Stereoselectivity and Structural Characterisation of an Imine Reductase (IRED) from Amycolatopsis orientalis

Gideon James Grogan; Godwin Aleku; Henry Man; Scott P. France; Friedemann Leipold; Shahed Hussain; Laura Toca-Gonzalez; Rebecca Marchington; Sam J. Hart; Johan P. Turkenburg; Nicholas J. Turner

doi:10.1021/acscatal.6b00782

Stereoselectivity and Structural Characterisation of an Imine Reductase (IRED) from Amycolatopsis orientalis

Gideon James Grogan, Godwin Aleku, Henry Man, Scott P. France, Friedemann Leipold, Shahed Hussain, Laura Toca-Gonzalez, Rebecca Marchington, Sam J. Hart, Johan P. Turkenburg, Nicholas J. Turner

Chemistry

Research output: Contribution to journal › Article › peer-review

Abstract

The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with e.e.s of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on the age of the enzyme after purification. The structure of AoIRED has been determined in an ‘open’ apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N- and C-terminal domains of participating monomers. Co-crystallisation with NADPH gave a ‘closed’ form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by co-crystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and reveals a binding site for the (R)-amine product which places the chiral carbon within 4 Å of the putative location of the C4 atom of NADPH that delivers hydride to the C=N bond of the substrate. The ternary complex has permitted structure-informed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A, of altered activity and stereoselectivity.

Original language	English
Pages (from-to)	3880–3889
Number of pages	10
Journal	ACS Catalysis
Volume	6
Issue number	6
Early online date	18 May 2016
DOIs	https://doi.org/10.1021/acscatal.6b00782
Publication status	E-pub ahead of print - 18 May 2016

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© 2016, American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

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Cite this

@article{4b5afbd2fc694b05a1b89352e3454b76,

title = "Stereoselectivity and Structural Characterisation of an Imine Reductase (IRED) from Amycolatopsis orientalis",

abstract = "The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with e.e.s of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on the age of the enzyme after purification. The structure of AoIRED has been determined in an {\textquoteleft}open{\textquoteright} apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N- and C-terminal domains of participating monomers. Co-crystallisation with NADPH gave a {\textquoteleft}closed{\textquoteright} form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by co-crystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and reveals a binding site for the (R)-amine product which places the chiral carbon within 4 {\AA} of the putative location of the C4 atom of NADPH that delivers hydride to the C=N bond of the substrate. The ternary complex has permitted structure-informed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A, of altered activity and stereoselectivity. ",

author = "Grogan, {Gideon James} and Godwin Aleku and Henry Man and France, {Scott P.} and Friedemann Leipold and Shahed Hussain and Laura Toca-Gonzalez and Rebecca Marchington and Hart, {Sam J.} and Turkenburg, {Johan P.} and Turner, {Nicholas J.}",

note = "{\textcopyright} 2016, American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher{\textquoteright}s self-archiving policy. Further copying may not be permitted; contact the publisher for details",

year = "2016",

month = may,

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doi = "10.1021/acscatal.6b00782",

language = "English",

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T1 - Stereoselectivity and Structural Characterisation of an Imine Reductase (IRED) from Amycolatopsis orientalis

AU - Grogan, Gideon James

AU - Aleku, Godwin

AU - Man, Henry

AU - France, Scott P.

AU - Leipold, Friedemann

AU - Hussain, Shahed

AU - Toca-Gonzalez, Laura

AU - Marchington, Rebecca

AU - Hart, Sam J.

AU - Turkenburg, Johan P.

AU - Turner, Nicholas J.

N1 - © 2016, American Chemical Society. This is an author-produced version of the published paper. Uploaded in accordance with the publisher’s self-archiving policy. Further copying may not be permitted; contact the publisher for details

PY - 2016/5/18

Y1 - 2016/5/18

N2 - The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with e.e.s of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on the age of the enzyme after purification. The structure of AoIRED has been determined in an ‘open’ apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N- and C-terminal domains of participating monomers. Co-crystallisation with NADPH gave a ‘closed’ form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by co-crystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and reveals a binding site for the (R)-amine product which places the chiral carbon within 4 Å of the putative location of the C4 atom of NADPH that delivers hydride to the C=N bond of the substrate. The ternary complex has permitted structure-informed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A, of altered activity and stereoselectivity.

AB - The imine reductase AoIRED from Amycolatopsis orientalis (Uniprot R4SNK4) catalyzes the NADPH-dependent reduction of a wide range of prochiral imines and iminium ions, predominantly with (S)-selectivity and with e.e.s of up to >99%. AoIRED displays up to 100-fold greater catalytic efficiency for 2-methyl-1-pyrroline (2MPN) compared to other IREDs, such as the enzyme from Streptomyces sp. GF3546, which also exhibits (S)-selectivity, and thus AoIRED is an interesting candidate for preparative synthesis. AoIRED exhibits unusual catalytic properties, with inversion of stereoselectivity observed between structurally similar substrates, and also, in the case of 1-methyl-3,4-dihydroisoquinoline, for the same substrate, dependent on the age of the enzyme after purification. The structure of AoIRED has been determined in an ‘open’ apo-form, revealing a canonical dimeric IRED fold in which the active site is formed between the N- and C-terminal domains of participating monomers. Co-crystallisation with NADPH gave a ‘closed’ form in complex with the cofactor, in which a relative closure of domains, and associated loop movements, has resulted in a much smaller active site. A ternary complex was also obtained by co-crystallization with NADPH and 1-methyl-1,2,3,4-tetrahydroisoquinoline [(MTQ], and reveals a binding site for the (R)-amine product which places the chiral carbon within 4 Å of the putative location of the C4 atom of NADPH that delivers hydride to the C=N bond of the substrate. The ternary complex has permitted structure-informed mutation of the active site, resulting in mutants including Y179A, Y179F and N241A, of altered activity and stereoselectivity.

U2 - 10.1021/acscatal.6b00782

DO - 10.1021/acscatal.6b00782

M3 - Article

SN - 2155-5435

VL - 6

SP - 3880

EP - 3889

JO - ACS Catalysis

JF - ACS Catalysis

IS - 6

ER -