Structure-function characterization of an aldo-keto reductase involved in detoxification of the mycotoxin, deoxynivalenol

Abstract

Deoxynivalenol (DON) is a mycotoxin, produced by filamentous fungi such as Fusarium graminearum, that causes significant yield losses of cereal grain crops worldwide. One of the most promising methods to detoxify this mycotoxin involves its enzymatic epimerization to 3-epi-DON. DepB plays a critical role in this process by reducing 3-keto-DON, an intermediate in the epimerization process, to 3-epi-DON. DepB_Rleg from Rhizobium leguminosarum is a member of the new aldo-keto reductase family, AKR18, and it has the unusual ability to utilize both NADH and NADPH as coenzymes, albeit with a 40-fold higher catalytic efficiency with NADPH compared to NADH. Structural analysis of DepB_Rleg revealed the putative roles of Lys-217, Arg-290, and Gln-294 in NADPH specificity. Replacement of these residues by site-specific mutagenesis to negatively charged amino acids compromised NADPH binding with minimal effects on NADH binding. The substrate-binding site of DepB_Rleg is larger than its closest structural homolog, AKR6A2, likely contributing to its ability to utilize a wide range of aldehydes and ketones, including the mycotoxin, patulin, as substrates. The structure of DepB_Rleg also suggests that 3-keto-DON can adopt two binding modes to facilitate 4-pro-R hydride transfer to either the re- or si-face of the C3 ketone providing a possible explanation for the enzyme's ability to convert 3-keto-DON to 3-epi-DON and DON in diastereomeric ratios of 67.2% and 32.8% respectively.

Figure 1

The Dep pathway. The C3 OH group of DON (shown in red) is stereochemically inverted to produce the diastereomer, 3-epi-DON. In the expanded image of this stereogenic center, R₁ and R₂ represent the priority groups attached to the sp² hybridized C3. Hydride attack on the re-face at the prochiral C3 center generates 3-epi-DON.

Figure 2

A 40% representative sequence similarity network (SSN) at a threshold of e⁻⁵⁷. (A) Nodes within each cluster contain a representative protein sequence of a collection of sequences that share 40% or more sequence identity. Clusters have been annotated and color-coded based on curated protein sequences from the AKR Database. Grey nodes represent AKRs that have no functional annotation. Metabolic pathways associated with the function of AKR enzymes have been color-coded to cross-reference with each cluster and numerically labeled to designate each AKR family number. The nodes of AKRs possessing biochemical data on coenzyme specificity and crystal structures have also been accordingly labeled as outlined in the legend. (B) SSN generated at a threshold of e⁻⁶⁷ depicting complete segregation of the AKR18 family from AKR6, AKR12 and AKR14 members. (C) Genome Neighborhood Diagram of DepB_Rleg. The GND was generated using the EFI-GNT server which depicts the coding sequence region of depB_Rleg along with putative functions of upstream and downstream genes.

Figure 3

Crystal structure of DepB_Rleg protomer and octameric assembly. (A) Cartoon representation of the protomer with a view looking down the top of the TIM barrel rendered in black and white with the β7 annotated in red. Loop A (orange) caps the top of the substrate-binding cleft, loop B (dashes) could not be modeled but its position has been highlighted in this figure, and finally, loop C (green) which extends into the active site of the opposing protomer in the dimer. A sequence logo highlighting the conservation of the catalytic residues has also been provided. (B) Surface and cartoon rendition of the DepB_Rleg octamer. This assembly was predicted using the PISA server. View rotated at a 90° angle depicting 3D domain swapping of loop C at the dimer interface.

Figure 4

Model of the coenzyme binding site highlighting the conservation of putative coenzyme binding residues. (A) NADP⁺ was modeled by superimposing the ternary complex of AKR6A2 with apo-DepB_Rleg. White sticks represent the residues from DepB_Rleg complex, while cyan sticks are residues corresponding to AKR6A2. Trp-206 has been re-oriented in the apo-DepB_Rleg structure to prevent a steric clash with the nicotinamide ring of NADP⁺. π-stacking interactions between the nicotinamide ring and Trp-206 are shaded in blue. In apo-DepB_Rleg, Lys-217 points away from the entrance of the coenzyme binding pocket. However, by analogy to Lys-254 in AKR6A2, this residue may interact with at least one of the oxygen atoms of 2’monophosphate. (B) MSA depicting conservation of residues near the 2’monophosphate of the modeled NADP⁺ (See Supplementary Table S1 for accession codes).

Figure 5

Model of the ternary complex of DepB_Rleg. (A) Binding orientation for 3-keto-DON for hydride attack at the re-face. π-stacking interactions between Phe-84 and the cyclohexene ring are shaded yellow. (B) Binding orientation for 3-keto-DON for hydride attack at the si-face. (C) Multiple sequence alignments depicting the sequence variability at positions corresponding to Phe-84 and Ala-123 in DepB_Rleg with that of various AKR family representatives.