Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of variants of monoamine oxidase from<i>Aspergillus niger</i>

Atkin, Kate E.; Reiss, Renate; Turner, Nicholas J.; Brzozowski, A.M.; Grogan, Gideon

doi:10.1107/s174430910800345x

Cited by 34 publications

(34 citation statements)

References 21 publications

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“…The mutant MAO-N D5 (Atkin et al 2008), which contains five amino acid mutations, has been used for the desymmetrisation of non-chiral amines such as the substituted pyrrolidine 3-azabicyclo [3,3,0]octane ). This amine is a building block in the hepatitis C viral serine protease inhibitor compound telaprevir (Online Resource, Fig.…”

Section: Introductionmentioning

confidence: 99%

Immobilised whole-cell recombinant monoamine oxidase biocatalysis

Zajkoska

Rosenberg

Heath

et al. 2014

Appl Microbiol Biotechnol

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This work demonstrates the first example of the immobilisation of MAO-N whole cells to produce a biocatalyst that remained suitable for repetitive use after 11 months of storage and stable up to 15 months after immobilisation. The production of Escherichia coli expressing recombinant MAO-N was scaled up to bioreactors under regulated, previously optimised conditions (10% DO, pH 7), and the amount of biomass was almost doubled compared to flask cultivation. Subsequently, pilot immobilisation of the whole-cell biocatalyst using LentiKats technology was performed. The amount of the immobilised biomass was optimised and the process was scaled up to a production level by immobilising 15 g of dry cell weight per litre of polyvinyl alcohol to produce 3 kg of whole-cell ready-to-use biocatalyst. The immobilised biocatalyst retained its initial activity over six consecutive biotransformations of the secondary amine model compound 3-azabicylo [3,3,0]octane, a building block of the hepatitis C drug telaprevir. Consecutive cultivation cycles in growth conditions not only increased the initial specific activity of biocatalyst produced on the industrial plant by more than 30%, but also significantly increased the rate of the biotransformation compared to the non-propagated biocatalyst.

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Section: Introductionmentioning

confidence: 99%

Immobilised whole-cell recombinant monoamine oxidase biocatalysis

Zajkoska

Rosenberg

Heath

et al. 2014

Appl Microbiol Biotechnol

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“…Following agarose gel analysis of the PCR products, the relevant bands were eluted from the gels using a PCR Cleanup kit® (Qiagen). The genes were then sub-cloned into the pET-YSBL-LIC-3C vector using previously published techniques [14]. The resultant plasmids were then used to transform E. coli XL1-Blue cells (Novagen), yielding colonies which in turn gave plasmids using standard miniprep procedures that were sequenced to confirm the identity and sequence of the genes.…”

Section: Gene Cloning Expression and Protein Purificationmentioning

confidence: 99%

“…RasADH was expressed from a strain of E. coli BL21 (DE3) that had been transformed with the gene encoding RasADH ligated into the pET-YSBLIC-3C vector [14]. After purification using nickel affinity and gel filtration chromatography, the pure protein was initially concentrated to 8 mg mL -1 .…”

Section: Structure Of Rasadhmentioning

confidence: 99%

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Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp. Reveal the Molecular Basis for Their Recognition of ‘Bulky–Bulky’ Ketones

et al. 2013

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Alcohol dehydrogenases (ADHs) are applied in industrial synthetic chemistry for the production of optically active secondary alcohols. However, the substrate spectrum of many ADHs is narrow, and few, for example, are suitable for the reduction of prochiral ketones in which the carbonyl group is bounded by two bulky and/or hydrophobic groups; so-called 'bulky-bulky' ketones. Recently two ADHs, RasADH from Ralstonia sp. DSM 6428, and SyADH from Sphingobium yanoikuyae DSM 6900, have been described, which are distinguished by their ability to accept bulky-bulky ketones as substrates. In order to examine the molecular basis of the recognition of these substrates the structures of the native and NADPH complex of RasADH, and the NADPH complex of SyADH have been determined and refined to resolutions of 1.5, 2.9 and 2.5 Å respectively. The structures reveal hydrophobic active site tunnels near the surface of the enzymes that are well-suited to the recognition of large hydrophobic substrates, as determined by modelling of the bulkybulky substrate n-pentyl phenyl ketone. The structures also reveal the bases for NADPH specificity and (S)-stereoselectivity in each of the biocatalysts for n-pentyl phenyl ketone and related substrates.

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“…The binding mode of (R)-mexiletine with MAO-N-D5 (Fig. 2) is ineffective because the hydrogen atom connected to the chiral carbon that participates in the catalytic process orients opposite to the isoalloxazine ring of the cofactor FAD, which is involved directly in the catalytic reaction via the N1 and N5 atoms [2]. To enable catalysis by MAO-N-D5, the hydrogen atom must invert its orientation to face the FAD isoalloxazine moiety, which corresponds to the (S)-form enantiomer of rac-mexiletine.…”

Section: Resultsmentioning

confidence: 98%

Semi-rational Directed Evolution of Monoamine Oxidase for Kinetic Resolution of rac-Mexiletine

Chen

et al. 2015

Appl Biochem Biotechnol

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Semi-rational directed evolution was applied to the D5 variant of monoamine oxidase from Aspergillus niger (MAO-N-D5) with the aim of deriving the more desirable (R)-mexiletine through the kinetic resolution of mexiletine enantiomers. Although MAO-N-D5 shows no activity towards rac-mexiletine, theoretical molecular docking studies revealed the potential binding conformations of both mexiletine enantiomers and MAO-N-D5. The key factors affecting the catalytic activity and specificity were identified. Based on the docking results, six residues in the binding pocket and along the binding pathway were selected as key sites for saturation mutagenesis of MAO-N-D5. Through several rounds of screening and combinatorial experiments, two active MAO variants with high enantioselectivities towards (S)-mexiletine evolved, namely A-1 (F210V/L213C, E = 101) and AC-1 (F210V/I367T, E = 69). Molecular simulation experiments indicated that the introduced activity of these variants may be due to the reduced steric hindrance in the binding pocket of the relatively small-sized amino acid residues, a synergetic effect of the entrance residue mutation, and the formation of a new disulfide bond.

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of variants of monoamine oxidase fromAspergillus niger

Cited by 34 publications

References 21 publications

Immobilised whole-cell recombinant monoamine oxidase biocatalysis

Immobilised whole-cell recombinant monoamine oxidase biocatalysis

Structures of Alcohol Dehydrogenases from Ralstonia and Sphingobium spp. Reveal the Molecular Basis for Their Recognition of ‘Bulky–Bulky’ Ketones

Semi-rational Directed Evolution of Monoamine Oxidase for Kinetic Resolution of rac-Mexiletine

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