Biochemical and structural characterization of tyrosine aminotransferase suggests broad substrate specificity and a two-state folding mechanism in Leishmania donovani

FEBS Open Bio
Santanu Sasidharan, Prakash Saudagar

Abstract

Tyrosine aminotransferase (TAT) is an aminotransferase with broad substrate specificity that catalyzes the transamination of aromatic amino acids in Leishmania donovani and plays a crucial role in the survival and pathogenicity of the parasite. In this study, we have biochemically characterized tyrosine aminotransferase from Leishmania donovani using in vitro and in silico techniques. Leishmania donovani tyrosine aminotransferase (LdTAT) was cloned into the pET28a(+) vector and expressed in the BL21 strain of Escherichia coli. The Ni-NTA-purified protein was then characterized biochemically, and its various kinetic parameters were investigated. The apparent Km value for the tyrosine-pyruvate pair was determined to be 3.5 ± 0.9 mm, and Vmax was analyzed to be at 11.7 ± 1.5 μm·min.μg-1 . LdTAT was found to exhibit maximum activity at 50 °C and at a pH of 8.0. Cofactor identification for LdTAT showed that pyridoxal-5-phosphate (PLP) binds with a Km value of 23.59 ± 3.99 μm and that the phosphate group is vital for the activity of the enzyme. Sequence analysis revealed that S151, Y256, K286, and P291 are conserved residues and form hydrogen bonds with PLP. Urea-based denaturation studies revealed a biphasic folding mechanism involv...Continue Reading

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Datasets Mentioned

BETA
MK426678
13386702
PM0081305

Methods Mentioned

BETA
PCR
Fluorescence

Software Mentioned

UCSF Chimera
Xmgrace
GROMACS
autodock
SASA
figtree
Modeller
gromacs package
RAMPAGE
ACPYPE

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