title: Broadened Substrate Specificity of 3-Hydroxyethyl Bacteriochlorophyllide a Dehydrogenase (BchC) Indicates a New Route for the Biosynthesis of Bacteriochlorophyll a.
abstract: Bacteriochlorophyll a biosynthesis requires formation of a 3-hydroxyethyl group on pyrrole ring A that gets subsequently converted into a 3-acetyl group by 3-vinyl bacteriochlorophyllide a hydratase (BchF) followed by 3-hydroxyethyl bacteriochlorophyllide a dehydrogenase (BchC). Heterologous overproduction of Chlorobaculum tepidum BchF revealed an integral transmembrane protein that was efficiently isolated by detergent solubilization. Recombinant C. tepidum BchC was purified as a soluble protein-NAD(+) complex. Substrate recognition of BchC was investigated using six artificial substrate molecules. Modification of the isocyclic E ring, omission of the central magnesium ion, zinc as an alternative metal ion, and a non-reduced B ring system were tolerated by BchC. According to this broadened in vitro activity, the chlorin 3-hydroxyethyl chlorophyllide a was newly identified as a natural substrate of BchC in a reconstituted pathway consisting of dark-operative protochlorophyllide oxidoreductase, BchF, and BchC. The established reaction sequence would allow for an additional new branching point for the synthesis of bacteriochlorophyll a. Biochemical and site-directed mutagenesis analyses revealed, in contrast to theoretical predictions, a zinc-independent BchC catalysis that requires NAD(+) as a cofactor. Based on these results, we are designating a new medium-chain dehydrogenase/reductase family (MDR057 BchC) as theoretically proposed from a recent bioinformatics analysis.
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany and.
authors.forename: Christiane
authors.initials: C
authors.lastname: Lange
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany and.
authors.forename: Svenja
authors.initials: S
authors.lastname: Kiesel
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany and.
authors.forename: Sabine
authors.initials: S
authors.lastname: Peters
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany and.
authors.forename: Simone
authors.initials: S
authors.lastname: Virus
authors.affiliation: Department Biology I, Botany, Ludwig-Maximilians-Universität München, Menzingerstrasse 67, D-80638 München, Germany.
authors.forename: Hugo
authors.initials: H
authors.lastname: Scheer
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany and.
authors.forename: Dieter
authors.initials: D
authors.lastname: Jahn
authors.affiliation: From the Institute of Microbiology, Technische Universität Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, Germany and j.moser@tu-bs.de.
authors.forename: Jürgen
authors.initials: J
authors.lastname: Moser
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D025541:Bacteriochlorophyll A / Q000096:biosynthesis* / Q000737:chemistry; D019414:Chlorobi / Q000737:chemistry / Q000201:enzymology*; D002735:Chlorophyllides / Q000737:chemistry / Q000378:metabolism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000737:chemistry* / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000235:genetics / Q000378:metabolism; D010788:Photosynthesis / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D025541:Bacteriochlorophyll A; D002735:Chlorophyllides; D011994:Recombinant Proteins; D009243:NAD; C034388:chlorophyllide a; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C023886:protochlorophyllide reductase
keywords: BchC; BchF; bacteriochlorophyll a; dehydrogenase; green sulfur bacterium; membrane protein; pathway reconstitution; photosynthesis; photosynthetic pigment; substrate specificity