The gene yhdA from Bacillus subtilis encoding a putative flavin mononucleotide (FMN)-dependent oxidoreductase was cloned and heterologously expressed in Escherichia coli. The purified enzyme has a noncovalently bound FMN cofactor, which is preferentially reduced by NADPH, indicating that YhdA is a NADPH:FMN oxidoreductase. The rate of NADPH oxidation is enhanced by the addition of external FMN, and analysis of initial rate measurements reveals the occurrence of a ternary complex in a bi-bi reaction mechanism. YhdA has also been shown to reductively cleave the -N=N- bond in azo dyes at the expense of NADPH, and hence, it possesses azoreductase activity, however, at a rate 100 times slower than that found for FMN. Using Cibacron Marine as a model compound, we could demonstrate that the dye is a competitive inhibitor of NADPH and FMN. The utilization of NADPH and the absence of a flavin semiquinone radical distinguish YhdA from flavodoxins, which adopt the same structural fold, i.e., a five-stranded beta sheet sandwiched by five alpha helices. The native molecular-mass of YhdA was determined to be 76 kDa, suggesting that the protein occurs as a tetramer, whereas the YhdA homologue in Saccharomyces cerevisiae (YLR011wp) forms a dimer in solution. Interestingly, the different oligomerization of these homologous proteins correlates to their thermostability, with YhdA exhibiting a melting point of 86.5 degrees C, which is 26.3 degrees C higher than that for the yeast protein. This unusually high melting point is proposed to be the result of increased hydrophobic packing between dimers and the additional presence of four salt bridges stabilizing the dimer-dimer interface. Study holds ProTherm entries: 20477, 20478 Extra Details: YhdA; azoreductase activity; hydrophobic packing; dimer-dimer interface
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:52 p.m.
|Number of data points||2|
|Proteins||NAD(P)H-dependent FMN reductase LOT6 ; FMN-dependent NADPH-azoreductase ; Vinculin ; FMN-dependent NADPH-azoreductase|
|Assays/Quantities/Protocols||Experimental Assay: Tm|
|Libraries||Mutations for sequence A:AMPVFHTRTIESILEPVAQQISHLVIMHEEGEVDGKAIPDLTAPVSAVQAAVSNLVRVGKETVQTTEDQILKRDMPPAFIKVENACTKLVRAAQMLQADPYSVPARDYLIDGSRGILSGTSDLLLTFDEAEVRKIIRVCKGILEYLTVAEVVETMEDLVTYTKNLGPGMTKMAKMIDERQQELTHQEHRVMLVNSMNTVKELLPVLISAMKIFVTTKNTKSQGIEEALKNRNFTVEKMSAEINEIIRVLQLTSWD/B:GRPLLQAAKGLAGAVSELLRSAQP ; Mutations for sequence MNMLVINGTPRKHGRTRIAASYIAALYHTDLIDLSEFVLPVFNGEAEQSELLKVQELKQRVTKADAIVLLSPEYHSGMSGALKNALDFLSSEQFKYKPVALLAVAGGGKGGINALNNMRTVMRGVYANVIPKQLVLDPVHIDVENATVAENIKESIKELVEELSMFAKAGNPGV|