title: Identification and characterization of a tetramethylpyrazine catabolic pathway in Rhodococcus jostii TMP1.
abstract: At present, there are no published data on catabolic pathways of N-heterocyclic compounds, in which all carbon atoms carry a substituent. We identified the genetic locus and characterized key reactions in the aerobic degradation of tetramethylpyrazine in Rhodococcus jostii strain TMP1. By comparing protein expression profiles, we identified a tetramethylpyrazine-inducible protein of 40 kDa and determined its identity by tandem mass spectrometry (MS-MS) de novo sequencing. Searches against an R. jostii TMP1 genome database allowed the identification of the tetramethylpyrazine-inducible protein-coding gene. The tetramethylpyrazine-inducible gene was located within a 13-kb genome cluster, denominated the tetramethylpyrazine degradation (tpd) locus, that encoded eight proteins involved in tetramethylpyrazine catabolism. The genes from this cluster were cloned and transferred into tetramethylpyrazine-nondegrading Rhodococcus erythropolis strain SQ1. This allowed us to verify the function of the tpd locus, to isolate intermediate metabolites, and to reconstruct the catabolic pathway of tetramethylpyrazine. We report that the degradation of tetramethylpyrazine is a multistep process that includes initial oxidative aromatic-ring cleavage by tetramethylpyrazine oxygenase, TpdAB; subsequent hydrolysis by (Z)-N,N'-(but-2-ene-2,3-diyl)diacetamide hydrolase, TpdC; and further intermediate metabolite reduction by aminoalcohol dehydrogenase, TpdE. Thus, the genes responsible for bacterial degradation of pyrazines have been identified, and intermediate metabolites of tetramethylpyrazine degradation have been isolated for the first time.
authors.affiliation: Department of Molecular Microbiology and Biotechnology, Institute of Biochemistry, Vilnius University, Vilnius, Lithuania. simonas.kutanovas@bchi.vu.lt
authors.forename: Simonas
authors.initials: S
authors.lastname: Kutanovas
authors.forename: Jonita
authors.initials: J
authors.lastname: Stankeviciute
authors.forename: Gintaras
authors.initials: G
authors.lastname: Urbelis
authors.forename: Daiva
authors.initials: D
authors.lastname: Tauraite
authors.forename: Rasa
authors.initials: R
authors.lastname: Rutkiene
authors.forename: Rolandas
authors.initials: R
authors.lastname: Meskys
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli; D005470:Fluorometry; D020869:Gene Expression Profiling; D006868:Hydrolysis; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D008969:Molecular Sequence Data; D011719:Pyrazines / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012240:Rhodococcus / Q000235:genetics* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D053719:Tandem Mass Spectrometry
chemical_list: D001426:Bacterial Proteins; D011719:Pyrazines; C017953:tetramethylpyrazine
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