QM/MM Study of the Catalytic Mechanism and Substrate Specificity of the Aromatic Substrate C-Methyltransferase Fur6.

In the field of medical chemistry and other organic chemistry, introducing a methyl group into a designed position has been difficult to achieve. However, owing to the vigorous developments in the field of enzymology, methyltransferases are considered potential tools for addressing this problem. Within the methyltransferase family, Fur6 catalyzes the methylation of C3 of 1,2,4,5,7-pentahydroxynaphthalene (PHN) using S-adenosyl-l-methionine (SAM) as the methyl donor. Here, we report the catalytic mechanism and substrate specificity of Fur6 based on computational studies. Our molecular dynamics (MD) simulation studies reveal the reactive form of PHN and its interactions with the enzyme. Our hybrid quantum mechanics/molecular mechanics (QM/MM) calculations suggest the reaction pathway of the methyl transfer step in which the energy barrier is 8.6 kcal mol-1. Our free-energy calculations with a polarizable continuum model (PCM) indicate that the final deprotonation step of the methylated intermediate occurs after it is ejected into the water solvent from the active center pocket of Fur6. Additionally, our studies on the protonation states, the highest occupied molecular orbital (HOMOs), and the energy barriers of the methylation reaction for the analogs of PHN demonstrate the mechanism of the specificity to PHN. Our study provides valuable insights into Fur6 chemistry, contributing to a deeper understanding of molecular mechanisms and offering an opportunity to engineer the enzyme to achieve high yields of the desired product(s).

Molecular Basis for Enzymatic Aziridine Formation via Sulfate Elimination.

Natural products containing an aziridine ring, such as mitomycin C and azinomycin B, exhibit antitumor activities by alkylating DNA via their aziridine rings; however, the biosynthetic mechanisms underlying the formation of these rings have not yet been elucidated. We herein investigated the biosynthesis of vazabitide A, the structure of which is similar to that of azinomycin B, and demonstrated that Vzb10/11, with no similarities to known enzymes, catalyzed the formation of the aziridine ring via sulfate elimination. To elucidate the detailed reaction mechanism, crystallization of Vzb10/11 and the homologous enzyme, AziU3/U2, in the biosynthesis of azinomycin B was attempted, and the structure of AziU3/U2, which had a new protein fold overall, was successfully determined. The structural analysis revealed that these enzymes adjusted the dihedral angle between the amino group and the adjacent sulfate group of the substrate to almost 180° and enhanced the nucleophilicity of the C6-amino group temporarily, facilitating the SN2-like reaction to form the aziridine ring. The present study reports for the first time the molecular basis for aziridine ring formation.

Biosynthesis of the nucleoside antibiotic angustmycins: identification and characterization of the biosynthetic gene cluster reveal unprecedented dehydratase required for exo-glycal formation.

The nucleoside antibiotic angustmycin, produced by some Streptomyces strains, is composed of adenine and C6 sugar and shows antibiotic and antitumor activities. In this study, we propose a biosynthetic pathway for angustmycin using a heterologous expression experiment coupled with in silico analysis of the angustmycin biosynthetic gene (agm) cluster. The biochemical characterization of Agm6 demonstrated its role in angustmycin biosynthesis as an unprecedented dehydratase.

Reconstitution of a Highly Reducing Type II PKS System Reveals 6π-Electrocyclization Is Required for o-Dialkylbenzene Biosynthesis.

Natural products containing an o-dialkylbenzene moiety exhibit a wide variety of bioactivities, including antibacterial, antifungal, antitumor, and antiangiogenic activities. However, the biosynthetic scheme of the o-dialkylbenzene moiety remains unclear. In this study, we identified the biosynthetic gene cluster (BGC) of compounds 1 and 2 in Streptomyces sp. SANK 60404, which contains a rare o-dialkylbenzene moiety, and successfully reconstituted the biosynthesis of 1 using 22 recombinant enzymes in vitro. Our study established a biosynthetic route for the o-tolyl group within the o-dialkylbenzene moiety, where the triene intermediate 3 loaded onto a unique acyl carrier protein (ACP) is elongated by a specific ketosynthase-chain length factor pair of a type II polyketide synthase system with the aid of a putative isomerase to be termed "electrocyclase" and a thioesterase-like enzyme in the BGC. The C2-elongated all-trans diketo-triene intermediate is subsequently isomerized to the 6Z configuration by the electrocyclase to allow intramolecular 6π-electrocyclization, followed by coenzyme FAD/FMN-dependent dehydrogenation. Bioinformatics analysis showed that the key genes are all conserved in BGCs of natural products containing an o-dialkylbenzene moiety, suggesting that the proposed biosynthetic scheme is a common strategy to form o-dialkylbenzenes in nature.

