Determinants and Expansion of Specificity in a Trichothecene UDP-Glucosyltransferase from Oryza sativa

Abstract

Family 1 UDP-glycosyltransferases (UGTs) in plants primarily form glucose conjugates of small molecules and, besides other functions, play a role in detoxification of xenobiotics. Indeed, overexpression of a barley UGT in wheat has been shown to control Fusarium head blight, which is a plant disease of global significance that leads to reduced crop yields and contamination with trichothecene mycotoxins such as deoxynivalenol (DON), T-2 toxin, and many other structural variants. The UGT Os79 from rice has emerged as a promising candidate for inactivation of mycotoxins because of its ability to glycosylate DON, nivalenol, and hydrolyzed T-2 toxin (HT-2). However, Os79 is unable to modify T-2 toxin (T-2), produced by pathogens such as Fusarium sporotrichioides and Fusarium langsethii. Activity toward T-2 is desirable because it would allow a single UGT to inactivate co-occurring mycotoxins. Here, the structure of Os79 in complex with the products UDP and deoxynivalenol 3-O-glucoside is reported together with a kinetic analysis of a broad range of trichothecene mycotoxins. Residues associated with the trichothecene binding pocket were examined by site-directed mutagenesis that revealed that trichothecenes substituted at the C4 position, which are not glycosylated by wild-type Os79, can be accommodated in the binding pocket by increasing its volume. The H122A/L123A/Q202L triple mutation, which increases the volume of the active site and attenuates polar contacts, led to strong and equivalent activity toward trichothecenes with C4 acetyl groups. This mutant enzyme provides the broad specificity required to control multiple toxins produced by different Fusarium species and chemotypes.

UDP-Glucosyltransferases from Rice, Brachypodium, and Barley: Substrate Specificities and Synthesis of Type A and B Trichothecene-3-O-β-d-glucosides

“…Although the masses of such glucosides were detectable by liquid chromatography coupled to high resolution mass spectrometry (LC-HRMS), even at high enzyme concentrations and long incubation times, the acceptor concentrations remained unchanged and preparative synthesis was out of reach. This is in agreement with two recent papers on structure, catalytic mechanism, and substrate binding of OsUGT79 [ 45 , 46 ] that reported OsUGT79 is inactive with T2 and FUSX.…”

“…Nevertheless, it is obvious that C-4 acetylated trichothecenes are generally recalcitrant to glucosylation at C-3-OH. This is due to steric hindrance at the active site, as recently shown in the case of OsUGT79 [ 45 ]. By increasing the volume of the active site through mutagenesis, the activity of OsUGT79 could be expanded to include also T2, FUSX, and DAS.…”

Enzymes for Detoxification of Various Mycotoxins: Origins and Mechanisms of Catalytic Action

“…Though it is not discussed by the authors, Gln143 is closely located to substrate/product (3.9 Å) and it may participate in catalytic mechanism. Moreover, replacement Gln143Ala (as well as Phe199Gln) results in a complete loss of enzyme activity according to recent studies of site-directed mutagenesis [137].…”

“…Besides, they are not active towards substrates containing a 4-acetoxy substituent as in the case of T-2 toxin ((2α,3α,4β,8α)-4,15-bis(acetyloxy)-3-hydroxy-12,13-epoxytrichothec-9-en-8-yl 3-methylbutanoate), 4-acetoxy-nivalenol, etc. Therefore, site-directed mutagenesis of OsUGT79 was carried out to increase its hydrophobic pocket [137]. The best activity towards T-2 toxin and diacetoxyscirpenol ((3α,4β)-4,15-bis(acetyloxy)-3-hydroxy-12,13-epoxytrichothec-9-en) was shown by a ternary mutant His122Ala/Leu123Ala/Gln202Ala.…”

Novel Soil Bacterium Strain Desulfitobacterium sp. PGC-3-9 Detoxifies Trichothecene Mycotoxins in Wheat via De-Epoxidation under Aerobic and Anaerobic Conditions

“…Previously reported de-epoxidation bacteria were also active on some trichothecene mycotoxins, but exhibited no activity on others carrying an additional acetyl group at the C3 or C4 position [ 11 , 19 ]. The limited volume of the active site in the DON-degradation enzyme likely prevents binding of the enzyme to the trichothecene mycotoxins carrying an additional acetyl group, as described for UDP-glycosyltransferases from plants that form glucose conjugates to detoxify trichothecene mycotoxins [ 35 ].…”