Molecular basis for the inhibition of β-hydroxyacyl-ACP dehydratase HadAB complex from Mycobacterium tuberculosis by flavonoid inhibitors

Dehydration is one of the key steps in the biosynthesis of mycolic acids and is vital to the growth of Mycobacterium tuberculosis (Mtb). Consequently, stalling dehydration cures tuberculosis (TB). Clinically used anti-TB drugs like thiacetazone (TAC) and isoxyl (ISO) as well as flavonoids inhibit the enzyme activity of the β-hydroxyacyl-ACP dehydratase HadAB complex. How this inhibition is exerted, has remained an enigma for years. Here, we describe the first crystal structures of the MtbHadAB complex bound with flavonoid inhibitor butein, 2’,4,4’-trihydroxychalcone or fisetin. Despite sharing no sequence identity from Blast, HadA and HadB adopt a very similar hotdog fold. HadA forms a tight dimer with HadB in which the proteins are sitting side-by-side, but are oriented anti-parallel. While HadB contributes the catalytically critical His-Asp dyad, HadA binds the fatty acid substrate in a long channel. The atypical double hotdog fold with a single active site formed by MtbHadAB gives rise to a long, narrow cavity that vertically traverses the fatty acid binding channel. At the base of this cavity lies Cys61, which upon mutation to Ser confers drug-resistance in TB patients. We show that inhibitors bind in this cavity and protrude into the substrate binding channel. Thus, inhibitors of MtbHadAB exert their effect by occluding substrate from the active site. The unveiling of this mechanism of inhibition paves the way for accelerating development of next generation of anti-TB drugs.     

Mrakia terrae sp. nov. and Mrakia soli sp. nov., Two Novel Basidiomycetous Yeast Species Isolated from Soil in Korea

Three strains, YP416^T, YP421^T, and Y422, were isolated from soil samples in Pocheon City, Gyeonggi province, South Korea. The strains belong to two novel yeast species in the genus Mrakia. Molecular phylogenetic analysis showed that the strain YP416^T was closely related to Mrakia niccombsii. Still, it differed by 9 nucleotide substitutions with no gap (1.51%) in the D1/D2 domain of the LSU rRNA gene and 14 nucleotide substitutions with 7 gaps (2.36%) in the ITS region. The strain YP421^T differed from the type strain of the most closely related species, Mrakia aquatica, by 5 nucleotide substitutions with no gap (0.81%) in the D1/D2 domain of the LSU rRNA gene and 9 nucleotide substitutions with one gap (1.43%) in the ITS region. The names Mrakia terrae sp. nov. and Mrakia soli sp. nov. are proposed, with type strains YP416^T (KCTC 27886^T) and YP421^T (KCTC 27890^T), respectively. MycoBank numbers of the strains YP416^T and YP421^T are MB 836844 and MB 836847, respectively.     

A Study on Spin-Labelled Oligonucleotide Synthesis and its Electron Spin Resonance Behavior in Solution

An oligonucleotide spin-labelled with 4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl (4-amino-TEMPO) at the internucleotide bond (d-Tp(L)TpTpTpT) prepared by oxidation of the pentanucleotide containing the H-phosphonate diester (d-Tp(H)TpTpTpT) in the presence of 4-amino-TEMPO, was separated and identified by high-performance, reverse-phase liquid chromatography combined with detection by electron spin resonance spectroscopy. This spin-labelled oligonucleotide produced a triplet with the slightly broadened M₁ = — I ESR component, while a triplet with almost equal intensities was obtained from the spin-label. The M₁ = —1 component from the labelled oligonucleotide was further broadened in the presence of poly(A) which forms a complementary double strand with this molecule.     

Molecular dynamics simulations of Hsp40 J-domain mutants identifies disruption of the critical HPD-motif as the key factor for impaired curing in vivo of the yeast prion [URE3]

Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.