Proanthocyanidins were purified from avocado (Persea americana) fruit, and their structures were analyzed by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and high-performance liquid chromatography-electrospray ionization-QTRAP mass spectrometry (HPLC-ESI-QTRAP MS) techniques. The results obtained from mass spectrometry (MS) analysis demonstrated that the proanthocyanidins were homo- and heteropolymers of procyanidins, prodelphinidins, propelargonidins, and procyanidin gallate. From the enzyme analysis, the results showed that they could inhibit the monophenolase and diphenolase activities of tyrosinase. The inhibition mechanism of the proanthocyanidins on the enzyme was further studied, and the results indicated that they were reversible and competitive inhibitors. Finally, the results acquired from molecular docking, fluorescence quenching, and copper ion interacting tests revealed that adjacent hydroxyl groups on the B ring of proanthocyanidins could chelate the dicopper catalytic center of the enzyme. In addtion, proanthocyanidins were proven to be an efficient quencher of substrates. This study would lay a scientific foundation for their use in agriculture, food, and nutrition industries.

No abstract

No abstract

Methionine adenosyltransferases (MATs) catalyze the formation of S-adenosyl-L-methionine (SAM) inside living cells. Recently, S-alkyl analogues of SAM have been documented as cofactor surrogates to label novel targets of methyltransferases. However, these chemically synthesized SAM analogues are not suitable for cell-based studies because of their poor membrane permeability. This issue was recently addressed under a cellular setting through a chemoenzymatic strategy to process membrane-permeable S-alkyl analogues of methionine (SAAM) into the SAM analogues with engineered MATs. Here we describe a general, sensitive activity assay for engineered MATs by converting the reaction products into S-alkyl-thioadenosines, followed by HPLC/MS/MS quantification. With this assay, 40 human MAT mutants were evaluated against seven SAAM as potential substrates. The structure-activity-relationship revealed that, besides better engaged SAAM binding by the MAT mutants (lower Km value in contrast to native MATs), the gained activity towards the bulky SAAM stems from their ability to maintain the desired linear SN2 transition state (reflected by higher kcat value). Here the I117A mutant of human MATI was identified as the most active variant for biochemical production of SAM analogues from diverse SAAM.

No abstract

No abstract

Mycothiol (MSH) is the major low molecular mass thiols in many Gram-positive bacteria such as Mycobacterium tuberculosis and Corynebacterium glutamicum. The physiological roles of MSH are believed to be equivalent to those of GSH in Gramnegative bacteria, but current knowledge of MSH is limited to detoxification of alkalating chemicals and protection from host cell defense/killing systems. Recently, an MSH-dependent maleylpyruvate isomerase (MDMPI) was discovered from C. glutamicum, and this isomerase represents one example of many putative MSH-dependent enzymes that take MSH as cofactor. In this report, fourteen mutants of MDMPI were generated. The wild type and mutant (H52A) MDMPIs were crystallized and their structures were solved at 1.75 and 2.05 Å resolution, respectively. The crystal structures reveal that this enzyme contains a divalent metal-binding domain and a C-terminal domain possessing a novel folding pattern (␣␤␣␤␤␣ fold). The divalent metal-binding site is composed of residues His 52 , Glu 144 , and His 148 and is located at the bottom of a surface pocket. Combining the structural and site-directed mutagenesis studies, it is proposed that this surface pocket including the metal ion and MSH moiety formed the putative catalytic center.Mycothiol, also known as MSH 4 and chemically 1D-myo-inosityl-2-(N-acetyl-L-cysteinyl)-amido-2-deoxy-␣-D-glucopyranoiside (1-4), is the major low molecule mass thiol in many groups of Gram-positive bacteria such as coryneform bacteria, mycobacteria, and streptomycetes (5, 6). These bacteria synthesize MSH but lack GSH molecule that plays important roles in many physiological processes. It is believed that MSH functions similarly to GSH in many microbial activities (6). However, the understanding of MSH physiological function was limited to detoxification of reactive oxygen/alkalating species and to protection of pathogens such as Mycobacterium tuberculosis from host cell defense systems (1,7,8). Very recently, a novel physiological role of MSH in assimilation of aromatic compounds was described and an MSH-dependent maleylpyruvate isomerase (MDMPI) was identified in Corynebacterium glutamicum (9). This MDMPI catalyzes the conversion of maleylpyruvate (substrate) to fumarylpyruvate (product) (Fig. 1).BLAST searches with MDMPI sequence against GenBank TM and other protein data bases revealed that MDMPI is not homologous to any functionally identified proteins but showed significant identities (27-36%) to a range of conserved hypothetical proteins from the genomes of Streptomyces coelicolor, Streptomyces avermitilis, Propionibacterium acnes, and Nocardia farcinica (10) (Fig. 2A). Earlier research on mycothiol biochemistry and biosynthesis has been conducted with mycobacteria such as M. tuberculosis, with the ultimate aim of defining new targets against tuberculosis, the resurgence of which has been a growing health concern in both developed and developing nations. The understanding of MSH-dependent enzyme structure is the key to the characterization of MSH-dependent enzyme...

No abstract