Substrate and cofactor promiscuity of the F₄₂₀ dependent reductases

Abstract

Aflatoxins are polyaromatic mycotoxins contaminating a range of food crops, resulting in health problems due to their carcinogenic and toxic properties. Aflatoxins can be metabolized by a group of M. smegmatis enzymes called the F420 dependent reductases (FDRs) which utilise the unusual cofactor F420H2. The FDRs fall into two distinct but related families termed the FDR-As and FDR-Bs, the nearest relatives of both being the structurally similar flavin mononucleotide (FMN) dependent pyridoxamine 5'- phosphate oxidase (PNPOx) enzymes.

This study analyses the substrate and cofactor switching ability of the FDR-A and -B enzymes. For the purpose of comparison, it was first necessary to identify and characterise a PNPOx from M. smegmatis. Phylogenetic analysis of published genome sequences identified MSMEG_5675 as a probable PNPOx. The protein was heterologously expressed and purified. It had an intense yellow colour suggesting bound FMN and catalysed the oxidation of pyridoxamine 5'-phosphate (PMP), showing a specific activity of 0.18 per second, consistent with those of other validated PNPOx's. MSMEG_5675 was thus confirmed to be an FMN dependent PNPOx enzyme. It showed no activity with either F420 or F420H2 as cofactor, confirming that the PNPOx and FDR families are functionally distinct.

The next goals were to test if FDR-A enzymes from other Actinomycetales species could degrade aflatoxins and to find other possible substrates which might be more relevant physiologically for these enzymes. Ten FDR-A enzymes from diverse Actinomycetales were heterologously expressed and nine were found to use F420H2 and reduce aflatoxin. One FDR-A enzyme which could not reduce aflatoxin belonged to a distinct clade (denoted FDR-AA), and subsequent expression and analysis of seven other FDR-AAs from M. smegmatis found that none could reduce aflatoxin. Certain FDR-A and FDR-B enzymes that could reduce aflatoxin also showed activity with coumarin and three furanocoumarins (angelicin, 8-methoxysporalen and imperatorin), but none of the FDR-AAs tested showed any of these activities. The shared feature of the compounds that were substrates for the FDR-As and -Bs was an alpha,beta-unsaturated lactone moiety. Several of the FDR enzymes were then tested with FMN to determine if they showed any cofactor promiscuity. They were all found to have very high preference for cofactor F420H2 in performing reduction reactions. However, three (MSMEG_3356, MSMEG_2027 and MSMEG_6848) were also found to degrade aflatoxins in the presence of FMN in place of F420.

Further characterisation of these three enzymes, including determining their reaction products, specific activities and binding constants with FMN, showed that these enzymes performed an oxidation reaction, which was opposite to the one demonstrated with F420. The last chapter is a general discussion of the evolution of FDR enzymes, the potential physiological substrates utilised by these enzymes, their role in the environment and their cofactor switching ability. The ability of these enzymes to show different reaction chemistries with different cofactors is also discussed. A priority for future research is also suggested. - provided by Candidate. Show more