Deletion and insertion of a 192-residue peptide in the active-site domain of glycosyl hydrolase family-2 β-galactosidases

The monomeric multimetal-binding β-galactosidase of Saccharopolyspora rectivirgula (srbg), a glycosyl hydrolase family-2 enzyme, has a unique sequence consisting of 192 amino acid residues with no similarity to known proteins. This 192-residue sequence (termed the “ι [iota] sequence”) appears to be inserted into a sequence homologous to the active-site domain of the Escherichia coli lacZ enzyme (lacZbg). To assess the effects of the t sequence at specific sites of β-galactosidase on the catalytic functioning and molecular properties of β-galactosidase, deletion or insertion mutants of β-galactosidases were constructed, expressed in LacZ − E. coli strains, and characterized: srbgΔ in which the ι sequence was deleted from srbg, and lacZbgI, in which the 192-residue ι sequence was inserted into the corresponding position (between Asp 591 and Phe 592) in the active-site domain of lacZbg. srbgΔ was a catalytically inactive, dimeric protein which retained multimetal-binding characteristics, suggesting that the ι sequence is very important for maintaining the structure necessary for the catalytic functioning and the monomeric structure of srbg but is not responsible for the unique metal ion requirements of srbg. On the other hand, lacZbgI existed as a mixture of a monomer, a tetramer, and higher multimers. The monomeric species was inactive, whereas the tetramer and other multimers were catalytically active ( V max K m value, 25% of that of lacZbg) and highly specific for β- d-galactoside. The tetrameric lacZbgI was activated by Mg 2+ and Mn 2+ with lowered metal affinities, and the stoichiometry of metal binding was unchanged from that of lacZbg. These results, along with the published stereo structure of lacZbg, suggest that, in lacZbgI, the inserted 192-residue ι peptide could fold independently of the lacZbg domains into a “sub-domain,” lying distant from the active site and subunit interfaces.