Feifei Ren

Multitarget Drug Discovery for Tuberculosis and Other Infectious Diseases

We report the discovery of a series of new drug leads that have potent activity against Mycobacterium tuberculosis as well as against other bacteria, fungi, and a malaria parasite. The compounds are analogues of the new tuberculosis (TB) drug SQ109 (1), which has been reported to act by inhibiting a transporter called MmpL3, involved in cell wall biosynthesis. We show that 1 and the new compounds also target enzymes involved in menaquinone biosynthesis and electron transport, inhibiting respiration and ATP biosynthesis, and are uncouplers, collapsing the pH gradient and membrane potential used to power transporters. The result of such multitarget inhibition is potent inhibition of TB cell growth, as well as very low rates of spontaneous drug resistance. Several targets are absent in humans but are present in other bacteria, as well as in malaria parasites, whose growth is also inhibited.

Squalene synthase as a target for Chagas disease therapeutics.

Trypanosomatid parasites are the causative agents of many neglected tropical diseases and there is currently considerable interest in targeting endogenous sterol biosynthesis in these organisms as a route to the development of novel anti-infective drugs. Here, we report the first x-ray crystallographic structures of the enzyme squalene synthase (SQS) from a trypanosomatid parasite, Trypanosoma cruzi, the causative agent of Chagas disease. We obtained five structures of T. cruzi SQS and eight structures of human SQS with four classes of inhibitors: the substrate-analog S-thiolo-farnesyl diphosphate, the quinuclidines E5700 and ER119884, several lipophilic bisphosphonates, and the thiocyanate WC-9, with the structures of the two very potent quinuclidines suggesting strategies for selective inhibitor development. We also show that the lipophilic bisphosphonates have low nM activity against T. cruzi and inhibit endogenous sterol biosynthesis and that E5700 acts synergistically with the azole drug, posaconazole. The determination of the structures of trypanosomatid and human SQS enzymes with a diverse set of inhibitors active in cells provides insights into SQS inhibition, of interest in the context of the development of drugs against Chagas disease.

Crystal structures of d-psicose 3-epimerase from Clostridium cellulolyticum H10 and its complex with ketohexose sugars

Abstractd-Psicose 3-epimerase (DPEase) is demonstrated to be useful in the bioproduction of d-psicose, a rare hexose sugar, from d-fructose, found plenty in nature. Clostridium cellulolyticum H10 has recently been identified as a DPEase that can epimerize d-fructose to yield d-psicose with a much higher conversion rate when compared with the conventionally used DTEase. In this study, the crystal structure of the C. cellulolyticum DPEase was determined. The enzyme assembles into a tetramer and each subunit shows a (β/α)8 TIM barrel fold with a Mn2+ metal ion in the active site. Additional crystal structures of the enzyme in complex with substrates/products (d-psicose, d-fructose, d-tagatose and d-sorbose) were also determined. From the complex structures of C. cellulolyticum DPEase with d-psicose and d-fructose, the enzyme has much more interactions with d-psicose than d-fructose by forming more hydrogen bonds between the substrate and the active site residues. Accordingly, based on these ketohexose-bound complex structures, a C3-O3 proton-exchange mechanism for the conversion between d-psicose and d-fructose is proposed here. These results provide a clear idea for the deprotonation/protonation roles of E150 and E244 in catalysis.

Functional and structural studies of pullulanase from Anoxybacillus sp. LM18‐11

Pullulanase is a debranching enzyme that specifically hydrolyzes the α‐1,6 glycosidic linkage of α‐glucans, and has wide industrial applications. Here, we report structural and functional studies of a new thermostable pullulanase from Anoxybacillus sp. LM18‐11 (PulA). Based on the hydrolysis products, PulA was classified as a type I pullulanase. It showed maximum activity at 60°C and pH 6.0. Kinetic study showed that the specific activity and Km for pullulan of PulA are 750 U mg−1 and 16.4 μmol L−1, respectively. PulA has a half‐life of 48 h at 60°C. The remarkable thermostability makes PulA valuable for industrial usage. To further investigate the mechanism of the enzyme, we solved the crystal structures of PulA and its complexes with maltotriose and maltotetraose at 1.75–2.22 Å resolution. The PulA structure comprises four domains (N1, N2, A, and C). A is the catalytic domain, in which three conserved catalytic residues were identified (D413, E442, and D526). Two molecules of oligosaccharides were seen in the catalytic A domain in a parallel binding mode. Interestingly, another two oligosaccharides molecules were found between the N1 domain and the loop between the third β‐strand and the third α‐helix in the A domain. Based on sequence alignment and the ligand binding pattern, the N1 domain is identified as a new type of carbohydrate‐binding motif and classified to the CBM68 family. The structure solved here is the first structure of pullulanase which has carbohydrate bound to the N1 domain. Proteins 2014; 82:1685–1693.

Structural and functional analysis of Bacillus subtilis YisP reveals a role of its product in biofilm production.

YisP is involved in biofilm formation in Bacillus subtilis and has been predicted to produce C30 isoprenoids. We determined the structure of YisP and observed that it adopts the same fold as squalene and dehydrosqualene synthases. However, the first aspartate-rich motif found in essentially all isoprenoid synthases is aspartate poor in YisP and cannot catalyze head-to-head condensation reactions. We find that YisP acts as a phosphatase, catalyzing formation of farnesol from farnesyl diphosphate, and that it is the first phosphatase to adopt the fold seen in the head-to-head prenyl synthases. Farnesol restores biofilm formation in a Δyisp mutant and modifies lipid membrane structure similarly to the virulence factor staphyloxanthin. This work clarifies the role of YisP in biofilm formation and suggests an intriguing possibility that many of the YisP-like homologs found in other bacteria may also have interesting products and functions.

Structural Insights into Enzymatic Degradation of Oxidized Polyvinyl Alcohol

The ever‐increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel α/β‐hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA‐degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid‐like β‐ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the CC bond of β‐diketone, although it has a catalytic triad similar to that of most α/β‐hydrolases. Analysis of the crystal structures suggests a double‐oxyanion‐hole mechanism, previously only found in thiolase cleaving β‐ketoacyl‐CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications.

Insights into TIM-barrel prenyl transferase mechanisms: crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus.

Well structured: As a new triose phosphate isomerase (TIM) barrel-fold prenyl transferase, PcrB catalyzes the production of heptaprenylglyceryl phosphate from heptaprenyl diphosphate and glycerol-1-phosphate. Crystal structures of PcrB from Bacillus subtilis and Staphylococcus aureus in complex with ligands were solved, and together with site-directed mutagenesis and bioinformatics analyses, clearly reveal the catalytic mechanism of the enzyme.