Phage Display on the Anti‐infective Target 1‐Deoxy‐d‐xylulose‐5‐phosphate Synthase Leads to an Acceptor–Substrate Competitive Peptidic Inhibitor

Enzymes of the 2‐C‐methyl‐d ‐erythritol‐4‐phosphate pathway for the biosynthesis of isoprenoid precursors are validated drug targets. By performing phage display on 1‐deoxy‐d ‐xylulose‐5‐phosphate synthase (DXS), which catalyzes the first step of this pathway, we discovered several peptide hits and recognized false‐positive hits. The enriched peptide binder P12 emerged as a substrate (d ‐glyceraldehyde‐3‐phosphate)‐competitive inhibitor of Deinococcus radiodurans DXS. The results indicate possible overlap of the cofactor‐ and acceptor‐substrate‐binding pockets and provide inspiration for the design of inhibitors of DXS with a unique and novel mechanism of inhibition.     

Biosynthesis of Epimers C2 and C2a in the Gentamicin C Complex

Gentamicin is a broad‐spectrum aminoglycoside antibiotic widely used to treat life‐threatening bacterial infections. The gentamicin C complex consists of gentamicin C1, gentamicin C1a, and epimers gentamicin C2 and gentamicin C2a. At present there is a generally accepted pathway of gentamicin biosynthesis, except for detailed understanding of the epimerization process involving gentamicins C2 and C2a. Here we have investigated the biosynthesis of these epimers. JI‐20B—an intermediate in the gentamicin biosynthetic pathway—and its epimer JI‐20Ba were generated by in‐frame deletion within genP, which encodes a phosphotransferase that catalyzes the first step of 3′,4′‐bisdehydroxylation in gentamicin biosynthesis. GenB1 and GenB2 are aminotransferases with different substrate specificities and enantioselectivities. JI‐20Ba, containing a 6′S chiral amine, a precursor of gentamicin C2a, was synthesized from G418 by GenQ/GenB1 through sequential oxidation/transamination at C‐6′. GenQ/GenB2 catalyzed the synthesis of JI‐20B, containing a 6′R chiral amine, a precursor of gentamicin C2, from G418. GenB2 catalyzed the epimerization of JI‐20Ba/JI‐20B and of gentamicins C2a/C2.     

Fabclavines: Bioactive Peptide–Polyketide‐Polyamino Hybrids from Xenorhabdus

The structure of the fabclavines—unique mixtures of nonribosomally derived peptide–polyketide hybrids connected to an unusual polyamino moiety—has been solved by detailed NMR and MS methods. These compounds have been identified in two different entomopathogenic Xenorhabdus strains, thereby leading also to the identification of the fabclavine biosynthesis gene cluster. Detailed analysis of these clusters and initial mutagenesis experiments allowed the prediction of a biosynthesis pathway in which the polyamino moiety is derived from an unusual type of fatty acid synthase that is normally involved in formation of polyunsaturated fatty acids. As fabclavines show broad‐spectrum activity against bacteria, fungi, and other eukaryotic cells, they might act as “protection factors” against all kinds of food competitors during the complex life cycle of Xenorhabdus, its nematode host, and their insect prey.     

Synthesis and characterization of cytidine derivatives that inhibit the kinase IspE of the non-mevalonate pathway for isoprenoid biosynthesis