Guanidyl modification of the 1-azabicyclo[3.1.0]hexane ring in ficellomycin essential for its biological activity.

The 1-azabicyclo[3.1.0]hexane ring is a key moiety in natural products for biological activities against bacteria, fungi, and tumor through DNA alkylation. Ficellomycin is a dipeptide that consists of l-valine and a non-proteinogenic amino acid with the 1-azabicyclo[3.1.0]hexane ring structure. Although the biosynthetic gene cluster of ficellomycin has been identified, the biosynthetic pathway currently remains unclear. We herein report the final stage of ficellomycin biosynthesis involving ring modifications and successive dipeptide formation. After the ring is formed, the hydroxy group of the ring is converted into the guanidyl unit by three enzymes, which include an aminotransferase with a novel inter ω-ω amino-transferring activity. In the last step, the resulting 1-azabicyclo[3.1.0]hexane ring-containing amino acid is connected with l-valine by an amino acid ligase to yield ficellomycin. The present study revealed a new machinery that expands the structural and biological diversities of natural products.

Recent advances in the biosynthesis of nucleoside antibiotics.

Nucleoside antibiotics are a diverse class of natural products with promising biomedical activities. These compounds contain a saccharide core and a nucleobase. Despite the large number of nucleoside antibiotics that have been reported, biosynthetic studies on these compounds have been limited compared with those on other types of natural products such as polyketides, peptides, and terpenoids. Due to recent advances in genome sequencing technology, the biosynthesis of nucleoside antibiotics has rapidly been clarified. This review covering 2009-2019 focuses on recent advances in the biosynthesis of nucleoside antibiotics.

Comprehensive Derivatization of Thioviridamides by Heterologous Expression.

New technology for the derivatization of peptide natural products is required for drug development. Despite the recent advances in the genome sequencing technique enabling us to search for the biosynthetic genes for wide variety of natural products, the technical methods to get access to them are limited. A class of RiPPs, a recently emerged natural product family such as thioviridamide, is one of those possessing such unexplored chemical space. In this paper, we report a streamlined method to generate new thioviridamide derivatives and to assess their biological activities. Heterologous expression of 42 constructs in an engineered Streptomyces avermitilis host gave 35 designed thioviridamide derivatives, along with several unprecedented analogues. Moreover, cytotoxicity assay revealed that several derivatives showed more potent activities than those of prethioviridamide. These results indicate that this strategy can become one of the potential ways to produce supreme unnatural products.

Glutamate Dehydrogenase from Thermus thermophilus Is Activated by AMP and Leucine as a Complex with Catalytically Inactive Adenine Phosphoribosyltransferase Homolog.

Glutamate dehydrogenase (GDH) from a thermophilic bacterium, Thermus thermophilus, is composed of two heterologous subunits, GdhA and GdhB. In the heterocomplex, GdhB acts as the catalytic subunit, whereas GdhA lacks enzymatic activity and acts as the regulatory subunit for activation by leucine. In the present study, we performed a pulldown assay using recombinant T. thermophilus, producing GdhA fused with a His tag at the N terminus, and found that TTC1249 (APRTh), which is annotated as adenine phosphoribosyltransferase but lacks the enzymatic activity, was copurified with GdhA. When GdhA, GdhB, and APRTh were coproduced in Escherichia coli cells, they were purified as a ternary complex. The ternary complex exhibited GDH activity that was activated by leucine, as observed for the GdhA-GdhB binary complex. Furthermore, AMP activated GDH activity of the ternary complex, whereas such activation was not observed for the GdhA-GdhB binary complex. This suggests that APRTh mediates the allosteric activation of GDH by AMP. The present study demonstrates the presence of complicated regulatory mechanisms of GDH mediated by multiple compounds to control the carbon-nitrogen balance in bacterial cells.IMPORTANCE GDH, which catalyzes the synthesis and degradation of glutamate using NAD(P)(H), is a widely distributed enzyme among all domains of life. Mammalian GDH is regulated allosterically by multiple metabolites, in which the antenna helix plays a key role to transmit the allosteric signals. In contrast, bacterial GDH was believed not to be regulated allosterically because it lacks the antenna helix. We previously reported that GDH from Thermus thermophilus (TtGDH), which is composed of two heterologous subunits, is activated by leucine. In the present study, we found that AMP activates TtGDH using a catalytically inactive APRTh as the sensory subunit. This suggests that T. thermophilus possesses a complicated regulatory mechanism of GDH to control carbon and nitrogen metabolism.

