3 research outputs found
Diversity in Functional Organization of Class I and Class II Biotin Protein Ligase
The cell envelope of Mycobacterium tuberculosis
(M.tuberculosis) is composed of a variety of lipids
including mycolic acids, sulpholipids, lipoarabinomannans, etc., which impart
rigidity crucial for its survival and pathogenesis. Acyl CoA carboxylase (ACC)
provides malonyl-CoA and methylmalonyl-CoA, committed precursors for fatty acid
and essential for mycolic acid synthesis respectively. Biotin Protein Ligase
(BPL/BirA) activates apo-biotin carboxyl carrier protein (BCCP) by biotinylating
it to an active holo-BCCP. A minimal peptide (Schatz), an efficient substrate
for Escherichia coli BirA, failed to serve as substrate for
M. tuberculosis Biotin Protein Ligase
(MtBPL). MtBPL specifically biotinylates
homologous BCCP domain, MtBCCP87, but not
EcBCCP87. This is a unique feature of
MtBPL as EcBirA lacks such a stringent
substrate specificity. This feature is also reflected in the lack of
self/promiscuous biotinylation by MtBPL. The N-terminus/HTH
domain of EcBirA has the self-biotinable lysine residue that is
inhibited in the presence of Schatz peptide, a peptide designed to act as a
universal acceptor for EcBirA. This suggests that when biotin
is limiting, EcBirA preferentially catalyzes, biotinylation of
BCCP over self-biotinylation. R118G mutant of EcBirA showed
enhanced self and promiscuous biotinylation but its homologue, R69A
MtBPL did not exhibit these properties. The catalytic
domain of MtBPL was characterized further by limited
proteolysis. Holo-MtBPL is protected from proteolysis by
biotinyl-5′ AMP, an intermediate of MtBPL catalyzed
reaction. In contrast, apo-MtBPL is completely digested by
trypsin within 20 min of co-incubation. Substrate selectivity and inability to
promote self biotinylation are exquisite features of MtBPL and
are a consequence of the unique molecular mechanism of an enzyme adapted for the
high turnover of fatty acid biosynthesis
The Presence of the Iron-Sulfur Motif Is Important for the Conformational Stability of the Antiviral Protein, Viperin
Viperin, an antiviral protein, has been shown to contain a CX3CX2C motif, which is conserved in the radical S-adenosyl-methionine (SAM) enzyme family. A triple mutant which replaces these three cysteines with alanines has been shown to have severe deficiency in antiviral activity. Since the crystal structure of Viperin is not available, we have used a combination of computational methods including multi-template homology modeling and molecular dynamics simulation to develop a low-resolution predicted structure. The results show that Viperin is an α -β protein containing iron-sulfur cluster at the center pocket. The calculations suggest that the removal of iron-sulfur cluster would lead to collapse of the protein tertiary structure. To verify these predictions, we have prepared, expressed and purified four mutant proteins. In three mutants individual cysteine residues were replaced by alanine residues while in the fourth all the cysteines were replaced by alanines. Conformational analyses using circular dichroism and steady state fluorescence spectroscopy indicate that the mutant proteins are partially unfolded, conformationally unstable and aggregation prone. The lack of conformational stability of the mutant proteins may have direct relevance to the absence of their antiviral activity