title: Structure and characterization of Ectothiorhodospira vacuolata cytochrome b(558), a prokaryotic homologue of cytochrome b(5).
abstract: A soluble cytochrome b(558) 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 b(5). As no prokaryotic homologue had been reported so far, we developed a protocol for the expression, purification, and crystallization of recombinant cytochrome b(558). The structure was solved by molecular replacement to a resolution of 1.65 A. It shows that cytochrome b(558) is indeed the first bacterial cytochrome b(5) 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 b(5) 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 b(5) domain. Thus, it may provide another example of a bacterial b(5) homologue.
authors.affiliation: Department of Biochemistry, Laboratory of Protein Biochemistry and Protein Engineering, University of Gent, B-9000 Gent, Belgium.
authors.forename: V
authors.initials: V
authors.lastname: Kostanjevecki
authors.forename: D
authors.initials: D
authors.lastname: Leys
authors.forename: G
authors.initials: G
authors.lastname: Van Driessche
authors.forename: T E
authors.initials: TE
authors.lastname: Meyer
authors.forename: M A
authors.initials: MA
authors.lastname: Cusanovich
authors.forename: U
authors.initials: U
authors.lastname: Fischer
authors.forename: Y
authors.initials: Y
authors.lastname: Guisez
authors.forename: J
authors.initials: J
authors.lastname: Van Beeumen
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002645:Chickens; D002850:Chromatography, Gel; D017124:Conserved Sequence; D018360:Crystallography, X-Ray; D003573:Cytochrome b Group / Q000737:chemistry* / Q000302:isolation & purification; D015786:Cytochromes b5 / Q000737:chemistry*; D020587:Ectothiorhodospira / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019255:NADPH Oxidases; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018629:Spectrometry, Mass, Secondary Ion
chemical_list: D003573:Cytochrome b Group; D011994:Recombinant Proteins; D006418:Heme; D015786:Cytochromes b5; C036888:cytochrome b558; D019255:NADPH Oxidases