J Biol Chem, Vol. 274, Issue 50, 35614-35620, December 10, 1999
Structure and Characterization of Ectothiorhodospira
vacuolata Cytochrome b558, a Prokaryotic
Homologue of Cytochrome b5*
Vesna
Kostanjeve
ki
,
David
Leys§,
Gonzalez
Van
Driessche,
Terrance E.
Meyer¶,
Michael A.
Cusanovich¶,
Ulrich
Fischer
,
Yves
Guisez, and
Jozef
Van
Beeumen**
From the Department of Biochemistry, Physiology, and
Microbiology, Laboratory of Protein Biochemistry and Protein
Engineering, University of Gent, B-9000 Gent, Belgium, the
¶ Department of Biochemistry, University of Arizona, Tucson,
Arizona 85721, and the Department of Marine Microbiology,
University of Bremen, D-28359 Bremen, Germany
A soluble cytochrome b558
from the purple phototropic bacterium Ectothiorhodospira
vacuolata was completely sequenced by a combination of automated
Edman degradation and mass spectrometry. The protein, with a measured
mass of 10,094.7 Da, contains 90 residues and binds a single protoheme.
Unexpectedly, the sequence shows homology to eukaryotic cytochromes
b5. As no prokaryotic homologue had been
reported so far, we developed a protocol for the expression,
purification, and crystallization of recombinant cytochrome
b558. The structure was solved by molecular
replacement to a resolution of 1.65 Å. It shows that cytochrome
b558 is indeed the first bacterial cytochrome
b5 to be characterized and differs from its
eukaryotic counterparts by the presence of a disulfide bridge and a
four-residue insertion in front of the sixth ligand (histidine).
Eukaryotes contain a variety of b5 homologues,
including soluble and membrane-bound multifunctional proteins as well
as multidomain enzymes such as sulfite oxidase, fatty-acid desaturase, nitrate reductase, and lactate dehydrogenase. A search of the Mycobacterium tuberculosis genome showed that a previously
unidentified gene encodes a fatty-acid desaturase with an N-terminal
b5 domain. Thus, it may provide another example
of a bacterial b5 homologue.
*
The work was supported in part by Grant GM 21277 from the
National Institutes of Health (to M. A. C.) and by Grant Fi 295/1 from the Deutsche Forschungsgemeinschaft (to U. F.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF183259.
The atomic coordinates and structure factors (code 1CXY, 009616)
have been deposited in the Protein Data Bank, Research Collaboratory
for Structural Bioinformatics, Rutgers University, New Brunswick, NJ
(http://www.rcsb.org/).
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