Identification and Characterization of a Tetramethylpyrazine Catabolic Pathway in Rhodococcus jostii TMP1

Abstract

bAt 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. P yrazines, monocyclic aromatic rings with two nitrogen atoms in para position, are a class of compounds that occur almost ubiquitously in nature. Various pyrazines can be synthesized both chemically and biologically, including tetramethylpyrazine (TTMP), which is produced by different bacteria (1, 2) or plants (3, 4). However, there is very little information available on the biodegradation of these N-heterocyclic compounds. While bacterial strains able to use various alkyl-substituted pyrazines as a sole carbon and energy source have been isolated and described (5-9), almost nothing is known about the degradation pathways of alkylpyrazines, including tetramethylpyrazine, in which all carbon atoms carry a substituent.Under aerobic conditions, alkylated pyrazines are metabolized via oxidative degradation, leading to the hydroxylation of the ring at a free position. Rhodococcus erythropolis DSM 6138 and Arthrobacter sp. strain DSM 6137 can use 2,5-dimethylpyrazine as a source of carbon and energy. The catabolism of 2,5-dimethylpyrazine by these microorganisms gives rise to the intermediate metabolite 2-hydroxy-3,6-dimethylpyrazine, which accumulates in the medium, indicating that ring hydroxylation occurs during the initial steps of degradation (5). However, no enzymes involved in this bioconversion were reported in the patent that describes the aforementioned reactions (5).The 2,5-dimethylpyraz...

Peer Review #2 of "Ketoreductase TpdE from Rhodococcus jostii TMP1: characterization and application in the synthesis of chiral alcohols (v0.2)"

“…We previously showed that TpdE is able to reduce carbonyl group of N-(butan-3-one-2yl)acetamide (Kutanovas et al, 2013). In this study we demonstrated that TpdE is also capable of reducing diacetyl when NADPH was used as a co-substrate.…”

Indole Biodegradation in Acinetobacter sp. Strain O153: Genetic and Biochemical Characterization

“…The opening of the epoxide to a diol was presumed to be a plausible pathway for indole-2,3-dihydrodiol formation (44). Moreover, the oxidation of several N-heterocyclic compounds catalyzed by monooxygenases when two oxygen atoms (one from oxygen, another from water) were inserted was described recently (45)(46)(47). A similar mechanism for bacterial degradation of indole acetic acid was described (48) involving the intermediate dioxindole-3-acetic acid (DOAA), a structural analogue of 3-hydroxyindolin-2-one in indole degradation.…”

Ketoreductase TpdE fromRhodococcus jostiiTMP1: characterization and application in the synthesis of chiral alcohols

“…To obtain a large amount of TpdE, the overexpression of the recombinant protein was performed as described previously ( Kutanovas et al, 2013 ) with slight modifications. E. coli BL21 (DE3) cells harbouring pET- tpdE were grown in BHI medium (supplemented with 50 mg/ml ampicillin) to an OD 600 of 0.8.…”

“…Previously, we have shown that the protein TpdE from Rhodococcus jostii TMP1 is involved in the catabolism of tetramethylpyrazine, where it reduces N -(butan-3-one-2-yl)acetamide ( Fig. 1 ) ( Kutanovas et al, 2013 ). Here we provide a biochemical characterization of TpdE.…”