The enzymes of the non-mevalonate pathway for isoprenoid biosynthesis are attractive targets for the development of novel drugs against malaria and tuberculosis. This pathway is used exclusively by the corresponding pathogens, but not by humans. A series of water-soluble, cytidine-based inhibitors that were originally designed for the fourth enzyme in the pathway, IspD, were shown to inhibit the subsequent enzyme, the kinase IspE (from Escherichia coli). The binding mode of the inhibitors was verified by co-crystal structure analysis, using Aquifex aeolicus IspE. The crystal structures represent the first reported example of a co-crystal structure of IspE with a synthetic ligand and confirmed that ligand binding affinity originates mainly from the interactions of the nucleobase moiety in the cytidine binding pocket of the enzyme. In contrast, the appended benzimidazole moieties of the ligands adopt various orientations in the active site and establish only poor intermolecular contacts with the protein. Defined binding sites for sulfate ions and glycerol molecules, components in the crystallization buffer, near the well-conserved ATP-binding Gly-rich loop of IspE were observed. The crystal structures of A. aeolicus IspE nicely complement the one from E. coli IspE for use in structure-based design, namely by providing invaluable structural information for the design of inhibitors targeting IspE from Mycobacterium tuberculosis and Plasmodium falciparum. Similar to the enzymes from these pathogens, A. aeolicus IspE directs the OH group of a tyrosine residue into a pocket in the active site. In the E. coli enzyme, on the other hand, this pocket is lined by phenylalanine and has a more pronounced hydrophobic character.     

In Vitro Reconstitution of a PKS Pathway for the Biosynthesis of Galbonolides in Streptomyces sp. LZ35

The galbonolides are 14‐membered macrolide antibiotics with a macrocyclic backbone similar to that of erythromycins. Galbonolides exhibit broad‐spectrum antifungal activities. Retro‐biosynthetic analysis suggests that the backbone of galbonolides is assembled by a type I modular polyketide synthase (PKS). Unexpectedly, the galbonolide biosynthetic gene cluster, gbn, in Streptomyces sp. LZ35 encodes a hybrid fatty acid synthase (FAS)‐PKS pathway. In vitro reconstitution revealed the functions of GbnA (an AT‐ACP didomain protein), GbnC (a FabH‐like enzyme), and GbnB (a novel multidomain PKS module without AT and ACP domains) responsible for assembling the backbone of galbonolides, respectively. To our knowledge, this study is the first biochemical characterization of a hybrid FAS‐PKS pathway for the biosynthesis of 14‐membered macrolides. The identification of this pathway provides insights into the evolution of PKSs and could facilitate the design of modular pools for synthetic biology.     

Targeting an Aromatic Hotspot in Plasmodium falciparum 1‐Deoxy‐d‐xylulose‐5‐phosphate Reductoisomerase with β‐Arylpropyl Analogues of Fosmidomycin

Blocking the 2‐C‐methyl‐d ‐erythrithol‐4‐phosphate pathway for isoprenoid biosynthesis offers new ways to inhibit the growth of Plasmodium spp. Fosmidomycin [(3‐(N‐hydroxyformamido)propyl)phosphonic acid, 1 ] and its acetyl homologue FR‐900098 [(3‐(N‐hydroxyacetamido)propyl)phosphonic acid, 2 ] potently inhibit 1‐deoxy‐d ‐xylulose‐5‐phosphate reductoisomerase (Dxr), a key enzyme in this biosynthetic pathway. Arylpropyl substituents were introduced at the β‐position of the hydroxamate analogue of 2 to study changes in lipophilicity, as well as electronic and steric properties. The potency of several new compounds on the P. falciparum enzyme approaches that of 1 and 2 . Activities against the enzyme and parasite correlate well, supporting the mode of action. Seven X‐ray structures show that all of the new arylpropyl substituents displace a key tryptophan residue of the active‐site flap, which had made favorable interactions with 1 and 2 . Plasticity of the flap allows substituents to be accommodated in many ways; in most cases, the flap is largely disordered. Compounds can be separated into two classes based on whether the substituent on the aromatic ring is at the meta or para position. Generally, meta‐substituted compounds are better inhibitors, and in both classes, smaller size is linked to better potency.     

