Characterization of regioselective flavonoid O-methyltransferase from the Streptomyces sp. KCTC 0041BP

Highlights

• A putative methyltransferase (GerMIII) from Streptomyces sp. KCTC 0041BP was tested in-vitro for the methylation of the substrates of diverse chemical structures.

• A single product peak observed with quercetin and luteolin methylation reactions were confirmed to be methylated at 4′-OH position by mass-spectrometry and NMR spectrometric analysis.

• The enzymatic properties of GerMIII and a mechanistic overview of the regiospecific modification were observed with quercetin.

Abstract

A flavonoid comprises polyphenol compounds with pronounced antiviral, antioxidant, anticarcinogenic, and anti-inflammatory effects. The flavonoid modification by methylation provides a greater stability and improved pharmacokinetic properties. The methyltransferase from plants or microorganisms is responsible for such substrate modifications in a regiospecific or a promiscuous manner. GerMIII, originally characterized as a putative methyltransferase in a dihydrochalcomycin biosynthetic gene cluster of the Streptomyces sp. KCTC 0041BP, was tested for the methylation of the substrates of diverse chemical structures. Among the various tested substrates, flavonoids emerged as the favored substrates for methylation. Further, among the flavonoids, quercetin is the most favorable substrate, followed by luteolin, myricetin, quercetin 3-O-β-D-glucoside, and fisetin, while only a single product was formed in each case. The products were confirmed by HPLC and mass-spectrometry analyses. A detailed NMR spectrometric analysis of the methylated quercetin and luteolin derivatives confirmed the regiospecific methylation at the 4′-OH position. Modeling and molecular docking provided further insight regarding the most favorable mechanism and substrate architecture for the enzymatic catalysis. Accordingly, a double bond between the C₂ and the C₃ and a single-ring-appended conjugate-hydroxyl group are crucial for the favorable enzymatic conversions of the GerMIII catalysis. Thus, in this study, the enzymatic properties of GerMIII and a mechanistic overview of the regiospecific modification that was implemented for the acceptance of quercetin as the most favorable substrate are presented.

Introduction

The natural products (NPs) that are derived from microbial or plant sources are a starting point for drug discovery, and they generally exhibit a wide range of pharmacophores and a high degree of stereochemistry [1]. These NPs comprise basic chemical skeletons, or modifications of their forms that have been produced by hydroxylation, glycosylation, and methylation [2]. Generally, methylation reactions are catalyzed by the O-methyltransferase (OMT) that catalyzes the transfer of the methyl group of S-(5′-Adenosyl)-L-methionine (SAM) to the hydroxyl groups of such NPs. Numerous OMTs that are either regioselective/stereoselective or substrate-promiscuous have been characterized from plants or microbial sources. Specifically, based on their size, amino-acid sequence, and cation dependency, plant OMTs are categorized into the following two major groups: Class I and Class II. The molecular-mass values of the Class-I OMTs (caffeoyl-coenzyme A OMTs [CCoAOMT]) from 26 to 29 KDa are lower, and this class methylate the lignin precursors viz caffeoyl-coenzyme A (CCoA) and 5-hydroxyferuloyl CoA in the presence of the magnesium (Mg) ion Mg²⁺. The molecular weights of the Class-II OMT (caffeic-acid OMT [COMT]) from 38 to 45 KDa, which can catalyze the methylation reaction of flavonoids as well as the 3-hydroxyl- and 5-hydroxyl-containing phenylpropanoid-derived lignin precursors without the presence of a metal cation, are higher [3]. The roles of these plant OMTs regarding the physiological and biochemical properties of plants are diverse. Similarly, the OMTs from microorganisms have been frequently characterized in the biosynthetic pathways of secondary metabolites, or they have been utilized for the biotechnological modifications of a number of compounds like flavonoids, alkaloids, and antibiotics. Consequently, an elevated interest in the understanding of the biological importance of microbial OMTs and the study of their enzymatic properties has emerged.

