Pyruvate carboxylase from Mycobacterium smegmatis: stabilization, rapid purification, molecular and biochemical characterization and regulation of the cellular level

This is the first report on the purification and characterization of an ana plerotic enzyme from a Mycobacterium. The anaplerotic reactions play import ant roles in the biochemical differentiation of mycobacteria into non-repli cating stages. We have purified and characterized a pyruvate carboxylase (P YC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided P YC with very high specific activities (up to 150 U/mg) that remained essent ially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs fo r activity and inhibited by ADP, by excess Mg2+, Co2+, and Mn2+, by asparta te, but not by glutamate and alpha-ketoglutarate. Supplementation of minima l growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observation s would not fit the idea that the M. smegmatis enzyme fulfills a straightfo rward anaplerotic function: in a closely related organism, Corynebacterium glutamicum, PYC is the major anaplerotic enzyme. Growth on glucose provided 2-fold higher cellular PYC level than that observed with glycerol. The PYC s of M. smegmatis and Mycobacterium tuberculosis were highly homologous to each other. In M. smegmatis, M. tuberculosis and M. lepra, pyc was flanked by a putative methylase and a putative integral membrane protein genes in a n identical operon-like arrangement. Thus, M. smegmatis could serve as a mo del for studying PYC-related physiological aspects of mycobacteria. Also, t he ease of purification and the extraordinary stability could make the M. s megmatis enzyme a model for studying the structure-function relationships o f PYCs in general. It should be noted that no crystal structure is availabl e for this enzyme of paramount importance in all three domains of life, arc haea, bacteria, and eukarya. (C) 2000 Elsevier Science B.V. All rights rese rved.