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Daniel V Santi

Publications and source records attributed to Daniel V Santi.

18 recordsLinked to original sources
PubMed2006

The role of protein dynamics in thymidylate synthase catalysis: variants of conserved 2'-deoxyuridine 5'-monophosphate (dUMP)-binding Tyr-261.

The enzyme thymidylate synthase (TS) catalyzes the reductive methylation of 2'-deoxyuridine 5'-monophosphate (dUMP) to 2'-deoxythymidine 5'-monophosphate. Using kinetic and X-ray crystallography experiments, we have examined the role of the highly conserved Tyr-261 in the catalytic mechanism of TS. While Tyr-261 is distant from the site of methyl transfer, mutants at this position show a marked decrease in enzymatic activity. Given that Tyr-261 forms a hydrogen bond with the dUMP 3'-O, we hypothesized that this interaction would be important for substrate binding, orientation, and specificity. Our results, surprisingly, show that Tyr-261 contributes little to these features of the mechanism of TS. However, the residue is part of the structural core of closed ternary complexes of TS, and conservation of the size and shape of the Tyr side chain is essential for maintaining wild-type values of kcat/Km. Moderate increases in Km values for both the substrate and cofactor upon mutation of Tyr-261 arise mainly from destabilization of the active conformation of a loop containing a dUMP-binding arginine. Besides binding dUMP, this loop has a key role in stabilizing the closed conformation of the enzyme and in shielding the active site from the bulk solvent during catalysis. Changes to atomic vibrations in crystals of a ternary complex of Escherichia coli Tyr261Trp are associated with a greater than 2000-fold drop in kcat/Km. These results underline the important contribution of dynamics to catalysis in TS.

Binding Sites↗
PubMed2006

Targeted covalent inactivation of protein kinases by resorcylic acid lactone polyketides.

Resorcylic acid lactones containing a cis-enone are susceptible to Michael addition reactions and are potent inhibitors of several protein kinases. A structural-bioinformatics analysis identified a conserved Cys residue in the ATP-binding site of the kinases reported to be inhibited by cis-enone resorcylic acid lactones but absent in those that are not. Mining of the kinome database revealed that a subset of some 46 kinases contained this Cys residue. Screening a panel of 124 kinases with the resorcylic acid lactone hypothemycin showed that 18 of 19 targets containing the conserved Cys were inhibited. Kinetic analyses showed time-dependent inhibition, a hallmark of covalent inactivation, and biochemical studies of the interaction of extracellular signal-regulated kinase (ERK)2 with hypothemycin confirmed covalent adduct formation. Resorcylic acid lactones are unique among kinase inhibitors in that they target mitogen-activated protein (MAP) kinase pathways at four levels: mitogen receptors, MAP kinase kinase (MEK)1/2 and ERK1/2, and certain downstream ERK substrates. Cell lines dependent on the activation of Tyr kinase mitogen receptor targets of the resorcylic acid lactones were unusually sensitive toward hypothemycin and showed the expected inhibition of kinase phosphorylation due to inhibition of the mitogen receptors and/or MEK1/2 and ERK1/2. Among cells without mitogen receptor targets, those harboring an ERK pathway-activating B-RAF V600E mutation were selectively and potently inhibited by hypothemycin. Hypothemycin also prevented stimulated activation of the p38 cascade through inhibition of the Cys-containing targets MEK3/6 and TGF-beta-activated kinase 1 and of the JNK/SAPK (c-Jun N-terminal kinase/stress-activated protein kinase) cascade through inhibition of MEK4/7.

Adenosine Triphosphate↗
PubMed2006

Activating hybrid modular interfaces in synthetic polyketide synthases by cassette replacement of ketosynthase domains.

Unnatural combinations of polyketide synthase modules often fail to make a polyketide product. The causes of these failures are likely complex and are not yet amenable to rational correction. One possible explanation is the inability of the ketosynthase (KS) domain to extend the ketide donated to it by the upstream module. We therefore addressed the problem by exchanging KS domains of the acceptor module in a combinatorial fashion and coexpressing these chimeric modules with ketide-donor modules that naturally interact with the transplanted KS. This approach was remarkably successful in activating previously unproductive bimodular combinations, and the results augur well for the ongoing development of molecular tools to design and produce novel polyketides.

Ligases↗
PubMed2005

Redesign, synthesis and functional expression of the 6-deoxyerythronolide B polyketide synthase gene cluster.

A generic design of Type I polyketide synthase genes has been reported in which modules, and domains within modules, are flanked by sets of unique restriction sites that are repeated in every module [1]. Using the universal design, we synthesized the six-module DEBS gene cluster optimized for codon usage in E. coli, and cloned the three open reading frames into three compatible expression vectors. With one correctable exception, the amino acid substitutions required for restriction site placements were compatible with polyketide production. When expressed in E. coli the codon-optimized synthetic gene cluster produced significantly more protein than did the wild-type sequence. Indeed, for optimal polyketide production, PKS expression had to be down-regulated by promoter attenuation to achieve balance with expression of the accessory proteins needed to support polyketide biosynthesis.

Escherichia coli↗
PubMed2005

Combinatorial polyketide biosynthesis by de novo design and rearrangement of modular polyketide synthase genes.