C-Methylation Catalyzed by Fom3, a Cobalamin-Dependent Radical S-adenosyl-l-methionine Enzyme in Fosfomycin Biosynthesis, Proceeds with Inversion of Configuration.

Fom3, a cobalamin-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase, catalyzes C-methylation at the C2 position of cytidylylated 2-hydroxyethylphosphonate (HEP-CMP) to afford cytidylylated 2-hydroxypropylphosphonate (HPP-CMP) in fosfomycin biosynthesis. In this study, the Fom3 reaction product HPP-CMP was reanalyzed by chiral ligand exchange chromatography to confirm its stereochemistry. The Fom3 methylation product was found to be ( S)-HPP-CMP only, indicating that the stereochemistry of the C-methylation catalyzed by Fom3 is ( S)-selective. In addition, Fom3 reaction was performed with ( S)-[2-2H1]HEP-CMP and ( R)-[2-2H1]HEP-CMP to elucidate the stereoselectivity during the abstraction of the hydrogen atom from C2 of HEP-CMP. Liquid chromatography-electrospray ionization mass spectrometry analysis of the 5'-deoxyadenosine produced showed that the 2H atom of ( R)-[2-2H1]HEP-CMP was incorporated into 5'-deoxyadenosine but that from ( S)-[2-2H1]HEP-CMP was not. Retention of the 2H atom of ( S)-[2-2H1]HEP-CMP in HPP-CMP was also observed. These results indicate that the 5'-deoxyadenosyl radical stereoselectively abstracts the pro-R hydrogen atom at the C2 position of HEP-CMP and the substrate radical intermediate reacts with the methyl group on cobalamin that is located on the opposite side of the substrate from SAM. Consequently, it was clarified that the C-methylation catalyzed by Fom3 proceeds with inversion of configuration.

Distinct roles for U-type proteins in iron-sulfur cluster biosynthesis revealed by genetic analysis of the Bacillus subtilis sufCDSUB operon.

The biosynthesis of iron-sulfur (Fe-S) clusters in Bacillus subtilis is mediated by the SUF-like system composed of the sufCDSUB gene products. This system is unique in that it is a chimeric machinery comprising homologues of E. coli SUF components (SufS, SufB, SufC and SufD) and an ISC component (IscU). B. subtilis SufS cysteine desulfurase transfers persulfide sulfur to SufU (the IscU homologue); however, it has remained controversial whether SufU serves as a scaffold for Fe-S cluster assembly, like IscU, or acts as a sulfur shuttle protein, like E. coli SufE. Here we report that reengineering of the isoprenoid biosynthetic pathway in B. subtilis can offset the indispensability of the sufCDSUB operon, allowing the resultant Δsuf mutants to grow without detectable Fe-S proteins. Heterologous bidirectional complementation studies using B. subtilis and E. coli mutants showed that B. subtilis SufSU is interchangeable with E. coli SufSE but not with IscSU. In addition, functional similarity in SufB, SufC and SufD was observed between B. subtilis and E. coli. Our findings thus indicate that B. subtilis SufU is the protein that transfers sulfur from SufS to SufB, and that the SufBCD complex is the site of Fe-S cluster assembly.

Fosfomycin Biosynthesis via Transient Cytidylylation of 2-Hydroxyethylphosphonate by the Bifunctional Fom1 Enzyme.

Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, etc. Its biosynthesis starts with the formation of a carbon-phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 N-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 C-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism via such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family.

Methylcobalamin-Dependent Radical SAM C-Methyltransferase Fom3 Recognizes Cytidylyl-2-hydroxyethylphosphonate and Catalyzes the Nonstereoselective C-Methylation in Fosfomycin Biosynthesis.

