The biosynthetic pathway for the production of branched amino acids, i.e.
valine, leucine and isoleucine, is unique to bacteria, fungi, algae and plants. This makes the key enzymes of this pathway very attractive targets for the design and development of herbicides, fungicides and antibiotics. One such enzyme, acetohydroxylate synthase (AHAS), catalyzes the first step in the biosynthesis of all branched amino acids (Friden et al.
; Jackson & Henderson, 1975
AHAS holoenzymes are assembled from large catalytic subunits and small regulatory subunits. In many enterobacteria there are three AHAS enzymes: AHAS I, II and III. The three AHAS isozymes in Escherichia coli are named IlvB, IlvG and IlvI, respectively, for the large subunits, and IlvN, IlvM and IlvH, respectively, for the corresponding small subunits. Although such genetic redundancy/variety is not always observed in other bacteria, AHAS III homologues exist ubiquitously among bacteria, especially in genomes encoding a single acetohydroxylate synthase (Fig. 1).
Figure 1 Sequence alignment of Mtb IlvH and its homologues from E. coli, Nitrosomonas europaea and Thermotoga maritima. The secondary structures corresponding to N. europaea IlvH (PDB entry 2pc6; Petkowski et al., 2007 ) are drawn at the top of the sequence (more ...)
In the absence of the small subunits, the large catalytic subunits of AHAS enzymes exhibit only partial activity in vitro
and are insensitive to product feedback inhibition (Vyazmensky et al.
). In the presence of their cognate small subunits, however, the holoenzymes gain full catalytic activity that is negatively regulated by the product valine (IlvB and IlvI). Interestingly, AHAS II, i.e.
the complex of IlvG and IlvM, is resistant to inhibition by valine.
The crystal structure of E. coli
IlvH shows two domains, each with a canonical βαββαβ ferrodoxin fold, connected by a short linker (Kaplun et al.
). The N-terminal domain is arranged at almost a right angle relative to the C-terminal domain, thereby creating an overall L-shaped structure. A higher-ordered structure of IlvH is also evident in the crystal: there are two IlvH homodimers in the asymmetric unit. In the IlvH dimer, the N-terminal domains of two IlvH molecules are juxtaposed with each other in an antiparallel fashion, with their β-sheets forming a contiguous and larger sheet. The two C-terminal domains are packed back-to-back against each other and are perpendicular to the extended β-sheet of the two N-terminal domains. Several lines of evidence lend support to the biological significance of the dimeric assembly of IlvH. Firstly, the dimerization interface is very large (4322 Å2
) and involves many highly conserved residues. Secondly, the IlvH dimer is structurally close to other members of the ACT-domain family, e.g.
3-phosphoglycerate dehydrogenase (3PGDH), for which the dimerization of functional domains is well known (Schuller et al.
; Dey et al.
). Thirdly, the valine-binding site has been predicted to lie between the two N-terminal domains in the IlvH dimer based on the interactions observed between 3PGDH and its feedback inhibitor l
-serine (Dey et al.
). Analysis of IlvH variants resulting from mutagenesis of the residues in the predicted valine-binding site seems to support this hypothesis (Jackson & Henderson, 1975
In the present study, we report the molecular cloning, protein purification and preliminary X-ray crystallographic analysis of Rv3002c from Mycobacterium tuberculosis
). The open reading frame of Rv3002c encodes the small regulatory subunit (IlvH) of Mtb
AHAS. There are presently two large subunits (Rv3003c and Rv3470c) and only one small regulatory subunit (Rv3002c) annotated in the sequenced Mtb
genome (TB Structural Genomics Consortium; http://www.webtb.org/
). Based on the identical orientation and close proximity of the Rv3002c and Rv3003c coding sequences in the Mtb
genome, as well as their similarity to E. coli
AHAS III, it is likely that Rv3002c is the small subunit that regulates the catalytic activity of Rv3003c. On the other hand, no known or predicted small regulatory subunit seems to exist for Rv3470. Interestingly, it has been shown that IlvM and some C-terminally truncated forms of IlvH are capable of cross-activating noncognate E. coli
isozymes in vitro
. However, these heterologously assembled AHAS holoenzymes are not sensitive to product feedback inhibition (Vyazmensky et al.
). It would be interesting to find out whether Mtb
IlvH can activate both Rv3470c and Rv3003c and/or confer product feedback regulation on their catalysis. Because the branched amino-acid biosynthetic pathway is absent in humans, the key enzymes of this pathway are potential targets for the design of novel antituberculosis therapies.