Alteration of the Donor/Acceptor Spectrum of the (S)-Amine Transaminase from Vibrio fluvialis

Abstract

To alter the amine donor/acceptor spectrum of an (S)-selective amine transaminase (ATA), a library based on the Vibrio fluvialis ATA targeting four residues close to the active site (L56, W57, R415 and L417) was created. A 3DM-derived alignment comprising fold class I pyridoxal-5′-phosphate (PLP)-dependent enzymes allowed identification of positions, which were assumed to determine substrate specificity. These positions were targeted for mutagenesis with a focused alphabet of hydrophobic amino acids to convert an amine:α-keto acid transferase into an amine:aldehyde transferase. Screening of 1200 variants revealed three hits, which showed a shifted amine donor/acceptor spectrum towards aliphatic aldehydes (mainly pentanal), as well as an altered pH profile. Interestingly, all three hits, although found independently, contained the same mutation R415L and additional W57F and L417V substitutions.

Biocatalytic Production of Amino Carbohydrates through Oxidoreductase and Transaminase Cascades

“…Moreover,c arbohydrate oxidoreductases with different regio-and substrate specificities beyondt he oxidation of the primary C-6 hydroxyl group (e.g.,p yranose dehydrogenases) can helpextend the range of available carbonyl-containingcarbohydrates to ketone-functionalized carbohydrates, which are also potentialsubstrates for w-TAs. [57] Our analysisd emonstrates biocatalytic cascades to aminated cyclic carbohydrates, including oligosaccharides (Scheme 1). [47,48,56] In the presents tudy, Cvi-w-TAwas investigated for its potentialto aminate aldol-and keto-carbohydrates initially formed through oxidation by FgrGaOx or the pyranose dehydrogenase from Agaricusb isporus (AbiPDH1,U niProt: Q3L1D1), [53] respectively.…”

“…Cvi-w-TAactivity on oxidized carbohydrates was also compared against the M1 variant of the w-TAf rom Vibrio fluvialis (Vfl-w-TA,U niProt: F2XBU9) engineered by the group of Bornscheuer for improved preference towards substrates other than pyruvate and generally improved activity in the neutral pH range. [57] Our analysisd emonstrates biocatalytic cascades to aminated cyclic carbohydrates, including oligosaccharides (Scheme 1). Corresponding pathways generate an ew class of renewable telechelic molecules that were missing from the arsenal of buildingb locks to new biobased polymers.…”

Improvement in the Thermostability of a β‐Amino Acid Converting ω‐Transaminase by Using FoldX

“…For this specific mutation, the distance to catalytically active K267 (Scheme ) is 13.5 Å but only 5.0 Å to Y159. Y159 is important for the coordination of the cofactor PLP at the active site and can be found in many S ‐selective ω‐TAs at the same position at approximately amino‐acid residue 150 . Such important sides are not automatically excluded by FoldX and have to be selected manually.…”

Fully automatized high‐throughput enzyme library screening using a robotic platform

“…These findings not only confirm the importance of the flipping arginine in dual substrate recognition, but we also identified mutants that can convert an aldehyde. Further investigation of the substrate scope of these interesting mutants has been reported elsewhere (Genz et al, 2015).…”

“…1 and 2). The suitability and general applicability of this platform is exemplified for four different enzymes with relevance for biocatalysis: a (S)-selective amine transaminase [VFL-ATA, from Vibrio fluvialis (Genz et al, 2015)], a Baeyer-Villiger monooxygenase [CHMO, cyclohexanone monooxygenase from Acinetobacter calcoaceticus (Donoghue et al, 1976)], two haloalkane dehalogenases [(HLD, DhaA from Rhodococcus rhodochrous (Newman et al, 1999), and DhlA from Xanthobacter autotrophicus (Janssen et al, 1989)] and porcine acylase I [(pAcyl, from Sus scrofa (Lindner et al, 2008; for details see Table SI, Supporting Information)]. In all four cases, mutant libraries were created and screened for variants with altered properties using the automated robotic platform (Figs.…”