Flavonoids are polyphenol compounds with important medicinal values including antioxidant, anticarcinogenic, antimicrobial, anti-inflammatory, and antiviral effects [4]. Generally, it is thought that O-methylation plays an important role in the deactivation of the reactive hydroxyl groups of flavonoids and the alteration of their solubility and intracellular compartmentation. Although the structural diversity among flavonoids is naturally high, the production of novel or biologically potent flavonoid derivatives is a research-focus area. Numerous researches on both the synthetic approach [5,6] and the metabolic engineering using OMTs derived from plant or microbial sources [7,8] have been completed. The chemical synthesis for the O-methylation of a specific OH group is challenging, however, because of the requisite harsh catalytic conditions, a long reaction time, the protection of unwanted groups, and the monetary expense of the reagents. Alternatively, microorganisms are capable of the O-methylation of compounds for the production of novel bioactive compounds under much milder reaction conditions in a one-step reaction. These approaches for the generation of O-methylated compounds either by in vitro reactions or the whole-cell biotransformation have been successful for the production of valuable medical compounds, especially regarding the methylated derivatives of hydroxylated molecules.

Due to the production capability of the versatile secondary metabolites, different Actinomyces spp. are being explored for the identification and characterization of the OMT genes that are involved in the methylation of diverse substrates. Due to the usage potential in the cosmetic, pharmaceutical, and food industries, flavonoids are commonly selected as substrates. The Streptomyces coelicolor OMT can modify flavonoids with a broad specificity [8]. Similarly, the identification of the methylation of various flavonoids at the 7-OH and the 4′-OH occurred in the in vitro and in silico studies of DnrK, an OMT of the Streptomyces peucetius doxorubicin-biosynthesis pathway [9]. The different OMTs that have been derived from the Streptomyces sp. are capable of the methylated modification of flavonoids, as listed in Table S1. Regioselectivity/sterioselectivity is the most desirable feature of any OMT, as this allows the targeted substrate to be modified for the attainment of the desired product. These types of enzymes are suitable for substrate modifications at the industrial scale, which requires rigorous screening and selection processes. SaOMT-2, which is from Streptomyces avermilitis, is a regioselective OMT that can convert different flavonoids into their corresponding 7-O-methylated form. SaOMT-2 was used for the biotechnological production of the antifungal compound sakuranetin [7].

Generally, flavonoids are subjected to autooxidation or are oxidatively degraded, while the methylation at the 4′-hydroxyl group of the B-ring of flavonoids confers a structural constraint for auto-oxidation; therefore, the importance of the regiospecific 4′-OMT is significant [10]. SOMT-2 (glycine max) has been reported as a 4′OMT [2] for which naringenin is the best substrate. Recently, the 4′OMT (Pa 4′OMT) of liverworts (plant source) was characterized, and it was used to effectively catalyze apigenin to acacetin [11]. A few of the OMTs from Streptomyces have been examined for the methylation of flavonoids, and to date, none of the Streptomyces methyltransferases (MTs) have been characterized as the regiospecific 4′OMT.

GerMIII (GenBank accession no. AY118081) is present on the biosynthetic gene cluster of dihydrochalcomycin, a macrolide antibiotic that is produced by the Streptomyces sp. KCTC 0041BP. It has not been functionally characterized, but it has been proposed as a putative sugar OMT [12]. In this study, GerMIII was heterologously expressed in E. coli and was screened against the substrates of diverse chemical structures. The enzyme can accept flavonoids as a suitable substrate, so the reactions were performed with various flavonoids, thereby leading to the selection of quercetin as the most favorable substrate. Based on the studies on the enzymatic properties and the product characterization, GerMIII was established as a novel regiospecific flavonoid 4′OMT. GerMIII showed the best activity for the conversion of quercetin to tamarixetin, with the conversion of 85% of 2 mM quercetin that was fed in vitro; this is the best conversion rate of any OMT for the 4′-OH methylation. Further, in silico modeling and docking studies were performed to obtain a deep insight regarding the interesting features of the GerMIII-modifying flavonoid in a regiospecific manner.