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Heme A synthase enzyme functions dissected by mutagenesis of Bacillus subtilis CtaA

Hederstedt, Lars LU ; Lewin, Anna LU and Throne-Holst, Mimmi (2005) In Journal of Bacteriology 187(24). p.8361-8369
Abstract
Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme 0 into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed an affinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro... (More)
Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme 0 into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed an affinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme 0, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Bacteriology
volume
187
issue
24
pages
8361 - 8369
publisher
American Society for Microbiology
external identifiers
  • wos:000233880000018
  • pmid:16321940
  • scopus:28844505581
ISSN
0021-9193
DOI
10.1128/JB.187.24.8361-8369.2005
language
English
LU publication?
yes
id
9670ba82-cbbd-4c26-a3fb-9c6f3f533071 (old id 210918)
date added to LUP
2007-08-14 14:07:49
date last changed
2017-08-10 10:00:09
@article{9670ba82-cbbd-4c26-a3fb-9c6f3f533071,
  abstract     = {Heme A, as a prosthetic group, is found exclusively in respiratory oxidases of mitochondria and aerobic bacteria. Bacillus subtilis CtaA and other heme A synthases catalyze the conversion of a methyl side group on heme 0 into a formyl group. The catalytic mechanism of heme A synthase is not understood, and little is known about the composition and structure of the enzyme. In this work, we have: (i) constructed a ctaA deletion mutant and a system for overproduction of mutant variants of the CtaA protein in B. subtilis, (ii) developed an affinity purification procedure for isolation of preparative amounts of CtaA, and (iii) investigated the functional roles of four invariant histidine residues in heme A synthase by in vivo and in vitro analyses of the properties of mutant variants of CtaA. Our results show an important function of three histidine residues for heme A synthase activity. Several of the purified mutant enzyme proteins contained tightly bound heme O. One variant also contained trapped hydroxylated heme 0, which is a postulated enzyme reaction intermediate. The findings indicate functional roles for the invariant histidine residues and provide strong evidence that the heme A synthase enzyme reaction includes two consecutive monooxygenations.},
  author       = {Hederstedt, Lars and Lewin, Anna and Throne-Holst, Mimmi},
  issn         = {0021-9193},
  language     = {eng},
  number       = {24},
  pages        = {8361--8369},
  publisher    = {American Society for Microbiology},
  series       = {Journal of Bacteriology},
  title        = {Heme A synthase enzyme functions dissected by mutagenesis of <em>Bacillus subtilis</em> CtaA},
  url          = {http://dx.doi.org/10.1128/JB.187.24.8361-8369.2005},
  volume       = {187},
  year         = {2005},
}