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abstracts
Phosphopantetheinyl transferases (PPTases) are essential enzymes that catalyze covalent attachment of the 4'-phosphopantetheine (4'-PP) moiety from coenzyme A (CoA) to a conserved serine residue on acyl (ACP) and peptidyl carrier proteins (PCP) [1]. This post-translational modification converts the inactive apo-carrier proteins to the functional form, shuttling the intermediates of biosynthetic reactions catalyzed by fatty acid synthases (FAS), polyketide synthases (PKS) and non-ribosomal peptide synthetases (NRPSs). In Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), two PPTases, AcpS (type-I) and PptT (type-II), are involved in the biosynthesis of essential lipids, virulence factors and siderophores, activating over 20 target proteins [2]. These two proteins have been shown to be independently essential, suggesting that PPTases could be targeted against tuberculosis [2, 3]. We have expressed the Mtb-PptT protein as a fusion protein with maltose binding protein (MBP). The use of the MBP-PptT fusion protein overcame stability and solubility problems and resulted in successful crystallization. The structure of Mtb-PptT in complex with CoA was determined from the crystal of the fusion protein, solved at 1.75 Å resolution. Excellent electron density is present for all parts of the CoA molecule, revealing a conserved CoA-binding mode. Conformational and charge distribution differences in the putative ACP binding cleft, however, suggest a different mode of ACP binding compared to other homologues. This is the first and only three-dimensional structure of a type-II PPTase from pathogenic bacteria, providing structural features that can be exploited in drug development when compared with its human counterpart.
abstracts
The proline utilization pathway in Mycobacterium tuberculosis (Mtb) has been recently identified as an important factor in Mtb persistence in vivo, suggesting that this pathway could be a valuable therapeutic target against tuberculosis (TB). In Mtb, two distinct enzymes perform the conversion of proline into glutamate; the first step is the oxidation of proline into Δ1-pyrroline-5-carboxylic acid (P5C) by the flavoenzyme proline dehydrogenase (PruB) and the second reaction involves converting the tautomeric form of P5C (glutamate-γ-semialdehyde) into glutamate using the NAD+-dependent Δ1-pyrroline-5-carboxylic dehydrogenase (PruA). Here we describe three-dimensional structures of Mtb-PruA, determined by X-ray crystallography both in its apo state and in complex with NAD+ at 2.5 and 2.1 Å resolution, respectively. The structure reveals a conserved NAD+ binding mode, common to other related enzymes. Conformational differences in the active site, however, linked to changes in the dimer interface, suggest possibilities for selective inhibition of Mtb-PruA despite reasonably high sequence identity with other PruA enzymes. Using recombinant PruA and PruB, the proline utilization pathway in Mtb has also been reconstituted in vitro. Functional validation using a novel NMR approach has demonstrated that the PruA and PruB enzymes are together sufficient to convert proline to glutamate, the first such demonstration for monofunctional proline utilization enzymes.
