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August 4, 2004 Crystal Structure of LeuA from Mycobacterium tuberculosis, a Key Enzyme in Leucine BiosynthesisN. Koon1,2, C.J. Squire1,2, and E.N. Baker1,2,3 The leucine biosynthetic pathway is essential for the growth of Mycobacterium tuberculosis, and is a potential target for the design of new anti-tuberculosis drugs. We have used multiwavelength anomalous diffraction (MAD) phasing to determine the high-resolution crystal structure of α-isopropylmalate synthase, the product of the leuA gene. The structure reveals a tightly associated dimer comprised of an (α/β)8 barrel catalytic domain joined by flexibly-hinged linker domains to a C-terminal regulatory domain. The structure shows how the substrate, the leucine precursor -ketoisovalerate, binds to an essential zinc atom in the catalytic domain, and a potential mechanism of feedback inhibition by leucine binding in the regulatory domain.
The biosynthetic pathways for the branched-chain amino acids leucine, isoleucine and valine (i.e. the series of enzyme reactions that produce these amino acids) are essential for the survival of the TB organism. These pathways are not present in humans, indicating that inhibitors targeted against enzymes from them could be useful as anti-TB drugs. We chose the enzyme α-isopropylmalate synthase (α-IPMS), the product of the leuA gene, for structural analysis. This enzyme catalyzes the first step in leucine biosynthesis, in which an acetyl group is transferred from acetyl-coenzymeA to the substrate α-ketoisovalerate (α-KIV), the leucine precursor. The enzyme was known to be dimeric (2 x 70 kDa), metal ion-dependent, and subject to feedback inhibition by the end product leucine, but its three-dimensional structure was unknown.
The α-IPMS monomer is folded into three domains (Figure 1), an (α/β)8 barrel catalytic domain, a helical linker domain and a C-terminal domain of novel fold. Two α-IPMS monomers form a domain-swapped dimer in which the linker domain of one monomer sits over the catalytic domain of the other. This domain swapping makes the active site almost completely enclosed, except for a tunnel that allows the entry of substrates. In the crystal structure, the substrate α-KIV is bound to the zinc (Figure 2A), and although acetyl-CoA is not present, two water molecules mark where the acetyl group would sit, adjacent to α-KIV. With this information we can propose a catalytic mechanism for α-IPMS, based on that of malate synthase, an enzyme that is evolutionarily unrelated but carries out a very similar reaction on a very similar substrate.
Several features make the α-IPMS structure suitable for drug development: the zinc ion as a template for inhibitor binding and surrounding invariant side chains inside the enclosed active site cavity. The structure also reveals an intriguing mechanism for feedback inhibition. Leucine binds in the regulatory C-terminal domain (Figure 2B) 70 Å away from the nearest active site, but this binding must be signalled via the flexible linker domain, which inserts two key residues into the active site. BEAMLINE FUNDING PUBLICATION FOR MORE INFORMATION |
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Tuberculosis (TB) is responsible for more deaths worldwide than
any other infectious disease – more than two million each year, with
an estimated one third of the world’s population infected. The
problem is compounded by the length of current treatments and by the
ability of Mycobacterium tuberculosis, the causative agent, to
survive in a dormant state inside activated macrophages in the lung
for long periods of time, emerging many years later as active
infections. Multi-drug resistance is also a growing problem. The
genome sequence of M. tuberculosis, completed in 1998, has
stimulated new approaches, including the formation of the TB
Structural Genomics Consortium (
The structure was solved using multiwavelength anomalous
diffraction (MAD) data from a selenomethionine-substituted α-IPMS
crystal, which was frozen and sent from New Zealand to beamline X8C
(Fedex crystallography!) for data collection. The data quality was
such that 70% of the 1288 residues in the dimer could be immediately
traced and autobuilt. The structure was then completed and refined
at 2.0 Å resolution. A spherical peak for a bound metal ion, marking
the active site of each monomer, was identified by a fluorescence
scan as zinc. We also subsequently prepared complexes with
α-KIV
(substrate) and leucine (feedback inhibitor). 