Nucleotide triphosphate promiscuity in Mycobacterium tuberculosis dethiobiotin synthetase

Abstract not availableWanisa Salaemae, Min Y. Yap, Kate L. Wegener, Grant W. Booker, Matthew C.J. Wilce, Steven W. Polya

Enzymatic Analysis of Recombinant Japanese Encephalitis Virus NS2B(H)-NS3pro Protease with Fluorogenic Model Peptide Substrates

Background Japanese encephalitis virus (JEV), a member of the Flaviviridae family, causes around 68,000 encephalitis cases annually, of which 20–30% are fatal, while 30–50% of the recovered cases develop severe neurological sequelae. Specific antivirals for JEV would be of great importance, particularly in those cases where the infection has become persistent. Being indispensable for flaviviral replication, the NS2B-NS3 protease is a promising target for design of anti-flaviviral inhibitors. Contrary to related flaviviral proteases, the JEV NS2B-NS3 protease is structurally and mechanistically much less characterized. Here we aimed at establishing a straightforward procedure for cloning, expression, purification and biochemical characterization of JEV NS2B(H)-NS3pro protease. Methodology/Principal Findings The full-length sequence of JEV NS2B-NS3 genotype III strain JaOArS 982 was obtained as a synthetic gene. The sequence of NS2B(H)-NS3pro was generated by splicing by overlap extension PCR (SOE-PCR) and cloned into the pTrcHisA vector. Hexahistidine-tagged NS2B(H)-NS3pro, expressed in E. coli as soluble protein, was purified to >95% purity by a single-step immobilized metal affinity chromatography. SDS-PAGE and immunoblotting of the purified enzyme demonstrated NS2B(H)-NS3pro precursor and its autocleavage products, NS3pro and NS2B(H), as 36, 21, and 10 kDa bands, respectively. Kinetic parameters, Km and kcat, for fluorogenic protease model substrates, Boc-GRR-amc, Boc-LRR-amc, Ac-nKRR-amc, Bz-nKRR-amc, Pyr-RTKR-amc and Abz-(R)4SAG-nY-amide, were obtained using inner filter effect correction. The highest catalytic efficiency kcat/Km was found for Pyr-RTKR-amc (kcat/Km: 1962.96±85.0 M−1 s−1) and the lowest for Boc-LRR-amc (kcat/Km: 3.74±0.3 M−1 s−1). JEV NS3pro is inhibited by aprotinin but to a lesser extent than DEN and WNV NS3pro. Conclusions/Significance A simplified procedure for the cloning, overexpression and purification of the NS2B(H)-NS3pro was established which is generally applicable to other flaviviral proteases. Kinetic parameters obtained for a number of model substrates and inhibitors, are useful for the characterization of substrate specificity and eventually for the design of high-throughput assays aimed at antiviral inhibitor discovery

The role of biotin in bacterial physiology and virulence: A novel antibiotic target for Mycobacterium tuberculosis

Tuberculosis (TB) is a global pandemic that ranks alongside HIV-AIDS and malaria as the leading cause of death by infectious disease, with the highest incidence rates observed in Southeast Asian, African, and Western Pacific countries ( 1 ). In 1993 the WHO declared TB to be a global health emergency and set the Millennium Development Goal of reducing the prevalence and mortality rates to 50% of those observed in 1990 by the 2015 deadline ( 2 ). Although the rates of new TB cases and mortality have declined over the past decade and are within reach of the 2015 target, the number of TB patients and the prevalence of drug-resistant strains are rising ( 3 ). Multidrug-resistant TB (MDR-TB) must be addressed now as a public health crisis to achieve the ambitious Millennium Development Goal target of complete elimination of TB as a public health concern by 2050 ( 4 ).Wanisa Salaemae, Grant W. Booker, and Steven W. Polya

Mycobacterium tuberculosis Dethiobiotin Synthetase Facilitates Nucleoside Triphosphate Promiscuity through Alternate Binding Modes

The penultimate step in the biosynthesis of biotin is the closure of the ureido heterocycle in a reaction requiring a nucleoside triphosphate (NTP). In Mycobacterium tuberculosis this reaction is catalyzed by dethiobiotin synthetase (MtDTBS). MtDTBS is unusual as it can employ multiple (NTPs), with a >100-fold preference for cytidine triphosphate (CTP). Here the molecular basis of NTP binding was investigated using a surface plasmon resonance-based ligand binding assay and X-ray crystallography. The biophysical and structural data revealed two discrete mechanisms by which MtDTBS binds NTPs: (i) A high affinity binding mode employed by CTP (KD 160 nM) that is characterized by a slow dissociation rate between enzyme and ligand (kd 5.3 × 10–2 s–1) and that is defined by an extended network of specific ligand–protein interactions involving both the cytidine and triphosphate moieties and (ii) a low affinity mode employed by the remaining NTPs (KD > 16.5 μM), that is characterized by weak interactions between protein and ligand. Previously intractable structures of MtDTBS in complex with ATP, GTP, UTP, and ITP were obtained to define the molecular basis of the low affinity ligand binding. Anchoring of the triphosphate moiety into the phosphate binding loop of MtDTBS allows the promiscuous utilization of multiple NTPs. Both high and low binding mechanisms showed conserved hydrogen bonding interactions involving the β-phosphate of NTPs and a high-affinity anion binding site within the phosphate binding loop. This study provides insights into enzymes that can likewise utilize multiple NTPs.Andrew P. Thompson, Wanisa Salaemae, Jordan L. Pederick, Andrew D. Abell, Grant W. Booker, John B. Bruning, Kate L. Wegener and Steven W. Polya

Inhibition of Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS): toward next-generation antituberculosis agents

Mycobacterium tuberculosis dethiobiotin synthase (MtDTBS) is a crucial enzyme involved in the biosynthesis of biotin in the causative agent of tuberculosis, M. tuberculosis. Here, we report a binder of MtDTBS, cyclopentylacetic acid 2 (K(D) = 3.4 ± 0.4 mM), identified viain silico screening. X-ray crystallography showed that 2 binds in the 7,8-diaminopelargonic acid (DAPA) pocket of MtDTBS. Appending an acidic group to the para-position of the aromatic ring of the scaffold revealed compounds 4c and 4d as more potent binders, with K(D) = 19 ± 5 and 17 ± 1 μM, respectively. Further optimization identified tetrazole 7a as a particularly potent binder (K(D) = 57 ± 5 nM) and inhibitor (Ki = 5 ± 1 μM) of MtDTBS. Our findings highlight the first reported inhibitors of MtDTBS and serve as a platform for the further development of potent inhibitors and novel therapeutics for the treatment of tuberculosis.Nicholas C. Schumann, Kwang Jun Lee, Andrew P. Thompson, Wanisa Salaemae, Jordan L. Pederick, Thomas Avery, Birgit I. Gaiser, James Hodgkinson-Bean, Grant W. Booker, Steven W. Polyak, John B. Bruning, Kate L. Wegener, and Andrew D. Abel