Characterization of a Novel Mesophilic CTP-Dependent Riboflavin Kinase and Rational Engineering to Create Its Thermostable Homologues**

Flavins play a central role in metabolism as molecules that catalyze a wide range of redox reactions in living organisms. Several variations in flavin biosynthesis exist among the domains of life, and their analysis has revealed many new structural and mechanistic insights till date. The cytidine triphosphate (CTP)-dependent riboflavin kinase in archaea is one such example. Unlike most kinases that use adenosine triphosphate, archaeal riboflavin kinases utilize CTP to phosphorylate riboflavin and produce flavin mononucleotide. In this study, we present the characterization of a new mesophilic archaeal CTP-utilizing riboflavin kinase homologue from Methanococcus maripaludis (MmpRibK), which is linked closely in sequence to the previously characterized thermophilic Methanocaldococcus jannaschii homologue. We reconstitute the activity of MmpRibK, determine its kinetic parameters and molecular factors that contribute to its unique properties, and finally establish the residues that improve its thermostability using computation and a series of experiments. Our work advances the molecular understanding of flavin biosynthesis in archaea by the characterization of the first mesophilic CTP-dependent riboflavin kinase. Finally, it validates the role of salt bridges and rigidifying amino acid residues in imparting thermostability to this unique structural fold that characterizes archaeal riboflavin kinase enzymes, with implications in enzyme engineering and biotechnological applications.     

A review of tobacco BY-2 cells as an excellent system to study the synthesis and function of sterols and other isoprenoids

In plants, two pathways are utilized for the synthesis of isopentenyl diphosphate (IPP), the universal precursor for isoprenoid biosynthesis. In this paper we review findings and observations made primarily with tobacco BY-2 cells (TBY-2), which have proven to be an excellent system in which to study the two biosynthetic pathways. A major advantage of these cells as an experimental system is their ability to readily take up specific inhibitors and stably- and/or radiolabeled precursors. This permits the functional elucidation of the role of isoprenoid end products and intermediates. Because TBY-2 cells undergo rapid cell division and can be synchronized within the cell cycle, they constitute a highly suitable test system for determination of those isoprenoids and intermediates that act as cell cycle inhibitors, thus giving an indication of which branches of the isoprenoid pathway are essential. Through chemical complementation, and use of precursors, intracellular compartmentation can be elucidated, as well as the extent to which the plastidial and cytoslic pathways contribute to the syntheses of specific groups of isoprenoids (e.g., sterols) via exchange of intermediates across membranes. These topics are discussed in the context of the pertinent literature.     

Some new aspects of isoprenoid biosynthesis in plants—A review

Plants are capable of synthesizing a myriad of isoprenoids and prenyl lipids. Much attention has been focused on 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), the enzyme that synthesizes mevalonate and is generally considered responsible for the regulation of substrate flux to isoprenoids. In contrast to vertebrates, where there seems to exist only one HMGR gene, in plants a small family of isogenes appears differentially expressed in regard to location and time. Much less is known in plants about the preceding steps,viz. the conversion of acetyl-CoA to HMG-CoA. An enzyme system has been isolated from radish that can catalyze this transformation, and which shows some unusual propertiesin vitro. The intracellular localization of the early steps of isoprenoid biosynthesis in plant cells is still a matter of debate. The various observations and hypotheses derived from incorporation and inhibition studies are somewhat contradictory, and an attempt is being made to rationalize various findings that do not at first seem compatible. There are good arguments in favor of an exclusively cytoplasmic formation of isopentenyl pyrophosphate (IPP) via mevalonic acid, but other studies and observations suggest an independent formation in plastids. Other possibilities are being considered, such as the existence of independent (compartmentalized) biosynthetic pathways of IPP formationvia the socalled Rohmer pathway. Substrate channeling through the formation of end product-specific multienzyme complexes (metabolons) with no release of substrate intermediates will also be discussed. Based in part on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids,” Atlanta, Georgia, May 1994.     

