Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli
Introduction
Many enzymes function in vivo in complexes with other proteins or cell components that modulate their catalytic activities. Previous studies of protein complexes formed by inorganic pyrophosphatase (PPase) used as a bait identified three cytosolic protein partners with enzymatic activities in Escherichia coli [1] — a putative 5-keto-4-deoxyuronate isomerase (later identified as a cupin-type phosphoglucose isomerase, cPGI [2]), class I fructose-1,6-bisphosphate aldolase (FbaB), and l-glutamate decarboxylase (GadA). The functional consequences of these interactions remain unclear.
The reactions catalyzed by these enzymes and some their characteristics are shown in Table 1. PPase catalyzes the hydrolysis of pyrophosphate (PPi), a byproduct of numerous biosynthetic reactions where nucleoside triphosphates are converted into nucleoside monophosphates [6], and is essential for cell viability. A recent elegant study by Serrano-Bueno et al. [7] demonstrated a dual cytosolic and nuclear localization for the PPase Ipp1p isoform in yeast, emphasizing the importance of the enzyme in nucleic acid metabolism. The E. coli PPase is a structurally and mechanistically well characterized homohexameric Mg2+-dependent enzyme, similar in many aspects to the homologous dimeric yeast PPase [8,9].
cPGI (formerly known as KduI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate and, less efficiently, the isomerization of glucuronate to fructuronate [2]. In solution, cPGI represents a mixture of inactive hexamers and active dimers, with its equilibrium shifted to dimer state by substrate binding and Zn2+ removal [2]. The physiological role of cPGI is unknown, as E. coli contains an alternative, non-cupin form of PGI [10]. It is noteworthy that Rothe et al. demonstrated the up-regulation of cPGI production by D-glucuronate and facilitation of hexuronate consumption by cell-free extracts of E. coli strains overexpressing the cPGI gene [11].
Decameric FbaB reversibly converts fructose-1,6-bisphosphate to dihydroxyacetone phosphate and glyceraldehyde-3-phosphate. This reaction is important for glycolysis and gluconeogenesis [12]. FbaB is a class I aldolase, whose active site lysine forms a covalent Schiff base intermediate with dihydroxyacetone phosphate [13]. FbaB is detected in cells growing on non-glucose carbon sources and is believed to function in gluconeogenesis [13]. E. coli growing on glucose contains an additional, class II aldolase (FbaA) with a slightly different catalytic mechanism [14].
GadA is a pyridoxal phosphate-dependent enzyme that converts l-glutamate to 4-aminobutirate and carbon dioxide. Together with GadB, which differs by five amino acid residues, GadA forms the most efficient acid resistance system in E. coli [15,16], allowing cells to survive for 2 h at pH 2.5, by maintaining an internal pH 5. At neutral pH, GadA is localized to the cytoplasm, but is found near the inner membrane at low pH.
In this study, we determined the effects of complex formation between these enzymes on their catalytic activities. Our results indicated that enzymes formed all possible binary complexes, with diverse effects on their constituent partners, including activation, inhibition, and change in oligomeric structure. The results suggest that interaction of PPase with its inducible partners (FbaB, GadA and cPGI) provide a dual mechanism of their regulation in cell at the levels of both activity and protein synthesis.
Section snippets
Enzymes
All enzymes were overproduced in a tag-free form in E. coli strain BL21 and purified as described previously [1]. Preparation purity was estimated as >95% using 12% polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate [17]. Stock enzyme solutions were made in 50 mM Tris-HCl buffer, pH 7.5, except for GadA, which was maintained in 0.1 M Na-acetate buffer, pH 4.6, containing 10 mM NaCl (this enzyme is inactivated at pH > 5.5 [18]). The PPase buffer also contained 1 mM MgCl2
Modulation of cPGI activity by the three partner proteins
cPGI existed in an equilibrium of active dimeric and inactive hexameric forms in the 0.025–5 μM concentration ranges used in this study [2], which complicated the effects of the other proteins on cPGI activity. Therefore, initial assays were performed at 0.035 μM cPGI concentration, where the dimeric form prevailed [2]. cPGI activity was assayed with 30 mM glucose-6-phosphate as substrate and was estimated by colorimetrically measuring the accumulation of ketose, fructose-6-phosphate. All
Discussion
Classical enzymology treats enzymes as individual entities. The main paradigm of protein chemistry had long been that nature selects amino acid sequences that fold into unique structures which participate in only function-dictated interactions with other proteins. However, a growing body of evidence indicates that a great many proteins, including enzymes, form dynamic associations with other proteins and cellular structures. The enzyme complexes investigated here are only some of the complexes
Declaration of Competing Interest
None.
Acknowledgements
We are indebted to Professor P.V. Sergiev for a gift of the E. coli knockout mutants and to anonymous reviewers for constructive criticism of the original version of the manuscript.
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