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

Pavlos Stampolidis, Naheed Kaderbhai, David Bryant, Ana Winters, Joe Gallagher, Mustak Ali Kaderbhai

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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.
Original languageEnglish
JournalArchives of Microbiology
DOIs
Publication statusPublished - 2013

Keywords

  • pyrroloquinoline quinone
  • lipanine hydroxylase
  • Escherichia coli
  • periplasmic space
  • quinohaemoprotein
  • protein export

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