Youli Xiao

Methylerythritol Phosphate Pathway of Isoprenoid Biosynthesis

Isoprenoids are a class of natural products with more than 55,000 members. All isoprenoids are constructed from two precursors, isopentenyl diphosphate and its isomer dimethylallyl diphosphate. Two of the most important discoveries in isoprenoid biosynthetic studies in recent years are the elucidation of a second isoprenoid biosynthetic pathway [the methylerythritol phosphate (MEP) pathway] and a modified mevalonic acid (MVA) pathway. In this review, we summarize mechanistic insights on the MEP pathway enzymes. Because many isoprenoids have important biological activities, the need to produce them in sufficient quantities for downstream research efforts or commercial application is apparent. Recent advances in both MVA and MEP pathway-based synthetic biology are also illustrated by reviewing the landmark work of artemisinic acid and taxadien-5α-ol production through microbial fermentations.

Revisiting the IspH catalytic system in the deoxyxylulose phosphate pathway: achieving high activity.

From two C(5) isoprene building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), the more than 30 000 members of the isoprenoid family are constructed in nature using two biosynthetic pathways, the mevalonate (MVA) pathway and the deoxyxylulose phosphate (DXP) pathway. IspH of the DXP pathway is a protein containing an iron-sulfur cluster and catalyzes a reductive dehydration reaction of the DXP pathway. In the literature, a wide range of Escherichia coli IspH activities have been reported (2.0 nmol min(-1) mg(-1) to 3.4 micromol min(-1) mg(-1)). For such a broad range of activities, reaction assays were carried out under many different conditions, preventing direct comparison of the activities and determination of the key factor responsible for such a dramatic difference in IspH activities. In this work, we systematically examined the role of redox mediators in IspH catalysis using E. coli IspH as the enzyme and dithionite as the ultimate electron source. Our studies not only suggest the importance of the iron-sulfur cluster but also improve the E. coli IspH activity by nearly 97-fold relative to that from the E. coli NADPH-flavodoxin reductase-flavodoxin system.

Mechanistic Studies of IspH in the Deoxyxylulose Phosphate Pathway: Heterolytic C-O Bond Cleavage at C4 Position

Isoprenoids are one of the largest and most structurally diverse groups of metabolites in nature. their biosyntheses require two precursors, isopentenyl diphosphate (ipp) and its isomer, dimethylallyl diphosphate (dmapp). there are two different pathways for the synthesis of ipp and dmapp: the deoxyxylulose phosphate (dxp) pathway and the mevalonic acid (mva) pathway. more importantly, these two pathways have a well-defined distribution among different kingdoms. most pathogenic bacteria and protozoan parasites utilize the dxp pathway, while animals synthesize their isoprenoid precursors from acety-coa via the mva pathway. plants have both dxp and mva pathways. thus, mechanistic studies on the dxp pathway enzymes may lead to the development of mechanism-based inhibitors as herbicides, broad-spectrum antibiotics, and antimalaria drugs. isph in the dxp pathway catalyzes the reductive dehydration of (e)-4-hydroxy-3-metho-2-butenyl diphosphate (hmbpp) to form ipp and dmapp, the last step in the dxp pathway. recent epr studies reported in literature suggest that isph is a unique iron-site-containing [4fe-4s] protein. in this study, we studied the isph-catalyzed reductive dehydration mechanism using two substrate analogues. our data reported herein provide evidence to not only support the integrity of the c1 position c-o bond during reaction, they also suggest a heterolytic c-o bond cleavage at the c4 position for isph-catatyzed reductive dehydration reaction. our kinetic studies also suggest that the c4 hydroxyl group is involved in substrate binding. because the isph-catalyzed reductive dehydration reaction does not fall into the two known classes of unique iron-site-containing [4fe-4s] proteins, aconitase-type and radical sam-type enzymes, isph may represent a new class of iron-sulfur-containing proteins.

Quaternary Ammonium Oxidative Demethylation: X-ray Crystallographic, Resonance Raman, and UV-Visible Spectroscopic Analysis of a Rieske-Type Demethylase.

