Mangiferin is a new potential antimalarial and anticancer drug for targeting serine hydroxymethyltransferase.

Mangiferin, a polyphenolic xanthone glycoside found in various botanical sources, including mango (Mangifera indica L.) leaves, can exhibit a variety of bioactivities. Although mangiferin has been reported to inhibit many targets, none of the studies have investigated the inhibition of serine hydroxymethyltransferase (SHMT), an attractive target for antimalarial and anticancer drugs. SHMT, one of the key enzymes in the deoxythymidylate synthesis cycle, catalyzes the reversible conversion of l-serine and (6S)-tetrahydrofolate (THF) into glycine and 5,10-methylene THF. Here, in vitro and in silico studies were used to probe how mangiferin isolated from mango leaves inhibits Plasmodium falciparum and human cytosolic SHMTs. The inhibition kinetics at pH 7.5 revealed that mangiferin is a competitive inhibitor against THF for enzymes from both organisms. Molecular docking and molecular dynamic (MD) simulations demonstrated the inhibitory effects of the deprotonated forms of mangiferin, specifically the C6-O- species and its resonance C9-O- species appearing at pH 7.5, combined with two docked poses, either a xanthone or glucose moiety, placed inside the THF-binding pocket. The MD analysis revealed that both C6-O- and its resonance-stabilized C9-O- species can favorably bind to SHMT in a similar fashion to THF, supporting the THF competitive inhibition of mangiferin. In addition, characterization of the proton dissociation equilibria of isolated mangiferin revealed that only three hydroxy groups of the xanthone moiety, C6-OH, C3-OH, and C7-OH, underwent varying degrees of deprotonation with pKa values of 6.38 ± 0.11, 8.21 ± 0.35, and 12.37 ± 0.30, respectively, while C1-OH remained protonated. Altogether, our findings demonstrate a new bioactivity of mangiferin and provide the basis for the future development of mangiferin as a potent antimalarial and anticancer drug.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.

A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase.

The oxygenase component (C2) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C2 with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (kOH) for 4-APA of 0.028 s-1 compared to 17 s-1 for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s-1 for 4-HPA versus 2.5 s-1 for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C2 reactivity toward a phenolic compound. Removing this hydroxyl group expands C2 activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.

Inhibitors of plasmodial serine hydroxymethyltransferase (SHMT): cocrystal structures of pyrazolopyrans with potent blood- and liver-stage activities.

Several of the enzymes related to the folate cycle are well-known for their role as clinically validated antimalarial targets. Nevertheless for serine hydroxymethyltransferase (SHMT), one of the key enzymes of this cycle, efficient inhibitors have not been described so far. On the basis of plant SHMT inhibitors from an herbicide optimization program, highly potent inhibitors of Plasmodium falciparum (Pf) and Plasmodium vivax (Pv) SHMT with a pyrazolopyran core structure were identified. Cocrystal structures of potent inhibitors with PvSHMT were solved at 2.6 Å resolution. These ligands showed activity (IC50/EC50 values) in the nanomolar range against purified PfSHMT, blood-stage Pf, and liver-stage P. berghei (Pb) cells and a high selectivity when assayed against mammalian cell lines. Pharmacokinetic limitations are the most plausible explanation for lack of significant activity of the inhibitors in the in vivo Pb mouse malaria model.

Distinct biochemical properties of human serine hydroxymethyltransferase compared with the Plasmodium enzyme: implications for selective inhibition.

UNLABELLED: Serine hydroxymethyltransferase (SHMT) catalyzes the transfer of a hydroxymethyl group from l-serine to tetrahydrofolate to yield glycine and 5,10-methylenetetrahydrofolate. Our previous investigations have shown that SHMTs from Plasmodium spp. (P. falciparum, Pf; P. vivax, Pv) are different from the enzyme from rabbit liver in that Plasmodium SHMT can use d-serine as a substrate. In this report, the biochemical and biophysical properties of the Plasmodium and the human cytosolic form (hcSHMT) enzymes including ligand binding and kinetics were investigated. The data indicate that, similar to Plasmodium enzymes, hcSHMT can use d-serine as a substrate. However, hcSHMT displays many properties that are different from those of the Plasmodium enzymes. The molar absorption coefficient of hcSHMT-bound pyridoxal-5'-phosphate (PLP) is much greater than PvSHMT-bound or PfSHMT-bound PLP. The binding interactions of hcSHMT and Plasmodium SHMT with d-serine are different, as only the Plasmodium enzyme undergoes formation of a quinonoid-like species upon binding to d-serine. Furthermore, it has been noted that hcSHMT displays strong substrate inhibition by tetrahydrofolate (THF) (at THF > 40 µm), compared with SHMTs from Plasmodium and other species. The pH-activity profile of hcSHMT shows higher activities at lower pH values corresponding to a pKa value of 7.8 ± 0.1. Thiosemicarbazide reacts with hcSHMT following a one-step model [k1 of 12 ± 0.6 m(-1) ·s(-1) and k-1 of (1.0 ± 0.6) × 10(-3) s(-1) ], while the same reaction with PfSHMT involves at least three steps. All data indicated that the ligand binding environment of SHMT from human and Plasmodium are different, indicating that it should be possible to develop species-selective inhibitors in future studies. DATABASE: serine hydroxymethyltransferase, EC 2.1.2.1; 5,10-methylenetetrahydrofolate dehydrogenase, EC 1.5.1.5.