Biophysical Characterization of Type III Pantothenate Kinase (PanK) from Acinetobacter baumannii.
Acinetobacter baumannii
coaX
coenzyme A
multidrug resistance
pantothenate kinase.
pathogen
Journal
Protein and peptide letters
ISSN: 1875-5305
Titre abrégé: Protein Pept Lett
Pays: Netherlands
ID NLM: 9441434
Informations de publication
Date de publication:
2021
2021
Historique:
received:
20
04
2020
revised:
11
06
2020
accepted:
12
06
2020
pubmed:
18
8
2020
medline:
12
6
2021
entrez:
18
8
2020
Statut:
ppublish
Résumé
Type-III Pantothenate kinase from the multi drug resistant bacteria, Acinetobacter baumannii (AbPanK) catalyzes the first step of the essential Coenzyme A biosynthesis pathway. AbPanK is an attractive drug target against the bacteria since it is an essential enzyme and its structure is significantly different from the human PanK. AbPanK was cloned, expressed, purified and crystallized. A good quality single crystal was used for X-ray intensity data collection. Dynamic light scattering was done for calculating the hydrodynamic radii and its oligomeric nature in the solution. Binding studies of this protein with its two substrates, Pantothenate and ATP were done using spectrofluorometer. Our results indicated that AbPanK shows a strong affinity with pantothenate with dissociation constant of 1.2 x 10- 8 M and moderate affinity towards ATP of 3.7x 10-3 M. This fact was further substantiated by the calculations of Km of both substrates using kinase assay kit. Dynamic light scattering studies have shown that it exists as homogenous solution with hydrodynamic radii corresponding to the molecular weight of 29.55 kDa. A low-resolution X-ray intensity data set was collected, which shows that AbPank crystallizes in P2 space group with cell dimensions of a= 165 Å, b= 260 Å, and, c= 197 Å and α= 90.0, β= 113.60, γ= 90.0. Recombinant Pantothenate kinase from Acinetobacter baumannii was purified to homogeneity and crystallized. The enzyme exhibits very low sequence identity (28%) to other corresponding enzymes. The recombinant enzyme was active and its binding affinities with its substrates pantothenate and ATP have been studied. This information would be very useful while designing the inhibitors of this enzyme in order to fight bacterial infections associated to this pathogen.
Sections du résumé
BACKGROUND
BACKGROUND
Type-III Pantothenate kinase from the multi drug resistant bacteria, Acinetobacter baumannii (AbPanK) catalyzes the first step of the essential Coenzyme A biosynthesis pathway. AbPanK is an attractive drug target against the bacteria since it is an essential enzyme and its structure is significantly different from the human PanK.
METHODS
METHODS
AbPanK was cloned, expressed, purified and crystallized. A good quality single crystal was used for X-ray intensity data collection. Dynamic light scattering was done for calculating the hydrodynamic radii and its oligomeric nature in the solution. Binding studies of this protein with its two substrates, Pantothenate and ATP were done using spectrofluorometer.
RESULTS
RESULTS
Our results indicated that AbPanK shows a strong affinity with pantothenate with dissociation constant of 1.2 x 10- 8 M and moderate affinity towards ATP of 3.7x 10-3 M. This fact was further substantiated by the calculations of Km of both substrates using kinase assay kit. Dynamic light scattering studies have shown that it exists as homogenous solution with hydrodynamic radii corresponding to the molecular weight of 29.55 kDa. A low-resolution X-ray intensity data set was collected, which shows that AbPank crystallizes in P2 space group with cell dimensions of a= 165 Å, b= 260 Å, and, c= 197 Å and α= 90.0, β= 113.60, γ= 90.0.
DISCUSSION
CONCLUSIONS
Recombinant Pantothenate kinase from Acinetobacter baumannii was purified to homogeneity and crystallized. The enzyme exhibits very low sequence identity (28%) to other corresponding enzymes.
CONCLUSION
CONCLUSIONS
The recombinant enzyme was active and its binding affinities with its substrates pantothenate and ATP have been studied. This information would be very useful while designing the inhibitors of this enzyme in order to fight bacterial infections associated to this pathogen.
Identifiants
pubmed: 32798368
pii: PPL-EPUB-109105
doi: 10.2174/0929866527666200813202445
doi:
Substances chimiques
Bacterial Proteins
0
Pantothenic Acid
19F5HK2737
Adenosine Triphosphate
8L70Q75FXE
Phosphotransferases (Alcohol Group Acceptor)
EC 2.7.1.-
pantothenate kinase
EC 2.7.1.33
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
450-458Informations de copyright
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