The Nitrogen Regulatory PII Protein (GlnB) and N -Acetylglucosamine 6-Phosphate Epimerase (NanE) Allosterically Activate Glucosamine 6-Phosphate Deaminase (NagB) in Escherichia coli

Abstract

Amino sugars are good sources of both ammonia and fructose-6-phosphate, produced by the glucosamine 6-phosphate deaminase, NagB. NagB is known to be allosterically regulated by -acetylglucosamine 6-phosphate (GlcNAc-6P) and the phosphocarrier protein of the bacterial phosphotransferase system, HPr, in We provide evidence that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein (U-PII) also allosterically activate NagB by direct protein-protein interactions. NanE is essential for neuraminic acid (NANA) and -acetylmannosamine (ManNAc) utilization, and PII is known to be a central metabolic nitrogen regulator. We demonstrate that uridylylated PII (but not underivatized PII) activates NagB>10-fold at low concentrations of substrate, whereas NanE increases NagB activity >2-fold. NanE activates NagB in the absence or presence of GlcNAc-6P, but HPr and U-PII activation requires the presence of GlcNAc-6P. Activation of NagB by HPr and uridylylated PII, as well as by NanE and HPr (but not by NanE and U-PII), is synergistic, and the modeling, which suggests specific residues involved in complex formation, provides possible explanations. Specific physiological functions for the regulation of NagB by its three protein activators are proposed. Each regulatory agent is suggested to mediate signal transduction in response to a different stimulus. The regulation of amino sugar utilization is important for the survival of bacteria in a competitive environment. NagB, a glucosamine 6-phosphate deaminase in , is essential for amino sugar utilization and is known to be allosterically regulated by-acetylglucosamine 6-phosphate (GlcNAc-6P) and the histidine-phosphorylatable phosphocarrier protein, HPr. We provide evidence here that NanE, GlcNAc-6P epimerase, and the uridylylated PII protein allosterically activate NagB by direct protein-protein interactions. NanE is essential for -acetylneuraminic acid (NANA) and-acetylmannosamine (ManNAc) utilization, and the PII protein is known to be a central metabolic nitrogen regulator. Regulatory links between carbon and nitrogen metabolism are important for adaptation of metabolism to different growth conditions.

Protein:Protein interactions in the cytoplasmic membrane apparently influencing sugar transport and phosphorylation activities of the e. coli phosphotransferase system

“…Our integral membrane interactome data suggested that many membrane proteins interact with many other membrane proteins, with a wide range of affinities [1]. Some of the high scoring interactions are likely to be permanent, high affinity interactions, while lower scoring interactions are more likely to be transient in nature [35, 36]. Among the observed interactions were many involving Enzyme IIC constituents of the PTS, particularly the fructose IIC protein, FruA, which also bears two additional domains, IIB and IIB′, where IIB′ is not involved in phosphoryl transfer, but plays a role in protein-protein interactions [21] (Table 2).…”

A novel transcription factor PunR and Nac are involved in purine and purine nucleoside transporter punC regulation in E. coli

“…The cultures were incubated at 24 o C overnight with continuous shaking, and cells were collected by centrifugation. The PunR recombinant protein, containing an N-terminal 6His tag, was puri ed by Ni-chelation chromatography from the soluble fraction as described (36,37). The insoluble fraction was solubilized in 7 M urea and puri ed on a Ni-NTA minicolumn with At-buffer (50 mM Tris-HCl buffer, pH 8, 0.5 mM NaCl, 5 mM imidazole, and 0.3% Brij) with 7M urea.…”

The uridylyltransferase GlnD and tRNA modification GTPase MnmE allosterically control Escherichia coli folylpoly-γ-glutamate synthase FolC

“…We have collaboratively published evidence for the existence of a network of protein-protein interactions (the interactome) in E. coli (3). This work provided the incentive and a guide for the research reported in this and previous works describing allosteric regulatory phenomena (1,2).…”

“…Carbon source availability is a signal for the recently identified global regulation of glycolysis and energy (ATP) balance by the phosphocarrier protein of the bacterial phosphotransferase system (PTS) HPr (1). Coregulation of carbon and nitrogen metabolism resulting, for example, in activation of glucosamine 6-P deaminase, NagB, by HPr, synergistically with uridylylated PII under limiting nitrogen conditions, is important for nutrient homeostasis (2). We have collaboratively published evidence for the existence of a network of protein-protein interactions (the interactome) in E. coli (3).…”