Isoprenoid Biosynthesis Inhibitors Targeting Bacterial Cell Growth

We synthesized potential inhibitors of farnesyl diphosphate synthase (FPPS), undecaprenyl diphosphate synthase (UPPS), or undecaprenyl diphosphate phosphatase (UPPP), and tested them in bacterial cell growth and enzyme inhibition assays. The most active compounds were found to be bisphosphonates with electron‐withdrawing aryl‐alkyl side chains which inhibited the growth of Gram‐negative bacteria (Acinetobacter baumannii, Klebsiella pneumoniae, Escherichia coli, and Pseudomonas aeruginosa) at ～1–4 μg mL^?1 levels. They were found to be potent inhibitors of FPPS; cell growth was partially “rescued” by the addition of farnesol or overexpression of FPPS, and there was synergistic activity with known isoprenoid biosynthesis pathway inhibitors. Lipophilic hydroxyalkyl phosphonic acids inhibited UPPS and UPPP at micromolar levels; they were active (～2–6 μg mL^?1) against Gram‐positive but not Gram‐negative organisms, and again exhibited synergistic activity with cell wall biosynthesis inhibitors, but only indifferent effects with other inhibitors. The results are of interest because they describe novel inhibitors of FPPS, UPPS, and UPPP with cell growth inhibitory activities as low as ～1–2 μg mL^?1.     

Production of Dehydrogingerdione Derivatives in Escherichia coli by Exploiting a Curcuminoid Synthase from Oryza sativa and a β‐Oxidation Pathway from Saccharomyces cerevisiae

Gingerol derivatives are bioactive compounds isolated from the rhizome of ginger. They possess various beneficial activities, such as anticancer and hepatoprotective activities, and are therefore attractive targets of bioengineering. However, the microbial production of gingerol derivatives has not yet been established, primarily because the biosynthetic pathway of gingerol is unknown. Here, we report the production of several dehydrogingerdione (a gingerol derivative) analogues from a recombinant Escherichia coli strain that has an “artificial” biosynthesis pathway for dehydrogingerdione that was not based on the original biosynthesis pathway of gingerol derivatives in plants. The system consists of a 4‐coumarate:CoA ligase from Lithospermum erythrorhizon, a fatty acid CoA ligase from Oryza sativa, a β‐oxidation system from Saccharomyces cerevisiae, and a curcuminoid synthase from O. sativa. To our knowledge, this is the first report of the microbial production of a plant metabolite the biosynthetic pathway of which has not yet been identified.     

Hydroxybenzaldoximes Are D‐GAP‐Competitive Inhibitors of E. coli 1‐Deoxy‐D‐Xylulose‐5‐Phosphate Synthase

1‐Deoxy‐D ‐xylulose 5‐phosphate (DXP) synthase is the first enzyme in the methylerythritol phosphate pathway to essential isoprenoids in pathogenic bacteria and apicomplexan parasites. In bacterial pathogens, DXP lies at a metabolic branch point, serving also as a precursor in the biosynthesis of vitamins B1 and B6, which are critical for central metabolism. In an effort to identify new bisubstrate analogue inhibitors that exploit the large active site and distinct mechanism of DXP synthase, a library of aryl mixed oximes was prepared and evaluated. Trihydroxybenzaldoximes emerged as reversible, low‐micromolar inhibitors, competitive against D ‐glyceraldehyde 3‐phosphate (D ‐GAP) and either uncompetitive or noncompetitive against pyruvate. Hydroxybenzaldoximes are the first class of D ‐GAP‐competitive DXP synthase inhibitors, offering new tools for mechanistic studies of DXP synthase and a new direction for the development of antimicrobial agents targeting isoprenoid biosynthesis.     

The Synechocystis sp. PCC6803 Sfp‐Type Phosphopantetheinyl Transferase Does Not Possess Characteristic Broad‐Range Activity

The cyanobacterium Synechocystis sp. PCC6803 harbours one phosphopantetheinyl transferase (PPTase), Sppt. Protein modelling supported previous bioinformatics analyses, which suggested that Sppt is a Sfp‐type PPTase with the potential to phosphopantetheinylate a broad range of carrier proteins from both primary and secondary metabolism. However, no natural products are synthesised by this species, which raises interesting evolutionary and functional questions. Phosphopantetheinylation assays and kinetic data demonstrate that Sppt was able to activate its cognate fatty acid synthesis carrier protein, SACP, but was unable to effectively activate various cyanobacterial carrier proteins from secondary metabolism or glycolipid biosynthesis pathways. To our knowledge, this is the first example of a PPTase with a Sfp‐type structure, but with activity more closely resembling AcpS‐type enzymes. The broad‐range PPTase from Nodularia spumigena NSOR10 was introduced into Synechocystis sp. PCC6803 and was shown to activate a noncognate carrier protein, in vivo. This engineered strain could provide a future biotechnological platform for the heterologous expression of cyanobacterial biosynthetic gene clusters.     

