Pyruvate carboxylase from Mycobacterium smegmatis: stabilization,rapid purification,molecular and biochemical characterization and regulation of the cellular level |
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Authors: | Biswarup Mukhopadhyay Endang Purwantini |
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Institution: | Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Avenue, Urbana, IL 61801, USA |
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Abstract: | This is the first report on the purification and characterization of an anaplerotic enzyme from a Mycobacterium. The anaplerotic reactions play important roles in the biochemical differentiation of mycobacteria into non-replicating stages. We have purified and characterized a pyruvate carboxylase (PYC) from Mycobacterium smegmatis and cloned and sequenced its gene. We have developed a very rapid and efficient purification protocol that provided PYC with very high specific activities (up to 150 U/mg) that remained essentially unchanged over a month. The enzyme was found to be a homomultimer of 121 kDa subunits, mildly thermophilic, absolutely dependent on acyl-CoAs for activity and inhibited by ADP, by excess Mg2+, Co2+, and Mn2+, by aspartate, but not by glutamate and α-ketoglutarate. Supplementation of minimal growth medium with aspartate did not lower the cellular PYC level, rather doubled it; with glutamate the level remained unchanged. These observations would not fit the idea that the M. smegmatis enzyme fulfills a straightforward 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 PYCs 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 an identical operon-like arrangement. Thus, M. smegmatis could serve as a model for studying PYC-related physiological aspects of mycobacteria. Also, the ease of purification and the extraordinary stability could make the M. smegmatis enzyme a model for studying the structure–function relationships of PYCs in general. It should be noted that no crystal structure is available for this enzyme of paramount importance in all three domains of life, archaea, bacteria, and eukarya. |
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Keywords: | Pyruvate carboxylase Rapid purification Stabilization of activity Regulation Pyridoxal 5′-phosphate PEP phosphoenolpyruvate α-KGA α-ketoglutarate TCA tricarboxylic acid MS malate synthase ICL isocitrate lyase OAD oxaloacetate decarboxylase PYC pyruvate carboxylase PPC PEP carboxylase PCC propionyl-CoA carboxylase BC biotin carboxylase BCC biotin carboxyl carrier BCCP biotin carboxyl carrier protein CT carboxyltransferase PCCα α-subunit of propionyl-CoA carboxylase (composed of BC and BCC domains) ACC acetyl-CoA carboxylase ACC/BC+BCC a subunit of ACC with both the BC and BCC domains PYCA A subunit of PYC carboxylase (composed of the ATP/bicarbonate binding or the BC domain) PYCB B subunit of PYC carboxylase (composed of CT and BCC domains) PLP pyridoxal 5′-phosphate ORF open reading frame |
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