Essential Role of Residue H49 for Activity of Escherichia coli 1-Deoxy--xylulose 5-Phosphate Synthase, the Enzyme Catalyzing the First Step of the 2-C-Methyl--erythritol 4-Phosphate Pathway for Isoprenoid Synthesis

Querol, Jordi; Rodríguez‐Concepción, Manuel; Boronat, Albert; Imperial, Santıago

doi:10.1006/bbrc.2001.5957

Cited by 36 publications

(25 citation statements)

References 32 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We previously reported that mutation of residue H49 by glutamine in E. coli DXS resulted in a mutant enzyme with undetectable activity [15]. Here we have analyzed the effect of H49Q and H49A point mutations by measuring their kinetic parameters k cat and k cat /K M .…”

Section: Kinetic Studiesmentioning

confidence: 98%

“…The coding region of the E. coli DXS, cloned into the expression vector pT7-7 [15], was amplified by PCR using Pfu DNA polymerase and primers T7 (5'-TAATACGACTCACTATAGG-3') and pET-23-XhoI (5'-CGCTCGAGTCCTGCCAGCCAGGCCTTGATTTTGGC-3'). After digestion with NdeI and XhoI, the amplified DNA fragment was ligated into the same sites of the expression vector pET-23(b).…”

Section: Construction Of the E Coli Dxs Expression Vector Pet-23-dxsmentioning

confidence: 99%

“…Point mutations H49Q, H49A, E370Q, E370A, D427N, D427A, H431Q and H431A were introduced into the E. coli DXS coding sequence by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) as described [15]. Primers used for introducing these point mutations are summarized in Table 1.…”

Section: Site-directed Mutagenesismentioning

confidence: 99%

See 2 more Smart Citations

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

Querol-Audí¹,

Boronat²,

Centelles³

et al. 2014

JBM

Self Cite

View full text Add to dashboard Cite

1-deoxy-D-xylulose 5-phosphate synthase (DXS) catalyzes the initial step of the 2-C-methyl-Derythritol 4-phosphate (MEP)pathway consisting in the condensation of (hydroxiethyl)thiamin derived from pyruvate with D-glyceraldehyde 3-phosphate (GAP) to yield 1-deoxy-D-xylulose 5-phosphate (DXP). The role of the conserved residues H49, E370, D427 and H431 of E. coli DXS was examined by site-directed mutagenesis and kinetic analysis of the purified recombinant enzyme mutants. Mutants at position H49 showed a severe reduction in their specific activities with a decrease of the kcat/KM ratio by two orders of magnitude lower than the wild-type DXS. According to available structural data residue H49 is perfectly positioned to abstract a proton from the donor substrate. Mutations in DXS E370 showed that this residue is also essential for catalytic activity. Three-dimensional structure supports its involvement in cofactor deprotonation, the first step in enzymatic thiamin catalysis. Results obtained with H431 mutant enzymes indicate that this residue plays a role contributing to transition state stabilization. Finally, mutants at position D427 also showed a severe specific activity decrease with a reduction of the kcat/KM ratio. A role in binding the substrate and selecting the stereoisomer is proposed for D427.

show abstract

Section: Kinetic Studiesmentioning

confidence: 98%

Section: Construction Of the E Coli Dxs Expression Vector Pet-23-dxsmentioning

confidence: 99%

See 1 more Smart Citation

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

Querol-Audí¹,

Boronat²,

Centelles³

et al. 2014

JBM

Self Cite

View full text Add to dashboard Cite

show abstract

“…3), it is not known whether they are functional DXS enzymes with a role in the MEP pathway. The deduced mature proteins lack stretches of amino acids that are present in all the bacterial and plant DXS enzymes (Lois et al, 1998, and a conserved His residue essential for DXS activity (Querol et al, 2001) is not present in the DXS3 protein (Fig. 3).…”

Section: Plant Genes and Enzymesmentioning

confidence: 99%

Elucidation of the Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis in Bacteria and Plastids. A Metabolic Milestone Achieved through Genomics

2002

Self Cite

View full text Add to dashboard Cite

“…1A). Also, as a positive control for essentiality, a pSC200 derivative was used to target the conserved essential gene dxs encoding a key enzyme for the synthesis of undecaprenyl phosphate (2,28), resulting in strain STC280.…”

mentioning

confidence: 99%

A Putative Gene Cluster for Aminoarabinose Biosynthesis Is Essential for Burkholderia cenocepacia Viability

Ortega¹,

et al. 2007

View full text Add to dashboard Cite

Using a conditional mutagenesis strategy we demonstrate here that a gene cluster encoding putative aminoarabinose (Ara4N) biosynthesis enzymes is essential for the viability of Burkholderia cenocepacia. Loss of viability is associated with dramatic changes in bacterial cell morphology and ultrastructure, increased permeability to propidium iodide, and sensitivity to sodium dodecyl sulfate, suggesting a general cell envelope defect caused by the lack of Ara4N.

show abstract

Essential Role of Residue H49 for Activity of Escherichia coli 1-Deoxy--xylulose 5-Phosphate Synthase, the Enzyme Catalyzing the First Step of the 2-C-Methyl--erythritol 4-Phosphate Pathway for Isoprenoid Synthesis

Cited by 36 publications

References 32 publications

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

Catalytically Important Residues in E. coli 1-Deoxy-D-Xylulose 5-Phosphate Synthase

Elucidation of the Methylerythritol Phosphate Pathway for Isoprenoid Biosynthesis in Bacteria and Plastids. A Metabolic Milestone Achieved through Genomics

A Putative Gene Cluster for Aminoarabinose Biosynthesis Is Essential for Burkholderia cenocepacia Viability

Contact Info

Product

Resources

About