An experimental study on carbon flow in Escherichia coli as a function of kinetic properties and expression levels of the enzyme phosphoglucomutase

Brautaset, Trygve; Petersen, Steffen B.; Valla, Svein

doi:10.1002/(sici)1097-0290(19980420)58:2/3<299::aid-bit27>3.3.co;2-h

Cited by 7 publications

(12 citation statements)

References 7 publications

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“…S1 shows a small alignment of 11 PGM sequences chosen because of previous structural, biochemical, or biological characterization. These include the four PGMs of known structure as well as PGM from A. xylinum , which has been biochemically characterized,19–21 and sequences from six human pathogens where this enzyme has been shown to play a role in infectivity 1, 3–5, 8, 50–52. Despite their general structural similarity and similar chain length, the sequence diversity of the PGM enzymes is quite high.…”

Section: Resultsmentioning

confidence: 99%

“…The coordinates for two bacterial PGMs from S. typhimurium (2FUV) and Thermus thermophilus (2Z0F) at resolutions of 2.0 and 2.5 Å have been deposited in the PDB, but never described in a publication. Several other PGM proteins have been biochemically characterized to various extents, including enzymes from Acetobacter xylinum ,19–21 Clostridium thermocellum ,22 Aedes aegypti ,23 yeast,24 wheat,25 and maize 26. Overall, however, detailed structure‐function studies of PGM enzymes are limited, especially for the bacterial proteins.…”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens

et al. 2011

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The crystal structure of the enzyme phosphoglucomutase from Salmonella typhimurium (StPGM) is reported at 1.7 Å resolution. This is the first high-resolution structural characterization of a bacterial protein from this large enzyme family, which has a central role in metabolism and is also important to bacterial virulence and infectivity. A comparison of the active site of StPGM with that of other phosphoglucomutases reveals conserved residues that are likely involved in catalysis and ligand binding for the entire enzyme family. An alternate crystal form of StPGM and normal mode analysis give insights into conformational changes of the C-terminal domain that occur upon ligand binding. A novel observation from the StPGM structure is an apparent dimer in the asymmetric unit of the crystal, mediated largely through contacts in an N-terminal helix. Analytical ultracentrifugation and small-angle X-ray scattering confirm that StPGM forms a dimer in solution. Multiple sequence alignments and phylogenetic studies show that a distinct subset of bacterial PGMs share the signature dimerization helix, while other bacterial and eukaryotic PGMs are likely monomers. These structural, biochemical, and bioinformatic studies of StPGM provide insights into the large α-D-phosphohexomutase enzyme superfamily to which it belongs, and are also relevant to the design of inhibitors specific to the bacterial PGMs.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens

et al. 2011

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“…This system should be useful both for high-level protein HCDC. Mermod et al (1986); Blatny et al (1997a,b); Brautaset et al, 1998;; Winther-Larsen et al, 2000a,b;Gimmestad et al (2003); Sletta et al (2004;2007 expression and for metabolic engineering studies. It should however be noticed that PprpB has CAPdependent activation and accordingly the carbon source used in the growth medium can severely affect background expression.…”

Section: Peptide Induced Expression Systemsmentioning

confidence: 99%

“…Relevant characteristics of this vector includes adjustable vector copy number (between 5 and 100 per chromosome), several cheap benzoic acid derivatives such as m-toluic acid can be used, they act in a dosedependent manner and inducer uptake is presumably passive (no transport system required) (Lambert and Stratford, 1999 and references therein) which further simplifies the use of the system across species barriers. pJB658 was used to control sugar metabolism in E. coli by fine-tuning the expression levels of a number of phosphoglucomutase mutant enzymes genes with different kinetic properties (Brautaset et al, 1998;. The same plasmid was used to control xanthan biosynthesis in a xanA-deficient X. campestris mutant strain by regulated low-level expression of the xanA gene, encoding a bifunctional phosphomannomutase/phosphoglucomutase (Winther-Larsen et al, 2000a).…”

