Analysis of Three Overexpression Systems for VanX, the Zinc(II) Dipeptidase Required for High-Level Vancomycin Resistance in Bacteria

Brandt, Jeffrey J.; Chatwood, Lisa L.; Crowder, Michael W.

doi:10.1006/prep.2000.1303

Cited by 4 publications

(3 citation statements)

References 25 publications

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“…The crystal structure of VanX has been shown to be a hexamer 6 . Although Brandt et al 30 judged that VanX assumed a dimeric form based on MALDI-TOF mass spectrometry, their spectrum also exhibited peaks corresponding to monomeric and tetrameric species, albeit at low levels. Wu et al 14 estimated the molecular mass of VanX to be 38 kDa using gel filtration chromatography.…”

Section: Discussionmentioning

confidence: 99%

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

Konuma,

Takai,

Tsuchiya

et al. 2023

Preprint

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Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin-resistant enterococci was a tremendous shock considering that vancomycin is the last resort for controlling methicillin-resistantStaphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, which is typically considered too large for NMR studies, we successfully assigned the main chain using an amino acid-selective unlabeling method. Because we found that the Zn2+-coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by mutating the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

show abstract

Section: Discussionmentioning

confidence: 99%

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

Konuma,

Takai,

Tsuchiya

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…The crystal structure of VanX has been shown to be a hexamer (Bussiere et al, 1998). Although Brandt et al (2000) judged that VanX assumed a dimeric form based on matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry, their spectrum also exhibited peaks corresponding to monomeric and tetrameric species, albeit at low levels. Wu et al (1995) estimated the molecular mass of VanX to be 38 kDa using gel‐filtration chromatography.…”

Section: Discussionmentioning

confidence: 99%

“…The crystal structure of VanX has been shown to be a hexamer (Bussiere et al, 1998). Although Brandt et al (2000) judged that VanX assumed a dimeric form based on matrix-assisted laser desorption/ionization timeof-flight mass spectrometry, their spectrum also exhibited F I G U R E 6 Comparison between the best structure, determined by High Ambiguity Driven protein-protein DOCKing (cyan), and the B/C subunits in the crystal structure (1r44) (pink).…”

Section: Vanx As a Homodimermentioning

confidence: 99%

Analysis of the homodimeric structure of a D‐Ala‐D‐Ala metallopeptidase, VanX, from vancomycin‐resistant bacteria

Konuma,

Takai,

Tsuchiya

et al. 2024

Protein Science

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Bacteria that have acquired resistance to most antibiotics, particularly those causing nosocomial infections, create serious problems. Among these, the emergence of vancomycin‐resistant enterococci was a tremendous shock, considering that vancomycin is the last resort for controlling methicillin‐resistant Staphylococcus aureus. Therefore, there is an urgent need to develop an inhibitor of VanX, a protein involved in vancomycin resistance. Although the crystal structure of VanX has been resolved, its asymmetric unit contains six molecules aligned in a row. We have developed a structural model of VanX as a stable dimer in solution, primarily utilizing nuclear magnetic resonance (NMR) residual dipolar coupling. Despite the 46 kDa molecular mass of the dimer, the analyses, which are typically not as straightforward as those of small proteins around 10 kDa, were successfully conducted. We assigned the main chain using an amino acid‐selective unlabeling method. Because we found that the zinc ion‐coordinating active sites in the dimer structure were situated in the opposite direction to the dimer interface, we generated an active monomer by replacing an amino acid at the dimer interface. The monomer consists of only 202 amino acids and is expected to be used in future studies to screen and improve inhibitors using NMR.

show abstract

VanX d-,d-dipeptidase

Lessard

2004

Handbook of Proteolytic Enzymes

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Analysis of Three Overexpression Systems for VanX, the Zinc(II) Dipeptidase Required for High-Level Vancomycin Resistance in Bacteria

Cited by 4 publications

References 25 publications

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

NMR analysis of the homodimeric structure of a D-Ala-D-Ala metallopeptidase, VanX, from vancomycin-resistant bacteria

Analysis of the homodimeric structure of a D‐Ala‐D‐Ala metallopeptidase, VanX, from vancomycin‐resistant bacteria

VanX d-,d-dipeptidase

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