A full-length insert for the Flavobacterium meningosepticum N4-(N-acetyl-beta-glucosaminyl)-L-asparagine amidase gene was located on a 2500-bp HindIII fragment and cloned into the plasmid vector pBluescript. DNA sequencing revealed an open reading frame of 1020 nucleotides encoding a putative 45-amino-acid leader sequence and a deduced precursor polypeptide of 295 amino acids. In F. meningosepticum this precursor polypeptide undergoes proteolytic processing by an as yet unknown mechanism to generate an alpha-subunit and a beta-subunit, which constitute the active form of the heterodimeric mature glycosylasparaginase. The Flavobacterium glycosylasparaginase gene was expressed in Escherichia coli and found to be enzymatically active. The recombinant enzyme was purified from crude lysates and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist of the typical alpha- and beta-subunits. The recombinant beta-subunit cross-reacted to antibody specific for the rat liver beta-subunit, and Edman analysis demonstrated that its amino-terminus corresponded exactly to that of the mature native glycosylasparagine beta-subunit. A comparison of the Flavobacterium glycosylasparaginase with a mammalian glycosylasparaginase revealed 30% structural identity and 60% overall similarity between the prokaryotic and eukaryotic forms of the enzyme. Even more striking was the conservation of the amino acid sequence in both proteins where the post-translational cleavage to generate the active enzyme occurs. Our data demonstrate that deglycosylation of asparagine-linked glycans via hydrolysis of the AspNHGlcNAc linkage is an important reaction which has been preserved during evolution.