Herein, the structure resulting from in situ turnover in a chemically challenging quaternary ammonium oxidative demethylation reaction was captured via crystallographic analysis and analyzed via single-crystal spectroscopy. Crystal structures were determined for the Rieske-type monooxygenase, stachydrine demethylase, in the unliganded state (at 1.6 Å resolution) and in the product complex (at 2.2 Å resolution). The ligand complex was obtained from enzyme aerobically cocrystallized with the substrate stachydrine (N,N-dimethylproline). The ligand electron density in the complex was interpreted as proline, generated within the active site at 100 K by the absorption of X-ray photon energy and two consecutive demethylation cycles. The oxidation state of the Rieske iron-sulfur cluster was characterized by UV-visible spectroscopy throughout X-ray data collection in conjunction with resonance Raman spectra collected before and after diffraction data. Shifts in the absorption band wavelength and intensity as a function of absorbed X-ray dose demonstrated that the Rieske center was reduced by solvated electrons generated by X-ray photons; the kinetics of the reduction process differed dramatically for the liganded complex compared to unliganded demethylase, which may correspond to the observed turnover in the crystal.

IspG converts an epoxide substrate analogue to (E)-4-hydroxy-3-methylbut-2-enyl diphosphate: implications for IspG catalysis in isoprenoid biosynthesis.

IspG is an intriguing enzyme in bacteria, parasite, and plant isoprenoid biosynthesis, and its catalytic mechanism remains elusive. We report here the synthesis of (2R,3R)-4-hydroxy-3-methyl-2,3-epoxybutanyl diphosphate (Epoxy-HMBPP), a proposed intermediate in one of the frequently cited mechanistic models. We have also demonstrated that this epoxide analogue is a catalytically competent IspG substrate. This study represents the first mechanistic study of this important enzyme.

Profiling of Multiple Targets of Artemisinin Activated by Hemin in Cancer Cell Proteome

The antimalarial drug artemisinin is found to have diverse biological activities ranging from anti-inflammatory to anticancer properties; however, as of today, the cellular targets and mechanism of action of this important compound have remained elusive. Here, we report the global protein target profiling of artemisinin in the HeLa cancer cell proteome using a chemical proteomics approach. In the presence of hemin, multiple proteins were targeted by artemisinin probe through covalent modification. Further studies revealed that reducing of hemin to heme by protein thiols was essential for endoperoxide activation and subsequent protein alkylation. Artemisinin may exert its synergistic therapeutic anticancer effects via modulation of a variety of cellular pathways through acting on multiple targets.

Methylerythritol cyclodiphosphate (MEcPP) in deoxyxylulose phosphate pathway: synthesis from an epoxide and mechanisms

In this communication, we reported another unique IspG-catalyzed transformation, the production of its substrate, MEcPP, from (2R,3R)-4-hydroxy-3-methyl-2,3-epoxybutanyl diphosphate (Epoxy-HMBPP) when reductants are excluded from the reaction mixture.

Study of IspH, a Key Enzyme in the Methylerythritol Phosphate Pathway Using Fluoro-Substituted Substrate Analogues

IspH, a [4Fe-4S]-cluster-containing enzyme, catalyzes the reductive dehydroxylation of 4-hydroxy-3-methyl-butenyl diphosphate (HMBPP) to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) in the methylerythritol phosphate pathway. Studies of IspH using fluoro-substituted substrate analogues to dissect the contributions of several factors to IspH catalysis, including the coordination of the HMBPP C(4)-OH group to the iron-sulfur cluster, the H-bonding network in the active site, and the electronic properties of the substrates, are reported.

IspG enzyme activity in the deoxyxylulose phosphate pathway: roles of the iron-sulfur cluster.

IspG is a [4Fe-4S] cluster-containing protein, and the [4Fe-4S](+) species is proposed to be the catalytically relevant species. However, attempts reported in the literature failed to detect the [4Fe-4S](+) species. In this study, using a potent reduction system, we have successfully detected the [4Fe-4S](+) species with X-band EPR spectroscopy. In addition, we have improved the Escherichia coli IspG activity to 550 nmol min(-1) mg(-1), which is approximately 20-fold greater than that of the NADPH-Fpr-FldA system in the literature.

IspG-Catalyzed Positional Isotopic Exchange in Methylerythritol Cyclodiphosphate of the Deoxyxylulose Phosphate Pathway: Mechanistic Implications

H(2)(18)O under the bridge: Recently, the deoxyxylulose phosphate (DXP) pathway was discovered to be a second pathway supplying isoprenoid biosynthetic precursors. One of steps is an IspG-catalyzed reductive deoxygenation of methylerythritol cyclodiphosphate (MEcPP) to 4-hydroxyl-3-methyl-2-(E)-1-diphosphate (HMBPP). Using [2-(13) C,(18) O]-MEcPP, we detected the positional isotopic exchange for the bridging oxygen in MEcPP.