Donor‐Promiskuität einer thermostabilen Transketolase durch gelenkte Evolution – effektive Komplementierung der 1‐Desoxy‐<scp>d</scp>‐ xylulose‐5‐phosphat‐Synthase‐Aktivität

Saravanan, T.; Junker, Sebastian; Kickstein, Michael; Hein, Sascha; Link, Marie‐Kristin; Ranglack, Jan; Witt, Samantha; Lorillière, Marion; Hecquet, Laurence; Fessner, Wolf‐Dieter

doi:10.1002/ange.201701169

Cited by 6 publications

(2 citation statements)

References 42 publications

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“…Following the principle of the microreversibility of TK gst reaction, we expected that the combination of L118I with the single position H102L or with the double‐site mutation H102L/H474 (S or G) could lead to the desired products with high efficiency. Indeed, the active site analysis showed that the double variant H102L/H474 (S or G) [10a] greatly improved the substrate affinity toward pyruvate analogs including oxobutyrate. The presence of smaller and less polar side chains in place of the two histidine allowed enlargement of active site for bigger hydrophobic donor substrates and kept the catalytic mechanism intact.…”

Section: Resultsmentioning

confidence: 99%

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Synthesis of Deoxysugars from Aliphatic α‐Ketoacids and Aldoses Catalyzed by Thermostable Transketolase Variants from Geobacillus stearothermophilus

et al. 2022

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We described a strategy for the enzymatic synthesis of 1-deoxy and 1,2 deoxyketoses from the aliphatic -ketoacids, pyruvate and 2-oxobutyrate, as donors, and natural aldoses of variable chain length as acceptors, catalyzed by thermostable transketolase variants from Geobacillus stearothermophilus (TKgst). Analytical studies have been carried out on a panel of TKgst variants with the appropriate substrates allowing to select the best combinations and to apply it to the preparative scale synthesis of 1-deoxy and 1,2 deoxy ketoses obtained with good to excellent isolated yields (61%-86%). To optimize the strategy, and as a proof of principle, the -ketoacids pyruvate and 2-oxobutyrate were generated in situ from the corresponding Daminoacids D-alanine and D-homoalanine respectively, using a thermostable D-aminoacid oxidase DAAO4536 that was selected from a screening of 55 putative DAAOs provided by Prozomix Limited.Hence, a one-pot one step procedure was performed at 50 °C by coupling DAAO4536 and the best TKgst variant H102L/L118I/H474S in the presence of Dalanine or D-homoalanine as -ketoacids precursors and D-erythrose as acceptor substrate. The corresponding 1-deoxy and 1,2-dideoxydeoxyketoses were isolated with good yields (64% and 72% respectively, out of two steps).

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Section: Resultsmentioning

confidence: 99%

“…Expression was carried out in E. coli BL21(DE3)pLysS strain. This strain was transformed by heat shock with the following TK gst variants: artificial wild type TK gst , [9] H102L/H474S, [10a] H102L/L118I/H474S, [10b] H102L/L118I/H474G, [10b] and H102L/L118I [10b] . DAAO Rg was overexpressed [16] with the plasmid pT7.…”

Section: Methodsmentioning

confidence: 99%

Synthesis of Deoxysugars from Aliphatic α‐Ketoacids and Aldoses Catalyzed by Thermostable Transketolase Variants from Geobacillus stearothermophilus

et al. 2022

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Transketolase Catalyzed Synthesis of N‐Aryl Hydroxamic Acids

2021

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Hydroxamic acids are metal‐chelating compounds that show important biological activity including anti‐tumor effects. We have recently engineered the transketolase from Geobacillus stearothermopilus (TKgst) to convert benzaldehyde as a non‐natural acceptor substrate. Realizing the structural and electronic similarity to nitrosobenzene, we studied the TK‐catalyzed conversion of nitrosoarenes to yield N‐arylated hydroxamic acids. Here we demonstrate that wild‐type and variants of this versatile TKgst enzyme indeed induce the rapid biocatalytic conversion of variously p‐, m‐ and o‐substituted nitrosoarenes to produce a variety of corresponding N‐aryl hydroxamic acids via creation of a carbon‐nitrogen instead of a carbon‐carbon bond. Further structural modifications can be introduced by varying the donor component, such as hydroxypyruvate or pyruvate.

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Nucleophile Promiscuity of Engineered Class II Pyruvate Aldolase YfaU from E. Coli

et al. 2018

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Pyruvate-dependent aldolases exhibit as tringent selectivity for pyruvate,l imiting application of their synthetic potential, which is ad rawback shared with other existing aldolases.S tructure-guided rational protein engineering rendered a2 -keto-3-deoxy-l-rhamnonate aldolase variant, fused with am altose-binding protein (MBP-YfaU W23V/L216A), capable of efficiently converting larger pyruvate analogues,for example,t hose with linear and branched aliphatic chains,i n aldol addition reactions.C ombination of these nucleophiles with N-Cbz-alaninal (Cbz = benzyloxycarbonyl) and N-Cbzprolinal electrophiles gave access to chiral building blocks,for example,d erivatives of (2S,3S,4R)-4-amino-3-hydroxy-2methylpentanoic acid (68 %, d.r.9 0:10) and the enantiomer of dolaproine (33 %, d.r.9 4:6) as well as ac ollection of unprecedented a-amino acid derivatives of the proline and pyrrolizidine type.C onversions varied between 6-93 %a nd diastereomeric ratios from 50:50 to 95:5 depending on the nucleophilic and electrophilic components.

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Donor‐Promiskuität einer thermostabilen Transketolase durch gelenkte Evolution – effektive Komplementierung der 1‐Desoxy‐d‐ xylulose‐5‐phosphat‐Synthase‐Aktivität

Cited by 6 publications

References 42 publications

Synthesis of Deoxysugars from Aliphatic α‐Ketoacids and Aldoses Catalyzed by Thermostable Transketolase Variants from Geobacillus stearothermophilus

Synthesis of Deoxysugars from Aliphatic α‐Ketoacids and Aldoses Catalyzed by Thermostable Transketolase Variants from Geobacillus stearothermophilus

Transketolase Catalyzed Synthesis of N‐Aryl Hydroxamic Acids

Nucleophile Promiscuity of Engineered Class II Pyruvate Aldolase YfaU from E. Coli

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