title: Structural Basis for β-Carboline Alkaloid Production by the Microbial Homodimeric Enzyme McbB.
abstract: The β-carboline (βC) alkaloids occur throughout nature and exhibit diverse biological activities. In contrast to βC alkaloid synthesis in plants, the biosynthesis in microorganisms remains poorly understood. The recently reported McbB from Marinactinospora thermotolerans is a novel enzyme proposed to catalyze the Pictet-Spengler (PS) reaction of L-tryptophan and oxaloacetaldehyde to produce the βC scaffold of marinacarbolines. In this study, we solved the crystal structure of McbB complexed with L-tryptophan at 2.48 Å resolution, which revealed the novel protein folding of McbB and the totally different structure from those of other PS condensation catalyzing enzymes, such as strictosidine synthase and norcoclaurine synthase from plants. Structural analysis and site-directed mutagenesis confirmed that the previously proposed catalytic Glu97 at the active-site center functions as an acid and base catalyst. Remarkably, the structure-based mutants R72A and H87A, with expanded active-site cavities, newly accepted bulky phenylglyoxal as the aldehyde substrate, to produce 1-benzoyl-3-carboxy-β-carboline.
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Mori
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shotaro
authors.initials: S
authors.lastname: Hoshino
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Shusaku
authors.initials: S
authors.lastname: Sahashi
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Wakimoto
authors.affiliation: Department of Medicinal Resources, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan.
authors.forename: Takashi
authors.initials: T
authors.lastname: Matsui
authors.affiliation: Department of Medicinal Resources, Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan. Electronic address: hmorita@inm.u-toyama.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Morita
authors.affiliation: Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: abei@mol.f.u-tokyo.ac.jp.
authors.forename: Ikuro
authors.initials: I
authors.lastname: Abe
mesh_terms: D000079:Acetaldehyde / Q000737:chemistry / Q000378:metabolism; D000192:Actinomycetales / Q000201:enzymology; D000470:Alkaloids / Q000096:biosynthesis* / Q000737:chemistry*; D002243:Carbolines / Q000737:chemistry* / Q000378:metabolism*; D019759:Carbon-Nitrogen Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D000470:Alkaloids; D002243:Carbolines; D014364:Tryptophan; D019759:Carbon-Nitrogen Lyases; D000079:Acetaldehyde