Dataset Information

Overcoming co-product inhibition in the nicotinamide independent asymmetric bioreduction of activated C=C-bonds using flavin-dependent ene-reductases.

ABSTRACT: Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation ("dismutation") of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co-product phenol could be circumvented via in situ co-product removal by scavenging the phenol using the polymeric adsorbent MP-carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a "coupled-substrate" approach via nicotinamide-free hydrogen transfer with >90% conversion and complete stereoselectivity.

SUBMITTER: Winkler CK

PROVIDER: S-EPMC4034509 | biostudies-literature | 2013 Dec

REPOSITORIES: biostudies-literature

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Overcoming co-product inhibition in the nicotinamide independent asymmetric bioreduction of activated C=C-bonds using flavin-dependent ene-reductases.

Winkler Christoph K CK Clay Dorina D van Heerden Esta E Faber Kurt K

Biotechnology and bioengineering 20130710 12

Eleven flavoproteins from the old yellow enzyme family were found to catalyze the disproportionation ("dismutation") of conjugated enones. Incomplete conversions, which were attributed to enzyme inhibition by the co-product phenol could be circumvented via in situ co-product removal by scavenging the phenol using the polymeric adsorbent MP-carbonate. The optimized system allowed to reduce an alkene activated by ester groups in a "coupled-substrate" approach via nicotinamide-free hydrogen transfe ...[more]

PMID: 23794404

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Project description:5,10-Methylenetetrahydrofolate reductase (MetF/MTHFR) is an essential enzyme in one-carbon metabolism for de novo biosynthesis of methionine. Our in vivo and in vitro analyses of MSMEG_6664/MSMEI_6484, annotated as putative MTHFR in Mycobacterium smegmatis, failed to reveal their function as MTHFRs. However, we identified two hypothetical proteins, MSMEG_6596 and MSMEG_6649, as noncanonical MTHFRs in the bacterium. MTHFRs are known to be oligomeric flavoproteins. Both MSMEG_6596 and MSMEG_6649 are monomeric proteins and lack flavin coenzymes. In vitro, the catalytic efficiency (kcat/Km ) of MSMEG_6596 (MTHFR1) for 5,10-CH2-THF and NADH was ∼13.5- and 15.3-fold higher than that of MSMEG_6649 (MTHFR2). Thus, MSMEG_6596 is the major MTHFR. This interpretation was further supported by better rescue of the E. coli Δmthfr strain by MTHFR1 than by MTHFR2. As identified by liquid chromatography-tandem mass spectrometry, the product of MTHFR1- or MTHFR2-catalyzed reactions was 5-CH3-THF. The M. smegmatis Δmsmeg_6596 strain was partially auxotrophic for methionine and grew only poorly without methionine or without being complemented with a functional copy of MTHFR1 or MTHFR2. Furthermore, the Δmsmeg_6596 strain was more sensitive to folate pathway inhibitors (sulfachloropyridazine, p-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are two noncanonical MTHFR proteins that are monomeric and lack flavin coenzyme. Both MTHFR1 and MTHFR2 are involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.IMPORTANCE MTHFR/MetF is an essential enzyme in a one-carbon metabolic pathway for de novo biosynthesis of methionine. MTHFRs are known to be oligomeric flavoproteins. Our in vivo and in vitro analyses of Mycobacterium smegmatis MSMEG_6664/MSMEI_6484, annotated as putative MTHFR, failed to reveal their function as MTHFRs. However, we identified two of the hypothetical proteins, MSMEG_6596 and MSMEG_6649, as MTHFR1 and MTHFR2, respectively. Interestingly, both MTHFRs are monomeric and lack flavin coenzymes. M. smegmatis deleted for the major mthfr (mthfr1) was partially auxotroph for methionine and more sensitive to folate pathway inhibitors (sulfachloropyridazine, para-aminosalicylic acid, sulfamethoxazole, and trimethoprim). The studies reveal that MTHFR1 and MTHFR2 are novel MTHFRs involved in de novo methionine biosynthesis and required for antifolate resistance in mycobacteria.

Dataset Information

Overcoming co-product inhibition in the nicotinamide independent asymmetric bioreduction of activated C=C-bonds using flavin-dependent ene-reductases.

Publications

Overcoming co-product inhibition in the nicotinamide independent asymmetric bioreduction of activated C=C-bonds using flavin-dependent ene-reductases.

Similar Datasets

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