Discovery of a sesamin-metabolizing microorganism and a new enzyme

Abstract

Sesamin is one of the major lignans found in sesame oil. Although some microbial metabolites of sesamin have been identified, sesamin-metabolic pathways remain uncharacterized at both the enzyme and gene levels. Here, we isolated microorganisms growing on sesamin as a sole-carbon source. One microorganism showing significant sesamin-degrading activity was identified as Sinomonas sp. no. 22. A sesamin-metabolizing enzyme named SesA was purified from this strain and characterized. SesA catalyzed methylene group transfer from sesamin or sesamin monocatechol to tetrahydrofolate (THF) with ring cleavage, yielding sesamin mono- or di-catechol and 5,10-methylenetetrahydrofolate. The kinetic parameters of SesA were determined to be as follows: Km for sesamin = 0.032 ± 0.005 mM, Vmax = 9.3 ± 0.4 (μmol⋅min(-1)⋅mg(-1)), and kcat = 7.9 ± 0.3 s(-1) Next, we investigated the substrate specificity. SesA also showed enzymatic activity toward (+)-episesamin, (-)-asarinin, sesaminol, (+)-sesamolin, and piperine. Growth studies with strain no. 22, and Western blot analysis revealed that SesA formation is inducible by sesamin. The deduced amino acid sequence of sesA exhibited weak overall sequence similarity to that of the protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation in most bacteria, archaea, and all eukaryotes. Only SesA catalyzes C1 transfer to THF with ring cleavage reaction among GcvT family proteins. Moreover, SesA homolog genes are found in both Gram-positive and Gram-negative bacteria. Our findings provide new insights into microbial sesamin metabolism and the function of GcvT family proteins.

Keywords: lignan; metabolism; sesamin; tetrahydrofolate.

Fig. 1.

Schematic representation of the bond cleavage location in the structure of substrates for SesA (A) and previously reported THF-dependent methyl transferases (B). The cleavage site of the bond between atoms within a substrate is indicated by a dashed line.

Fig. 2.

HPLC analysis of reaction products of sesamin. Chromatogram of the reaction mixture after incubation of sesamin with a cell-free extract of strain no. 22. The products are indicated as A and B.

Fig. 3.

SesA activities for plant-derived methylenedioxyphenyl compounds and their derivatives. The specific activities for each substance and structures of reaction products are indicated.

Fig. 4.

Sesamin metabolic pathway and proposed reaction mechanism. (A) Proposed sesamin metabolic pathway in strain no. 22. (B) DNA sequence of region encoding SesA and its flanking region. The thfl gene exhibits 60% amino acid sequence identity with that of Mycobacterium bovis (UniProtKB accession code P0A5T7), and the thf2 gene exhibits 69% amino acid sequence identity with that of Clavibacter michiganensis (UniProtKB accession code B0RD2). (C) Proposed reaction mechanism for SesA. In C, “B” represents a base.