title: Genes encoding norcoclaurine synthase occur as tandem fusions in the Papaveraceae.
abstract: Norcoclaurine synthase (NCS) catalyzes the enantioselective Pictet-Spengler condensation of dopamine and 4-hydroxyphenylacetaldehyde as the first step in benzylisoquinoline alkaloid (BIA) biosynthesis. NCS orthologs in available transcriptome databases were screened for variants that might improve the low yield of BIAs in engineered microorganisms. Databases for 21 BIA-producing species from four plant families yielded 33 assembled contigs with homology to characterized NCS genes. Predicted translation products generated from nine contigs consisted of two to five sequential repeats, each containing most of the sequence found in single-domain enzymes. Assembled contigs containing tandem domain repeats were detected only in members of the Papaveraceae family, including opium poppy (Papaver somniferum). Fourteen cDNAs were generated from 10 species, five of which encoded NCS orthologs with repeated domains. Functional analysis of corresponding recombinant proteins yielded six active NCS enzymes, including four containing either two, three or four repeated catalytic domains. Truncation of the first 25 N-terminal amino acids from the remaining polypeptides revealed two additional enzymes. Multiple catalytic domains correlated with a proportional increase in catalytic efficiency. Expression of NCS genes in Saccharomyces cereviseae also produced active enzymes. The metabolic conversion capacity of engineered yeast positively correlated with the number of repeated domains.
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Jing
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Eun-Jeong
authors.initials: EJ
authors.lastname: Lee
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Limei
authors.initials: L
authors.lastname: Chang
authors.affiliation: Department of Biological Sciences, University of Calgary, Calgary, Alberta T2N 1N4, Canada.
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Facchini
mesh_terms: D000470:Alkaloids / Q000096:biosynthesis; D000595:Amino Acid Sequence; D044182:Benzylisoquinolines / Q000378:metabolism; D055162:Biocatalysis; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D020134:Catalytic Domain; D003001:Cloning, Molecular; D020451:Contig Mapping; D018076:DNA, Complementary / Q000378:metabolism; D016208:Databases, Factual; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D028101:Papaveraceae; D010802:Phylogeny; D010940:Plant Proteins / Q000737:chemistry / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D000470:Alkaloids; D044182:Benzylisoquinolines; D018076:DNA, Complementary; D010940:Plant Proteins; D011994:Recombinant Proteins; D019731:Carbon-Nitrogen Ligases; C450500:norcoclaurine synthase