A methylcobalamin (MeCbl)-dependent radical S-adenosyl-l-methionine (SAM) methyltransferase Fom3 was found to catalyze the C-methylation of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to give cytidylyl-2-hydroxypropylphosphonate (HPP-CMP), although it was originally proposed to catalyze the C-methylation of 2-hydroxyethylphosphonate to give 2-hydroxypropylphosphonate in the biosynthesis of a unique C-P bond containing antibiotic fosfomycin in Streptomyces. Unexpectedly, the Fom3 reaction product from HEP-CMP was almost a 1:1 diastereomeric mixture of HPP-CMP, indicating that the C-methylation is not stereoselective. Presumably, only the CMP moiety of HEP-CMP is critical for substrate recognition; on the other hand, the enzyme does not fix the 2-hydroxy group of the substrate and either of the prochiral hydrogen atoms at the C2 position can be abstracted by the 5'-deoxyadenosyl radical generated from SAM to form the substrate radical intermediates, which react with MeCbl to afford the corresponding products. This strict substrate recognition mechanism with no stereoselectivity of a MeCbl-dependent radical SAM methyltransferase is remarkable in natural product biosynthetic chemistry, because such a hidden clue for selective substrate recognition is likely to be found in the other biosynthetic pathways.

Characterization of bafilomycin biosynthesis in Kitasatospora setae KM-6054 and comparative analysis of gene clusters in Actinomycetales microorganisms.

Bafilomycins A1, C1 and B1 (setamycin) produced by Kitasatospora setae KM-6054 belong to the plecomacrolide family, which exhibit antibacterial, antifungal, antineoplastic and immunosuppressive activities. An analysis of gene clusters from K. setae KM-6054 governing the biosynthesis of bafilomycins revealed that it contains five large open reading frames (ORFs) encoding the multifunctional polypeptides of bafilomycin polyketide synthases (PKSs). These clustered PKS genes, which are responsible for bafilomycin biosynthesis, together encode 11 homologous sets of enzyme activities, each catalyzing a specific round of polyketide chain elongation. The region contains an additional 13 ORFs spanning a distance of 73 287 bp, some of which encode polypeptides governing other key steps in bafilomycin biosynthesis. Five ORFs, BfmB, BfmC, BfmD, BfmE and BfmF, were involved in the formation of methoxymalonyl-acyl carrier protein (ACP). Two possible regulatory genes, bfmR and bfmH, were found downstream of the above genes. A gene-knockout analysis revealed that BfmR was only a transcriptional regulator for the transcription of bafilomycin biosynthetic genes. Two genes, bfmI and bfmJ, were found downstream of bfmH. An analysis of these gene-disruption mutants in addition to an enzymatic analysis of BfmI and BfmJ revealed that BfmJ activated fumarate and BfmI functioned as a catalyst to form a fumaryl ester at the C21 hydroxyl residue of bafilomycin A1. A comparative analysis of bafilomycin gene clusters in K. setae KM-6054, Streptomyces lohii JCM 14114 and Streptomyces griseus DSM 2608 revealed that each ORF of both gene clusters in two Streptomyces strains were quite similar to each other. However, each ORF of gene cluster in K. setae KM-6054 was of lower similarity to that of corresponding ORF in the two Streptomyces species.

Novel features of the ISC machinery revealed by characterization of Escherichia coli mutants that survive without iron-sulfur clusters.

Biological assembly of iron-sulfur (Fe-S) clusters is mediated by complex systems consisting of multiple proteins. Escherichia coli possesses two distinct systems called the ISC and SUF machineries encoded by iscSUA-hscBA-fdx-iscX and sufABCDSE respectively. Deletion of both pathways results in absence of the biosynthetic apparatus for Fe-S clusters, and consequent lethality, which has hampered detailed genetic studies. Here we report that modification of the isoprenoid biosynthetic pathway can offset the indispensability of the Fe-S cluster biosynthetic systems and show that the resulting Δisc Δsuf double mutants can grow without detectable Fe-S cluster-containing proteins. We also constructed a series of mutants in which each isc gene was disrupted in the deletion background of sufABCDSE. Phenotypic analysis of the mutants revealed that Fdx, an essential electron-transfer Fe-S protein in the ISC machinery, is dispensable under anaerobic conditions, which is similar to the situation with IscA. Furthermore, we found that several suppressor mutations in IscU, an Fe-S scaffold protein responsible for the de novo Fe-S cluster assembly, could bypass the essential role of the chaperone system HscA and HscB. These findings pave the way toward a detailed molecular analysis to understand the mechanisms involved in Fe-S cluster biosynthesis.

Two ATP-binding cassette transporters involved in (S)-2-aminoethyl-cysteine uptake in thermus thermophilus.

