21], yeasts (OYE1-OYE3 [86,87], CYE [62]) and plants [67], outlined in Tables 1 and 2. Nicotinamide coenzyme biomimetics (NCBs) previously used in OYE-catalysed hydrogenations to replace NAD(P)H [52,55,58].…”

Section: Phylogenetic Classification Of Oyesmentioning

confidence: 99%

“…Nevertheless, the enzymes AtOPR1-AtOPR3 from Arabidopsis thaliana [65,66], and LeOPR1-LeOPR3 from Solanum lycopersicum (tomato) [67,68], were characterised according to their structure, function and physiological role. Two thirds of the characterised OYEs have been obtained from various classes of bacteria including proteobacteria (28%) [69,70], actinobacteria (5%) [22,71,72], bacteroidetes (5%) [73,74], firmicutes (10%) [75][76][77], deinococcus-thermus (3%) [78][79][80], and cyanobacteria (17%) [21,81].…”

Section: Phylogenetic Classification Of Oyesmentioning

confidence: 99%

Old Yellow Enzyme-Catalysed Asymmetric Hydrogenation: Linking Family Roots with Improved Catalysis

et al. 2017

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Asymmetric hydrogenation of activated alkenes catalysed by ene-reductases from the old yellow enzyme family (OYEs) leading to chiral products is of potential interest for industrial processes. OYEs' dependency on the pyridine nucleotide coenzyme can be circumvented through established artificial hydride donors such as nicotinamide coenzyme biomimetics (NCBs). Several OYEs were found to exhibit higher reduction rates with NCBs. In this review, we describe a new classification of OYEs into three main classes by phylogenetic and structural analysis of characterized OYEs. The family roots are linked with their use as chiral catalysts and their mode of action with NCBs. The link between bioinformatics (sequence analysis), biochemistry (structure-function analysis), and biocatalysis (conversion, enantioselectivity and kinetics) can enable an early classification of a putative ene-reductase and therefore the indication of the binding mode of various activated alkenes.

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“…In the case of other characterized class III OYEs such as LacER [36] and YqiG [15,34], a similar pH profile was determined albeit with a wider pH working range as indicated by the reported optimum activities in the range of pHopt 8-9 and pHopt 6-9, respectively. Notably, OYE enzymes belonging to other subclasses exhibit similar pH profiles as reported for Ppo-Er1, e.g., the "classical" XenB [45] and NemA [45] with a pHopt of 6-7.5, the "thermophilic-like" YqjM [46] and Chr-OYE3 [47] with a pHopt of 6-8, and the class IV enzyme Ppo-Er3 [15] with a pHopt of 7-8.5. [44].…”

Section: Expression and Characterization Of Ppo-er1mentioning

confidence: 62%

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

2020

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Ene reductases enable the asymmetric hydrogenation of activated alkenes allowing the manufacture of valuable chiral products. The enzymes complement existing metal- and organocatalytic approaches for the stereoselective reduction of activated C=C double bonds, and efforts to expand the biocatalytic toolbox with additional ene reductases are of high academic and industrial interest. Here, we present the characterization of a novel ene reductase from Paenibacillus polymyxa, named Ppo-Er1, belonging to the recently identified subgroup III of the old yellow enzyme family. The determination of substrate scope, solvent stability, temperature, and pH range of Ppo-Er1 is one of the first examples of a detailed biophysical characterization of a subgroup III enzyme. Notably, Ppo-Er1 possesses a wide temperature optimum (Topt: 20–45 °C) and retains high conversion rates of at least 70% even at 10 °C reaction temperature making it an interesting biocatalyst for the conversion of temperature-labile substrates. When assaying a set of different organic solvents to determine Ppo-Er1′s solvent tolerance, the ene reductase exhibited good performance in up to 40% cyclohexane as well as 20 vol% DMSO and ethanol. In summary, Ppo-Er1 exhibited activity for thirteen out of the nineteen investigated compounds, for ten of which Michaelis–Menten kinetics could be determined. The enzyme exhibited the highest specificity constant for maleimide with a kcat/KM value of 287 mM−1 s−1. In addition, Ppo-Er1 proved to be highly enantioselective for selected substrates with measured enantiomeric excess values of 92% or higher for 2-methyl-2-cyclohexenone, citral, and carvone.

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Identification and characterization of a novel “thermophilic-like” Old Yellow Enzyme from the genome of Chryseobacterium sp. CA49

Cited by 16 publications

References 50 publications

Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

Functional characterization and stability improvement of a ‘thermophilic-like’ ene-reductase from Rhodococcus opacus 1CP

Old Yellow Enzyme-Catalysed Asymmetric Hydrogenation: Linking Family Roots with Improved Catalysis

Characterization of the Novel Ene Reductase Ppo-Er1 from Paenibacillus Polymyxa

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