A search for novel substrate activity in mutants of L‐Alanine Dehydrogenase

Abstract

The enzyme L‐Alanine dehydrogenase (AlaDH) from Mycobacterium tuberculosis catalyzes the reversible conversion of L‐alanine to pyruvate. AlaDH, however, belongs to the formate‐glycerate dehydrogenase superfamily, whose other members generally reversibly interconvert carbonyls to alcohols, not amines as in the AlaDHs. In a case of convergent evolution, enzymes in the Phe/Glu/Val/Leu amino acid dehydrogenase (AADH) superfamily catalyze the same reaction as AlaDH on larger amino acids, but bare no structural resemblance to AlaDH. Can AlaDH be altered in a way that will allow it to act on different amino acid substrates, or has evolution make it too specific to alanine to alter its activity? Our objective is to find novel activity on various substrates in the highly specific AlaDH. Previously, it has been shown that the replacement of the 94‐phenylalanine binding pocket residue with alanine and serine leads to novel activity on leucine and norleucine in comparison to the wild type, as well as greatly increased activity on norvaline and methionine. Here we present the additional mutation of 133‐methionine to alanine. We successfully express and purify this double mutant, and find additional novel activity.