Thermus thermophilus exhibits hypersensitivity to a lysine analog, (S)-2-aminoethyl-cysteine (AEC). Cosmid libraries were constructed using genomes from two AEC-resistant mutants, AT10 and AT14, and the cosmids that conferred AEC resistance on the wild-type strain were isolated. When the cosmid library for mutant AT14 was screened, two independent cosmids, conferring partial AEC resistance to the wild type, were obtained. Two cosmids carried a common genomic region from TTC0795 to TTC0810. This region contains genes encoding an ATP-binding cassette (ABC) transporter consisting of TTC0806/TTC0795, using TTC0807 as the periplasmic substrate-binding protein. Sequencing revealed that AT14 carries mutations in TTC0795 and TTC0969, causing decreases in the thermostability of the products. TTC0969 encodes the nucleotide-binding protein of a different ABC transporter consisting of TTC0967/TTC0968/TTC0969/TTC0970 using TTC0966 as the periplasmic substrate-binding protein. By similar screening for cosmids constructed for the mutant AT10, mutations were found at TTC0807 and TTC0969. Mutation in either of the transporter components gave partial resistance to AEC in the wild-type strain, while mutations of both transporters conferred complete AEC resistance. This result indicates that both transporters are involved in AEC uptake in T. thermophilus. To elucidate the mechanism of AEC uptake, crystal structures of TTC0807 were determined in several substrate-binding forms. The structures revealed that TTC0807 recognizes various basic amino acids by changing the side-chain conformation of Glu19, which interacts with the side-chain amino groups of the substrates.

Different biosynthetic pathways to fosfomycin in Pseudomonas syringae and Streptomyces species.

Fosfomycin is a wide-spectrum antibiotic that is used clinically to treat acute cystitis in the United States. The compound is produced by several strains of streptomycetes and pseudomonads. We sequenced the biosynthetic gene cluster responsible for fosfomycin production in Pseudomonas syringae PB-5123. Surprisingly, the biosynthetic pathway in this organism is very different from that in Streptomyces fradiae and Streptomyces wedmorensis. The pathways share the first and last steps, involving conversion of phosphoenolpyruvate to phosphonopyruvate (PnPy) and 2-hydroxypropylphosphonate (2-HPP) to fosfomycin, respectively, but the enzymes converting PnPy to 2-HPP are different. The genome of P. syringae PB-5123 lacks a gene encoding the PnPy decarboxylase found in the Streptomyces strains. Instead, it contains a gene coding for a citrate synthase-like enzyme, Psf2, homologous to the proteins that add an acetyl group to PnPy in the biosynthesis of FR-900098 and phosphinothricin. Heterologous expression and purification of Psf2 followed by activity assays confirmed the proposed activity of Psf2. Furthermore, heterologous production of fosfomycin in Pseudomonas aeruginosa from a fosmid encoding the fosfomycin biosynthetic cluster from P. syringae PB-5123 confirmed that the gene cluster is functional. Therefore, two different pathways have evolved to produce this highly potent antimicrobial agent.

Metabolic engineering for the production of prenylated polyphenols in transgenic legume plants using bacterial and plant prenyltransferases.

Prenylated polyphenols are secondary metabolites beneficial for human health because of their various biological activities. Metabolic engineering was performed using Streptomyces and Sophora flavescens prenyltransferase genes to produce prenylated polyphenols in transgenic legume plants. Three Streptomyces genes, NphB, SCO7190, and NovQ, whose gene products have broad substrate specificity, were overexpressed in a model legume, Lotus japonicus, in the cytosol, plastids or mitochondria with modification to induce the protein localization. Two plant genes, N8DT and G6DT, from Sophora flavescens whose gene products show narrow substrate specificity were also overexpressed in Lotus japonicus. Prenylated polyphenols were undetectable in these plants; however, supplementation of a flavonoid substrate resulted in the production of prenylated polyphenols such as 7-O-geranylgenistein, 6-dimethylallylnaringenin, 6-dimethylallylgenistein, 8-dimethylallynaringenin, and 6-dimethylallylgenistein in transgenic plants. Although transformants with the native NovQ did not produce prenylated polyphenols, modification of its codon usage led to the production of 6-dimethylallylnaringenin and 6-dimethylallylgenistein in transformants following naringenin supplementation. Prenylated polyphenols were not produced in mitochondrial-targeted transformants even under substrate feeding. SCO7190 was also expressed in soybean, and dimethylallylapigenin and dimethylallyldaidzein were produced by supplementing naringenin. This study demonstrated the potential for the production of novel prenylated polyphenols in transgenic plants. In particular, the enzymatic properties of prenyltransferases seemed to be altered in transgenic plants in a host species-dependent manner.

