Inositol monophosphatase activity from the Escherichia coli suhB gene product

Abstract

The suhB gene is located at 55 min on the Escherichia coli chromosome and encodes a protein of 268 amino acids. Mutant alleles of suhB have been isolated as extragenic suppressors for the protein secretion mutation (secY24), the heat shock response mutation (rpoH15), and the DNA synthesis mutation (dnaB121) (K. Shiba, K. Ito, and T. Yura, J. Bacteriol. 160:696-701, 1984; R. Yano, H. Nagai, K. Shiba, and T. Yura, J. Bacteriol. 172:2124-2130, 1990; S. Chang, D. Ng, L. Baird, and C. Georgopoulos, J. Biol. Chem. 266:3654-3660, 1991). These mutant alleles of suhB cause cold-sensitive cell growth, indicating that the suhB gene is essential at low temperatures. Little work has been done, however, to elucidate the role of the product of suhB in a normal cell and the suppression mechanisms of the suhB mutations in the aforementioned mutants. The sequence similarity shared between the suhB gene product and mammalian inositol monophosphatase has prompted us to test the inositol monophosphatase activity of the suhB gene product. We report here that the purified SuhB protein showed inositol monophosphatase activity. The kinetic parameters of SuhB inositol monophosphatase (K m ‫؍‬ 0.071 mM; V max ‫؍‬ 12.3 mol/min per mg) are similar to those of mammalian inositol monophosphatase. The ssyA3 and suhB2 mutations, which were isolated as extragenic suppressors for secY24 and rpoH15, respectively, had a DNA insertion at the 5 proximal region of the suhB gene, and the amount of SuhB protein within mutant cells decreased. The possible role of suhB in E. coli is discussed.The suhB gene was first identified as the locus for the ssyA3(Cs) mutation that was isolated as an extragenic suppressor for the secY24(Ts) mutant (31). The ssyA3 mutation restored the defect in protein secretion caused by the secY24 mutation (33) and rescued the temperature-sensitive cell growth of secY24 mutant cells. The ssyA3 mutation caused cold-sensitive cell growth by itself and impaired protein synthesis was observed in the ssyA3 mutant at low temperatures (31). The cold-sensitive growth of the ssyA3 mutant was rescued by introduction of the wild-type copy of suhB, indicating that ssyA3 is a mutant allele of suhB (40). The suhB2(Cs) mutation is another allele of suhB and was isolated as an extragenic suppressor of the rpoH15(Ts) mutation. The rpoH15 mutant gene produces an altered 32 protein and causes a defect in the induction of heat shock proteins at higher temperatures (42). The suhB2(Cs) mutation rescued the temperature-sensitive cell growth of the rpoH15 mutant and partially restored heat shock induction in the mutant cell (40). A coldsensitive mutation was also mapped in the suhB locus as an extragenic suppressor for the dnaB121(Ts) mutation that inhibits replication of phage (2). Thus, mutant alleles of suhB have effects on diverse cell activities, including protein export, stress response, and DNA replication.The suhB gene encodes a protein of 268 amino acids (40), and the primary structure of its translated product shows a high level of similarity t...

Use of modular, synthetic scaffolds for improved production of glucaric acid in engineered E. coli

“…As a result, the production cells grew considerably faster than the 11 non-scaffolded pathway expressed to levels required to achieve comparable titers. In the 12 present study, the modular architecture was utilized for the glucaric acid pathway to shown to possess activity against myo-inositol-1-phosphate (Matsuhisa et al, 1995). We 17 have over-expressed the suhB gene in an attempt to improve glucaric acid productivity; 18 however, the titers have always decreased in response to suhB (data not shown).…”

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

“…(Fig. 1a, boxed sequence) (Matsuhisa et al, 1995). We investigated whether the suhB Bc deletion mutant (herein DsuhB Bc ) has a growth defect.…”

“…The mechanism of action of SuhB Ec remains unclear. In vitro, purified SuhB Ec has IMPase activity (Matsuhisa et al, 1995). However, a SuhB Ec mutant protein D87N that lacks IMPase activity remains fully functional in complementing a defective suhB mutant (Chen & Roberts, 2000).…”

“…BCAL2157 encodes a 29 kDa protein that has high sequence similarity to SuhB from E. coli (SuhB Ec ). The primary amino acid sequence of SuhB Ec has a high level of similarity to mammalian proteins of the inositol monophosphatase (IMPase) family (Matsuhisa et al, 1995). IMPases are essential enzymes for the biosynthesis of phosphatidylinositides (PtdIns), since inositol is synthesized as myo-inositol 1-phosphate, which has to be converted to free myo-inositol by an IMPase (Eisenberg, 1967) prior to accepting the phosphatidic acid moiety from CDP-diglyceride for the generation of PtdIns (Paulus & Kennedy, 1960).…”

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

“…coli has five Li ϩ -sensitive phosphatases as follows: CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a 3Ј-phosphoadenosine 5Ј-phosphatase involved in the cysteine biosynthesis pathway (25,26), whereas SuhB is an inositol monophosphatase (IMPase) that is also known as a suppressor of temperature-sensitive growth phenotypes in E. coli (27,28). Fbp is required for growth on gluconeogenic substrates and probably represents the main gluconeogenic FBPase (12).…”