Co-expression of lupanine hydroxylase and pyrroloquinoline quinone 2 leads to assembled and active recombinant lupanine hydroxylase in the 3 Escherichia coli periplasm

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Co-expression of lupanine hydroxylase and pyrroloquinoline quinone 2 leads to assembled and active recombinant lupanine hydroxylase in the 3 Escherichia coli periplasm. / Stampolidis, Pavlos; Kaderbhai, Naheed; Bryant, David; Winters, Ana; Gallagher, Joe; Kaderbhai, Mustak Ali.

In: Archives of Microbiology, 2013.

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@article{11cf0d95ee2645d59b732e539b791ba1,
title = "Co-expression of lupanine hydroxylase and pyrroloquinoline quinone 2 leads to assembled and active recombinant lupanine hydroxylase in the 3 Escherichia coli periplasm",
abstract = "Lupanine hydroxylase (LH) is a quinohaemoprotein responsible for the conversion of the 25 alkaloid, lupanine to 17-hydroxylupanine. Previous attempts to express the enzyme in 26 Escherichia coli required in vitro addition of the co-factor pyrroloquinoline quinone 27 (PQQ) and posed some impediments on subsequent structural studies for further 28 characterization of the enzyme. An E. coli clone with LH and cytochrome c maturation 29 operon was transformed with a third plasmid containing the PQQ operon from Klebsiella 30 pneumoniae , luh gene and resulted in the production of periplasmically-targeted, 31 correctly folded, PQQ and haem inserted active enzyme. 32 Interestingly, LH was less active than the in vitro incorporated PQQ-LH, presumably due 33 to the incorporation of PQQ precursors in the periplasm. This is a first report of an active 34 LH enzyme with in vivo incorporation of PQQ in E. coli and provides the necessary tool 35 for further enzyme structural characterization. ",
keywords = "pyrroloquinoline quinone, lipanine hydroxylase, Escherichia coli, periplasmic space, quinohaemoprotein, protein export",
author = "Pavlos Stampolidis and Naheed Kaderbhai and David Bryant and Ana Winters and Joe Gallagher and Kaderbhai, {Mustak Ali}",
year = "2013",
doi = "10.13140/RG.2.1.4937.7449",
language = "English",
journal = "Archives of Microbiology",
issn = "0302-8933",
publisher = "Springer Nature",

}

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TY - JOUR

T1 - Co-expression of lupanine hydroxylase and pyrroloquinoline quinone 2 leads to assembled and active recombinant lupanine hydroxylase in the 3 Escherichia coli periplasm

AU - Stampolidis, Pavlos

AU - Kaderbhai, Naheed

AU - Bryant, David

AU - Winters, Ana

AU - Gallagher, Joe

AU - Kaderbhai, Mustak Ali

PY - 2013

Y1 - 2013

N2 - Lupanine hydroxylase (LH) is a quinohaemoprotein responsible for the conversion of the 25 alkaloid, lupanine to 17-hydroxylupanine. Previous attempts to express the enzyme in 26 Escherichia coli required in vitro addition of the co-factor pyrroloquinoline quinone 27 (PQQ) and posed some impediments on subsequent structural studies for further 28 characterization of the enzyme. An E. coli clone with LH and cytochrome c maturation 29 operon was transformed with a third plasmid containing the PQQ operon from Klebsiella 30 pneumoniae , luh gene and resulted in the production of periplasmically-targeted, 31 correctly folded, PQQ and haem inserted active enzyme. 32 Interestingly, LH was less active than the in vitro incorporated PQQ-LH, presumably due 33 to the incorporation of PQQ precursors in the periplasm. This is a first report of an active 34 LH enzyme with in vivo incorporation of PQQ in E. coli and provides the necessary tool 35 for further enzyme structural characterization.

AB - Lupanine hydroxylase (LH) is a quinohaemoprotein responsible for the conversion of the 25 alkaloid, lupanine to 17-hydroxylupanine. Previous attempts to express the enzyme in 26 Escherichia coli required in vitro addition of the co-factor pyrroloquinoline quinone 27 (PQQ) and posed some impediments on subsequent structural studies for further 28 characterization of the enzyme. An E. coli clone with LH and cytochrome c maturation 29 operon was transformed with a third plasmid containing the PQQ operon from Klebsiella 30 pneumoniae , luh gene and resulted in the production of periplasmically-targeted, 31 correctly folded, PQQ and haem inserted active enzyme. 32 Interestingly, LH was less active than the in vitro incorporated PQQ-LH, presumably due 33 to the incorporation of PQQ precursors in the periplasm. This is a first report of an active 34 LH enzyme with in vivo incorporation of PQQ in E. coli and provides the necessary tool 35 for further enzyme structural characterization.

KW - pyrroloquinoline quinone

KW - lipanine hydroxylase

KW - Escherichia coli

KW - periplasmic space

KW - quinohaemoprotein

KW - protein export

UR - http://hdl.handle.net/2160/43710

U2 - 10.13140/RG.2.1.4937.7449

DO - 10.13140/RG.2.1.4937.7449

M3 - Article

JO - Archives of Microbiology

JF - Archives of Microbiology

SN - 0302-8933

ER -

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