Investigating Deformylase and Deacylase Activity of Mammalian and Bacterial Sirtuins

Lysine acylation constitutes a major group of post‐translational modifications of proteins, and is found in the proteomes of organisms from all kingdoms of life. Sirtuins are considered the main erasers of these modification marks, and thus contribute to acylation‐dependent regulation of enzyme activity, and potentially of protein quality control. We have established a substrate scaffold to enable the analysis of sirtuin activity with a broad range of acyl‐lysine modifications, including hydrophobic fatty acids. Characterization of the deacylase activity of the bacterial SrtN, which is encoded by the yhdZ gene of Bacillus subtilis, showed that this enzyme is capable of removing a broad range of acyl groups. These investigations further showed that SrtN and human SIRT1 are efficient lysine‐deformylases, thereby providing a first clue as to how this nonenzymatic modification might be removed from affected proteins.     

Head‐to‐Head Prenyl Synthases in Pathogenic Bacteria

Many organisms contain head‐to‐head isoprenoid synthases; we investigated three such types of enzymes from the pathogens Neisseria meningitidis, Neisseria gonorrhoeae, and Enterococcus hirae. The E. hirae enzyme was found to produce dehydrosqualene, and we solved an inhibitor‐bound structure that revealed a fold similar to that of CrtM from Staphylococcus aureus. In contrast, the homologous proteins from Neisseria spp. carried out only the first half of the reaction, yielding presqualene diphosphate (PSPP). Based on product analyses, bioinformatics, and mutagenesis, we concluded that the Neisseria proteins were HpnDs (PSPP synthases). The differences in chemical reactivity to CrtM were due, at least in part, to the presence of a PSPP‐stabilizing arginine in the HpnDs, decreasing the rate of dehydrosqualene biosynthesis. These results show that not only S. aureus but also other bacterial pathogens contain head‐to‐head prenyl synthases, although their biological functions remain to be elucidated.     

醇脱氢酶在聚乙烯膜表面的固定化研究

以聚丙烯酸（PAA）改性的聚乙烯（PE）膜为载体，研究了醇脱氢酶（ADH）的两种固定化路线，并以甲醛为底物考察了固定化酶的催化性能。路线1用聚乙烯亚胺（PEI）进一步改性，使用戊二醛（GA）固定化ADH。最优固定化pH为6.0，温度为5～15℃，酶浓度为1.0 mg/ml，GA浓度为0.01%(质量)；固定化酶的最适反应pH为6.5，温度为15～30℃，反应速率最高为9.6 μmol/(L·min)；重复利用10次后可保持47.3%的活性。路线2以PAA-PE为载体，用1-(3-二甲氨基丙基)-2-乙基碳二亚胺盐酸盐（EDC）和N-羟基琥珀酰亚胺（NHS）为活化剂，固定化ADH。EDC和NHS最优摩尔比为1∶0.5，固定化时间为24 h；固定化酶的最适反应pH为6.5，温度为20～37℃，反应速率为15.58 μmol/(L·min)；重复利用10次后可保持53.8%的活性。     

Pyruvate-Kinase-Coupled Glycosyltransferase Assays: Limitations,Struggles and Problem Resolution

Enzyme assays involving coupled pyruvate kinase (PK) have been used for many years to monitor the activity of major classes of enzymes including glycosyltransferases. Numerous potent inhibitors have been discovered and kinetically characterized thanks to this technology. However, when inhibitors of these important enzymes are screened, PK inhibitors or activators are very often observed. In this study we report solutions to resolve the problems encountered either during the screening or during the kinetic characterization of glycosyltransferase inhibitors by means of PK-coupled assays. The enzyme under study—WaaC—is an important glycosyltransferase involved in the bacterial lipopolysaccharide (LPS) biosynthesis pathway. Firstly we showed that alternative kinases such as nucleoside 5-diphosphate kinase (NDPK), myokinase (MK), and ADPdependent hexokinase that catalyze similar reactions to PK are prone to the same troubles. Moreover, an ADP chemosensor was used as an alternative but the sensitivity was not sufficient to allow a proper screening. Finally, we found that a stepwise PK/luciferase assay resolved the problems encountered with PK inhibitors and that a WaaC HPLC assay allowed the identification of WaaC inhibitors acting as PK activators, thus allowing false positive and false negative results linked to the coupling to PK to be eliminated.     

