Title:Biophysical Characterization of Type III Pantothenate Kinase (PanK) from Acinetobacter baumannii
Volume: 28
Issue: 4
Author(s): Ankita Singla, Pradeep Sharma, Akshita Gupta, Naseer Iqbal , Chitra Rani, T.P. Singh and Sujata Sharma*
Affiliation:
- Department of Biophysics, All India Institute of Medical Sciences, New Delhi-110029,India
Keywords:
Acinetobacter baumannii, pathogen, multidrug resistance, coaX, coenzyme A, pantothenate kinase.
Abstract: 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: 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: 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: 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: 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.
