Biodegradation of the methylenedioxyphenyl group in piperine and its derivatives: discovery of a novel methylenetransferase in an actinomycete

Abstract

ABSTRACT Piperine is a methylenedioxyphenyl (MDP) group-containing alkaloid that is found in black pepper. Although piperine has multiple physiological effects on the human body that make it a suitable supplement, little is known about its natural metabolism. In this study, we isolated microorganisms that can grow using piperine as the sole nitrogen source. One of these microorganisms (strain no. 14) was identified as Rhodococcus ruber . In this strain, a homolog of a sesamin-metabolizing enzyme (SesA) named PipA was discovered. We succeeded in the cloning and expression of PipA in Escherichia coli . PipA was found to be able to decompose the MDP group to produce catechols using tetrahydrofolate as a coenzyme. This enzyme showed the highest specificity toward one of the piperine derivatives, piperonylic acid, among all the substrate candidates examined in the study. The following kinetic parameters of PipA were determined: K m = 0.024 ± 0.004 mM; V max = 1.314 ± 0.059 (μmol·min −1 ·mg −1 ); and k cat = 1.752 ± 0.079 s −1 . PipA also showed weak activity toward piperine, piperic acid, 3,4-methylenedioxycinnamic acid, and piperonal. A partial amino-acid sequence of PipA exhibited similarity to that of a large protein family of glycine cleavage T-proteins (GcvTs), which catalyze glycine degradation and are present in both prokaryotes and eukaryotes. This study provides new insights into the unique functions of GcvT members and the piperine metabolism in nature. IMPORTANCE Pepper is a spice that has been used worldwide since the Age of Discovery. The substance that is responsible for the spiciness in pepper is piperine, a type of alkaloid. It has never been reported how piperine is degraded by microorganisms. In this study, we discovered a bacterium in the soil that is capable of catabolizing piperine as its sole nitrogen source. Furthermore, we discovered the enzyme involved in piperine metabolism. This enzyme decomposed the methylenedioxyphenyl group, which is the common structure in various plant-derived bioactive compounds such as sesamin, piperonal, safrole, and berberin. By utilizing this enzyme, piperine can be converted into a useful antioxidant compound. The findings about previously unknown metabolic pathways in nature can lead to the discovery of new enzymes and provide methods for the enzymatic synthesis of useful compounds.