Immobilization of alcohol dehydrogenase on the surface of polyethylene membrane

Using polyacrylic acid (PAA) modified polyethylene (PE) membrane as a carrier, two immobilization routes of alcohol dehydrogenase (ADH) were studied, and the catalytic performance of immobilized enzyme was investigated using formaldehyde as a substrate. In the first route, PAA-PE membrane was further modified by polyethyleneimine (PEI) and then ADH was covalently linked by glutaraldehyde (GA) to the surface of PEI/PAA-PE. The results show that the optimal immobilization pH was 6.0, immobilization temperature was 5—15℃, ADH and GA concentrations were 1.0mg/ml and 0.01%(mass). For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 15—30℃, and the highest reaction rate was 9.6 μmol/(L·min), the remaining activity was 47.3% after 10 use cycles. In the second route, ADH was immobilized on PAA-PE membrane with 1-(3-dimethylaminopropyl)-2-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) as activators. The results show that the optimal molar ratio of EDC and NHS was 1∶0.5, and the immobilization time was 24 h. For immobilized enzyme, the optimal reaction pH was 6.5, temperature was 20—37℃, and the highest reaction rate was 15.58 μmol/(L·min), 53.8% activity was remained after 10 cycles.     

ValC, a new type of C7-Cyclitol kinase involved in the biosynthesis of the antifungal agent validamycin A

The gene valC, which encodes an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin A biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation experiments with a replicating plasmid harboring full-length valC restored the production of validamycin A, thus suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone--but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose--to afford their 7-phosphate derivatives. The results provide new insights into the activity of this enzyme and also confirm the existence of two different pathways leading to the same end-product: the valienamine moiety common to acarbose and validamycin A.     

A proposed mechanism for the reductive ring opening of the cyclodiphosphate MEcPP, a crucial transformation in the new DXP/MEP pathway to isoprenoids based on modeling studies and feeding experiments

Experimental and theoretical investigations concerning the second-to-last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants are reported. The proposed intrinsic or late intermediates 4-oxo-DMAPP (12) and 4-hydroxy-DMAPP (11) were synthesized in deuterium- or tritium-labeled form according to new protocols especially adapted to work without protection of the diphosphate moiety. When the labeled compounds MEcPP (7), 11, and 12 were applied to chromoplast cultures, aldehyde 12 was not incorporated. This finding is in agreement with a mechanistic and structural model of the responsible enzyme family: a three-dimensional model of the fragment L271-A375 of the enzyme GcpE of Streptomyces coelicolor including NADPH, the Fe(4)S(4) cluster, and MEcPP (7) as ligand has been developed based on homology modeling techniques. The model has been accepted by the Protein Data Bank (entry code 1OX2). Supported by this model, semiempirical PM3 calculations were performed to analyze the likely catalysis mechanism of the reductive ring opening of MEcPP (7), hydroxyl abstraction, and formation of HMBPP (8). The mechanism is characterized by a proton transfer (presumably from a conserved arginine 286) to the substrate, accompanied by a ring opening without high energy barriers, followed by the transfer of two electrons delivered from the Fe(4)S(4) cluster, and finally proton transfer from a carboxylic acid side chain to the hydroxyl group to be removed from the ligand as water. The proposed mechanism is in agreement with all known experimental findings and the arrangement of the ligand within the enzyme. Thus, a very likely mechanism for the second to last step of the DXP/MEP pathway in isoprenoid biosynthesis in plants is presented. A principally similar mechanism is also expected for the reductive dehydroxylation of HMBPP (8) to IPP (9) and DMAPP (10) in the last step.