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Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: Biophysical properties and interactions with cytochrome p450 BioI

Lawson, RJ; von Wachenfeldt, Claes LU ; Haq, I; Perkins, J and Munro, AW (2004) In Biochemistry 43(39). p.12390-12409
Abstract
The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B. subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/serniquinone couple = -105... (More)
The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B. subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/serniquinone couple = -105 mV/-105 mV) with respect to the hydroquinone (midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV). Apoflavodoxin forms were generated by trichloroacetic acid treatment. Circular dichroism studies indicated that flavin mononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN. Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins. Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderate destabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuP apoflavodoxin has similar (or slightly higher) stability compared to the holoprotein. Differential scanning calorimetry reveals further differences in structural stability. YkuP has a lower melting temperature than YkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic. Optical and fluorimetric titrations with oxidized flavodoxins revealed strong affinity (K-d values consistently < 5 muM) for their potential redox partner P450 Biol, YkuN showing tighter binding. Stopped-flow reduction studies indicated that the maximal electron-transfer rate (k(red)) to fatty acid-bound P450 Biol occurs from YkuN and YkuP at similar to2.5 s(-1), considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuN or YkuP, fatty acid-bound P450 Biol, and E. coli NADPH-flavodoxin reductase indicated that both flavodoxins supported lipid hydroxylation by P450 Biol with turnover rates of up to similar to100 min(-1) with lauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supported monohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary product was observed with both palmitic acid and palmitoleic acid as substrates. (Less)
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author
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publishing date
type
Contribution to journal
publication status
published
subject
in
Biochemistry
volume
43
issue
39
pages
12390 - 12409
publisher
The American Chemical Society
external identifiers
  • wos:000224231500002
  • scopus:4744337841
ISSN
0006-2960
DOI
10.1021/bi049131t
language
English
LU publication?
yes
id
946a4734-4153-4c55-b104-648bc9932fcb (old id 265475)
date added to LUP
2007-10-24 08:55:39
date last changed
2017-12-10 03:40:33
@article{946a4734-4153-4c55-b104-648bc9932fcb,
  abstract     = {The two flavodoxins (YkuN and YkuP) from Bacillus subtilis have been cloned, overexpressed in Escherichia coli and purified. DNA sequencing, mass spectrometry, and flavin-binding properties showed that both YkuN and YkuP were typical short-chain flavodoxins (158 and 151 amino acids, respectively) and that an error in the published B. subtilis genome sequence had resulted in an altered reading frame and misassignment of YkuP as a long-chain flavodoxin. YkuN and YkuP were expressed in their blue (neutral semiquinone) forms and reoxidized to the quinone form during purification. Potentiometry confirmed the strong stabilization of the semiquinone form by both YkuN and YkuP (midpoint reduction potential for oxidized/serniquinone couple = -105 mV/-105 mV) with respect to the hydroquinone (midpoint reduction potential for semiquinone/hydroquinone couple = -382 mV/-377 mV). Apoflavodoxin forms were generated by trichloroacetic acid treatment. Circular dichroism studies indicated that flavin mononucleotide (FMN) binding led to considerable structural rearrangement for YkuP but not for YkuN. Both apoflavodoxins bound FMN but not riboflavin avidly, as expected for short-chain flavodoxins. Structural stability studies with the chaotrope guanidinium chloride revealed that there is moderate destabilization of secondary and tertiary structure on FMN removal from YkuN, but that YkuP apoflavodoxin has similar (or slightly higher) stability compared to the holoprotein. Differential scanning calorimetry reveals further differences in structural stability. YkuP has a lower melting temperature than YkuN, and its endotherm is composed of a single transition, while that for YkuN is biphasic. Optical and fluorimetric titrations with oxidized flavodoxins revealed strong affinity (K-d values consistently &lt; 5 muM) for their potential redox partner P450 Biol, YkuN showing tighter binding. Stopped-flow reduction studies indicated that the maximal electron-transfer rate (k(red)) to fatty acid-bound P450 Biol occurs from YkuN and YkuP at similar to2.5 s(-1), considerably faster than from E. coli flavodoxin. Steady-state turnover with YkuN or YkuP, fatty acid-bound P450 Biol, and E. coli NADPH-flavodoxin reductase indicated that both flavodoxins supported lipid hydroxylation by P450 Biol with turnover rates of up to similar to100 min(-1) with lauric acid as substrate. Interprotein electron transfer is a likely rate-limiting step. YkuN and YkuP supported monohydroxylation of lauric acid and myristic acid, but secondary oxygenation of the primary product was observed with both palmitic acid and palmitoleic acid as substrates.},
  author       = {Lawson, RJ and von Wachenfeldt, Claes and Haq, I and Perkins, J and Munro, AW},
  issn         = {0006-2960},
  language     = {eng},
  number       = {39},
  pages        = {12390--12409},
  publisher    = {The American Chemical Society},
  series       = {Biochemistry},
  title        = {Expression and characterization of the two flavodoxin proteins of Bacillus subtilis, YkuN and YkuP: Biophysical properties and interactions with cytochrome p450 BioI},
  url          = {http://dx.doi.org/10.1021/bi049131t},
  volume       = {43},
  year         = {2004},
}