Constitutive inorganic pyrophosphatase as a reciprocal regulator of three inducible enzymes in Escherichia coli

https://doi.org/10.1016/j.bbagen.2020.129762Get rights and content

Highlights

  • Four recombinant enzymes of Escherichia coli form all six possible binary complexes

  • Fructose-1,6-bisphosphate aldolase forms 1:1 and 1:2 complexes with partner enzymes

  • Pyrophosphatase is inhibited whereas all other enzymes are activated in the complexes

  • Effect on pyrophosphatase allows a feedback regulation of its partner biosynthesis

Abstract

Background

Previous studies have demonstrated the formation of stable complexes between inorganic pyrophosphatase (PPase) and three other Escherichia coli enzymes – cupin-type phosphoglucose isomerase (cPGI), class I fructose-1,6-bisphosphate aldolase (FbaB) and l-glutamate decarboxylase (GadA).

Methods

Here, we determined by activity measurements how complex formation between these enzymes affects their activities and oligomeric structure.

Results

cPGI activity was modulated by all partner proteins, but none was reciprocally affected by cPGI. PPase activity was down-regulated upon complex formation, whereas all other enzymes were up-regulated. For cPGI, the activation was partially counteracted by a shift in dimer ⇆ hexamer equilibrium to inactive hexamer. Complex stoichiometry appeared to be 1:1 in most cases, but FbaB formed both 1:1 and 1:2 complexes with both GadA and PPase, FbaB activation was only observed in the 1:2 complexes. FbaB and GadA induced functional asymmetry (negative kinetic cooperativity) in hexameric PPase, presumably by favoring partial dissociation to trimers.

Conclusions

These four enzymes form all six possible binary complexes in vitro, resulting in modulated activity of at least one of the constituent enzymes. In five complexes, the effects on activity were unidirectional, and in one complex (FbaB⋅PPase), the effects were reciprocal. The effects of potential physiological significance include inhibition of PPase by FbaB and GadA and activation of FbaB and cPGI by PPase. Together, they provide a mechanism for feedback regulation of FbaB and GadA biosynthesis.

General significance

These findings indicate the complexity of functionally significant interactions between cellular enzymes, which classical enzymology treats as individual entities, and demonstrate their moonlighting activities as regulators.

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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