Section: Broad-host-range Expression Systems Based On Xyls/pmmentioning

confidence: 99%

Positively regulated bacterial expression systems

Brautaset

Lale

Valla

2008

Microbial Biotechnology

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SummaryRegulated promoters are useful tools for many aspects related to recombinant gene expression in bacteria, including for high‐level expression of heterologous proteins and for expression at physiological levels in metabolic engineering applications. In general, it is common to express the genes of interest from an inducible promoter controlled either by a positive regulator or by a repressor protein. In this review, we discuss established and potentially useful positively regulated bacterial promoter systems, with a particular emphasis on those that are controlled by the AraC‐XylS family of transcriptional activators. The systems function in a wide range of microorganisms, including enterobacteria, soil bacteria, lactic bacteria and streptomycetes. The available systems that have been applied to express heterologous genes are regulated either by sugars (l‐arabinose, l‐rhamnose, xylose and sucrose), substituted benzenes, cyclohexanone‐related compounds, ε‐caprolactam, propionate, thiostrepton, alkanes or peptides. It is of applied interest that some of the inducers require the presence of transport systems, some are more prone than others to become metabolized by the host and some have been applied mainly in one or a limited number of species. Based on bioinformatics analyses, the AraC‐XylS family of regulators contains a large number of different members (currently over 300), but only a small fraction of these, the XylS/Pm, AraC/PBAD, RhaR‐RhaS/rhaBAD, NitR/PnitA and ChnR/Pb regulator/promoter systems, have so far been explored for biotechnological applications.

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“…Ray and coworkers have extensively characterized the mechanism of rabbit PGM (Ray et al 1989, 1990, 1991). Other members of the family have also been studied mechanistically to varying degrees, including PMM/PGM from P. aeruginosa (Naught and Tipton 2001; Naught et al 2003); PGM from maize (Manjunath et al 1998), wheat (Davies et al 2003), Arabidopsis (Periappuram et al 2000), and A. xylinum (Brautaset et al 1998, 2000); PNGM from Escherichia coli and P. aeruginosa (Jolly et al 1999, 2000; Tavares et al 2000); and PAGM from humans (Mio et al 2000).…”

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confidence: 99%

Evolutionary trace analysis of the α‐D‐phosphohexomutase superfamily

2004

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The ␣-D-phosphohexomutase superfamily is composed of four related enzymes that catalyze a reversible, intramolecular phosphoryl transfer on their sugar substrates. The enzymes in this superfamily play important and diverse roles in carbohydrate metabolism in organisms from bacteria to humans. Recent structural and mechanistic studies of one member of this superfamily, phosphomannomutase/phosphoglucomutase (PMM/ PGM) from Pseudomonas aeruginosa, have provided new insights into enzyme mechanism and substrate recognition. Here we use sequence-sequence and sequence-structure comparisons via evolutionary trace analysis to examine 71 members of the ␣-D-phosphohexomutase superfamily. These analyses show that key residues in the active site, including many of those involved in substrate contacts in the P. aeruginosa PMM/PGM complexes, are conserved throughout the enzyme family. Several important regions show class-specific differences in sequence that appear to be correlated with differences in substrate specificity exhibited by subgroups of the family. In addition, we describe the translocation of a 20-residue segment containing the catalytic phosphoserine of phosphoacetylglucosamine mutase, which uniquely identifies members of this subgroup.Keywords: phosphohexomutase; evolutionary trace; enzyme superfamily; carbohydrate metabolismThe ␣-D-phosphohexomutase enzyme superfamily is widespread and diverse. Two related and well-characterized proteins make up the majority of the family: the highly specific PGM, which only uses glucose as a substrate, and the less specific PMM/PGM, which can use either glucose or mannose. Two other enzymes, PNGM and PAGM, are also members of the superfamily, although to date these two proteins have been less extensively characterized. The ␣-Dphosphohexomutases play varied roles in carbohydrate metabolism and other biosynthetic pathways. PGM is best known for its role in providing substrates that enter the glycolytic pathway. The PMM/PGM proteins are primarily bacterial and participate in the biosynthesis of a variety of carbohydrates, such as lipopolysaccharide and alginate (Shankar et al. 1995;Rocchetta et al. 1999). PNGM and PAGM are involved in the biosynthesis of UDP-N-acetylglucosamine, which is an essential common precursor for bacterial cell wall components and is also required for the posttranslational N-acetylglucosamine modification of eukaryotic proteins (Jolly et al. 1999;Mio et al. 2000).Despite differences in substrate specificity, the enzymes in this superfamily that have been characterized to date appear to use the same mechanism. They catalyze the reversible conversion of 1-phospho to 6-phosphosugars via a bisphosphorylated sugar intermediate. Active enzyme is phosphorylated at a conserved serine residue and binds one Mg 2+ ion. The reaction mechanism entails two phosphoryl transfer reactions: first, from the enzyme to substrate, and second, from the reaction intermediate back to the enzyme. The initial phosphoryl transfer is from phosphoserine to bound substrate, cre...

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An experimental study on carbon flow in Escherichia coli as a function of kinetic properties and expression levels of the enzyme phosphoglucomutase

Cited by 7 publications

References 7 publications

Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens

Crystal structure of a bacterial phosphoglucomutase, an enzyme involved in the virulence of multiple human pathogens

Positively regulated bacterial expression systems

Evolutionary trace analysis of the α‐D‐phosphohexomutase superfamily

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