The NMR structure of protein-glutaminase from Chryseobacterium proteolyticum

Abstract

Protein deamidation, the hydrolysis of side chain amido groups of protein-bound glutaminyl or asparaginyl residues to release ammonia, has received focused attention especially in food industries since protein deamidation is regarded as a promising method to improve functional properties of food proteins. Deamidation generally decreases an isoelectric point of proteins due to increase in number of negatively charged carboxyl groups and enhances protein solubility. In addition, deamidation leads to alteration of the tertiary structures of proteins with an improved amphiphilic character that is useful as an emulsifier or a foaming agent. Therefore, deamidation of food proteins have been investigated by various methods including mild acid treatment, anion-catalyzed deamidation, dry heating under mild alkaline conditions, and thermal treatment. Although deamidation by these treatments improved protein functionalities, there were undesired side-effects, such as concomitant peptide bond cleavages, that were unavoidably brought about by the chemical/ physical treatments. Therefore, enzymatic methods have advantages due to their selectivity and mild treatments. The possibilities of the use of transglutaminases, peptidoglutaminases, and proteases have been explored for this purpose. These enzymes, however, are not suitable because the primary catalytic reactions of transglutaminases and proteases are not deamidation itself, and primary substrates of peptidoglutaminases are not proteins. Protein-glutaminase (PG) is an enzyme produced from the microorganism Chryseobacterium proteolyticum strain 9670 (Yamaguchi et al. 2001). PG catalyzes only the deamidation of the side chain amido group of protein-bound glutaminyl residues to release ammonia without catalyzing the transglutamination and hydrolysis of asparaginyl residues or producing other undesirable changes in protein structures. PG is a monomeric single polypeptide with pI = 10.0 and a molecular weight of 19,860 and is synthesized as a prepro-form, containing a 21-amino-acid signal polypeptide, a 114-amino acid pro-region, and a sequence for the mature enzyme. PG with the pro-region (pro-PG) has no enzymatic activities, but when pro-PG is removed by an extracellular protease, an active enzyme is produced in C. proteolyticum. However, the amount of PG produced by C. proteolyticum is too small to be used for industrial application that limits the application of PG to deamidation of food proteins. Recently, we have constructed the high expression system of PG with Corynebacterium glutamicum, which enables us to prepare an amount of stable-isotope labeled PG for NMR experiments (Kikuchi et al. 2009). Here, we report the solution structure of mature PG determined by NMR and discuss the catalytic mechanism of PG on the structural basis.