Functional characterization of the promiscuous prenyltransferase responsible for furaquinocin biosynthesis: identification of a physiological polyketide substrate and its prenylated reaction products.

Furaquinocin is a natural polyketide-isoprenoid hybrid (meroterpenoid) that exhibits antitumor activity and is produced by the Streptomyces sp. strain KO-3988. Bioinformatic analysis of furaquinocin biosynthesis has identified Fur7 as a possible prenyltransferase that attaches a geranyl group to an unidentified polyketide scaffold. Here, we report the identification of a physiological polyketide substrate for Fur7, as well as its reaction product and the biochemical characterization of Fur7. A Streptomyces albus transformant (S. albus/pWHM-Fur2_del7) harboring the furaquinocin biosynthetic gene cluster lacking the fur7 gene did not produce furaquinocin but synthesized the novel intermediate 2-methoxy-3-methyl-flaviolin. After expression and purification from Escherichia coli, the recombinant Fur7 enzyme catalyzed the transfer of a geranyl group to 2-methoxy-3-methyl-flaviolin to yield 6-prenyl-2-methoxy-3-methyl-flaviolin and 7-O-geranyl-2-methoxy-3-methyl-flaviolin in a 10:1 ratio. The reaction proceeded independently of divalent cations. When 6-prenyl-2-methoxy-3-methyl-flaviolin was added to the culture medium of S. albus/pWHM-Fur2_del7, furaquinocin production was restored. The promiscuous substrate specificity of Fur7 was demonstrated with respect to prenyl acceptor substrates and prenyl donor substrates. The steady-state kinetic constants of Fur7 with each prenyl acceptor substrate were also calculated.

New glycosylated derivatives of versipelostatin, the GRP78/Bip molecular chaperone down-regulator, from Streptomyces versipellis 4083-SVS6.

Four novel glycosylated derivatives of versipelostatin (1), versipelostatins B-E (2-5), were isolated from the culture broth of Streptomyces versipellis 4083-SVS6. The inhibitory activities of the isolated compounds against the expression of molecular chaperone GRP78 induced by 2-deoxyglucose were evaluated. Of the five versipelostatin family members, 1 and 4 were the more potent with IC(50) values of 3.5 and 4.3 microM. These results suggest that the alpha-L-oleandropyranosyl (1-->4)-beta-D-digitoxopyranosyl residue in the sugar moiety may play an important role in down-regulating GRP78 expression induced by 2-deoxyglucose.

Crystal structure of fosfomycin resistance kinase FomA from Streptomyces wedmorensis.

The fosfomycin resistance protein FomA inactivates fosfomycin by phosphorylation of the phosphonate group of the antibiotic in the presence of ATP and Mg(II). We report the crystal structure of FomA from the fosfomycin biosynthetic gene cluster of Streptomyces wedmorensis in complex with diphosphate and in ternary complex with the nonhydrolyzable ATP analog adenosine 5'-(beta,gamma-imido)-triphosphate (AMPPNP), Mg(II), and fosfomycin, at 1.53 and 2.2 angstroms resolution, respectively. The polypeptide exhibits an open alphabetaalpha sandwich fold characteristic for the amino acid kinase family of enzymes. The diphosphate complex shows significant disorder in loops surrounding the active site. As a result, the nucleotide-binding site is wide open. Binding of the substrates is followed by the partial closure of the active site and ordering of the alpha2-helix. Structural comparison with N-acetyl-L-glutamate kinase shows several similarities in the site of phosphoryl transfer: 1) preservation of architecture of the catalytical amino acids of N-acetyl-L-glutamate kinase (Lys9, Lys216, and Asp150 in FomA); 2) good superposition of the phosphate acceptor groups of the substrates, and 3) good superposition of the diphosphate molecule with the beta- and gamma-phosphates of AMPPNP, suggesting that the reaction could proceed by an associative in-line mechanism. However, differences in conformations of the triphosphate moiety of AMPPNP molecules, the long distance (5.1 angstroms) between the phosphate acceptor and donor groups in FomA, and involvement of Lys18 instead of Lys9 in binding with the gamma-phosphate may indicate a different reaction mechanism. The present work identifies the active site residues of FomA responsible for substrate binding and specificity and proposes their roles in catalysis.