Purification and cloning of the two domain glyoxalase I from wheat bran

Investigation of proteins extracted from wheat bran lead to the isolation o f a 37 kDa polypeptide extracted from a polyacrylamide gel. Extensive inter nal peptide sequence information of this protein identified it as a glyoxal ase I. Glyoxalase I activity in crude wheat bran extract was measured to 1 U/mg protein (1U = 1 mu mol S-lactoyl glutathione formed/min). Degenerate p rimers were designed and used for PCR-RACE-based cloning of the correspondi ng composite cDNA sequence (AJ243528). The wheat bran glyoxalase I amino ac id sequence is very similar to the translated sequence of a RNA transcript induced by desiccation of the resurrection grass Sporobulus stapfianus, sug gesting a role for glyoxalase in de- or rehydration of plant tissue. The 37 kDa wheat enzyme belongs to a group of monomeric glyoxalases and is compos ed of two similar halves each representing the full-length human glyoxalase I enzyme. A survey of glyoxalase I sequences, including one (not previousl y reported) from Drosophila melanogaster, is presented and alignments of th ese sequences show that amino acid residues involved in co-ordinating zinc or interaction with the substrate are conserved. The alignments indicate a non-linear evolution of glyoxalase I enzymes. (C) 2000 Elsevier Science Ire land Ltd. All rights reserved.