Type I polyketide synthase (PKS) genes consist of modules approximately 3-6 kb long, which encode the structures of 2-carbon units in polyketide products. Alteration or replacement of individual PKS modules can lead to the biosynthesis of 'unnatural' natural products but existing techniques for this are time consuming. Here we describe a generic approach to the design of synthetic PKS genes where facile cassette assembly and interchange of modules and domains are facilitated by a repeated set of flanking restriction sites. To test the feasibility of this approach, we synthesized 14 modules from eight PKS clusters and associated them in 154 bimodular combinations spanning over 1.5-million bp of novel PKS gene sequences. Nearly half the combinations successfully mediated the biosynthesis of a polyketide in Escherichia coli, and all individual modules participated in productive bimodular combinations. This work provides a truly combinatorial approach for the production of polyketides.

Amino Acid Sequence↗
PubMed2005

GeMS: an advanced software package for designing synthetic genes.

A user-friendly, advanced software package for gene design is described. The software comprises an integrated suite of programs-also provided as stand-alone tools-that automatically performs the following tasks in gene design: restriction site prediction, codon optimization for any expression host, restriction site inclusion and exclusion, separation of long sequences into synthesizable fragments, T(m) and stem-loop determinations, optimal oligonucleotide component design and design verification/error-checking. The output is a complete design report and a list of optimized oligonucleotides to be prepared for subsequent gene synthesis. The user interface accommodates both inexperienced and experienced users. For inexperienced users, explanatory notes are provided such that detailed instructions are not necessary; for experienced users, a streamlined interface is provided without such notes. The software has been extensively tested in the design and successful synthesis of over 400 kb of genes, many of which exceeded 5 kb in length.

Base Sequence↗
PubMed2004

Total synthesis of long DNA sequences: synthesis of a contiguous 32-kb polyketide synthase gene cluster.

To exploit the huge potential of whole-genome sequence information, the ability to efficiently synthesize long, accurate DNA sequences is becoming increasingly important. An approach proposed toward this end involves the synthesis of approximately 5-kb segments of DNA, followed by their assembly into longer sequences by conventional cloning methods [Smith, H. O., Hutchinson, C. A., III, Pfannkoch, C. & Venter, J. C. (2003) Proc. Natl. Acad. Sci. USA 100, 15440-15445]. The major current impediment to the success of this tactic is the difficulty of building the approximately 5-kb components accurately, efficiently, and rapidly from short synthetic oligonucleotide building blocks. We have developed and implemented a strategy for the high-throughput synthesis of long, accurate DNA sequences. Unpurified 40-base synthetic oligonucleotides are built into 500- to 800-bp "synthons" with low error frequency by automated PCR-based gene synthesis. By parallel processing, these synthons are efficiently joined into multisynthon approximately 5-kb segments by using only three endonucleases and "ligation by selection." These large segments can be subsequently assembled into very long sequences by conventional cloning. We validated the approach by building a synthetic 31,656-bp polyketide synthase gene cluster whose functionality was demonstrated by its ability to produce the megaenzyme and its polyketide product in Escherichia coli.

Base Sequence↗
PubMed2004

Synthesis and biological activities of novel 17-aminogeldanamycin derivatives.

Geldanamycin interferes with the action of heat shock protein 90 (Hsp90) by binding to the N-terminal ATP binding site and inhibiting an essential ATPase activity. In a program directed toward finding potent, water soluble inhibitors of Hsp90, we prepared a library of over sixty 17-alkylamino-17-demethoxygeldanamycin analogs, and compared their affinity for Hsp90, ability to inhibit growth of SKBr3 mammalian cells, and in selected cases, water solubility. Over 20 analogs showed cell growth inhibition potencies similar to that of 17-allylamino-17-demethoxygeldanamycin (17-AAG), the front-runner geldanamycin analog that is currently in multiple clinical trials. Many of these analogs showed water solubility properties that were desirable for formulation. One of the most potent and water-soluble analogs in the series was 17-(2-dimethylaminoethyl)amino-17-demethoxygeldanamycin (17-DMAG), which was independently prepared by the NCI and will soon enter clinical trials. Importantly, the binding affinity of these analogs to the molecular target Hsp90 does not correlate well with their cytotoxicity in SKBr3 cells.

Amino Acid Sequence↗
PubMed2004

A sensitive and robust method for quantification of intracellular short-chain coenzyme A esters.

A procedure for the analysis of short-chain intracellular coenzyme A (CoA) esters and adenine nucleotide pools in microbial cells is described. The simultaneous isolation of bacterial cells from media, quenching of their metabolism, and extraction of metabolites was accomplished by centrifugation of cells through a layer of silicone oil into a denser solution of trichloroacetic acid. The acid was neutralized by extraction into Freon containing tri-n-octylamine to provide a salt-free solution of cell metabolites. After high-performance liquid chromatography separation, CoA, CoA esters, and adenine-containing nucleotides were derivatized by postcolumn reaction with bromoacetaldehyde to form the fluorescent 1,N6-ethenoadenine adducts which were analyzed by a fluorescence detector at picomolar levels.

Chromatography, High Pressure Liquid↗
PubMed2003

Filter binding assay for the geldanamycin-heat shock protein 90 interaction.

A filter binding assay to measure affinity of [3H-allyl]17-allylamino geldanamycin ([3H]AAG) for the ATP binding site of the N-terminal domain of human Hsp90alpha (hHsp90alpha9-236) was developed. Diethylaminoethyl cellulose or glass fiber filters impregnated with polyethyleneimine were used to capture the [3H]AAG-Hsp90 complex, and conditions which washed >98% of free [3H]AAG from the filters were developed. The complex formed at a rapid rate (k(on)=2.5 x 10(7)Lmol(-1) x s(-1)) and dissociated with a half-life of 2.3 min (k(off)=5 x 10(-3) x s(-1)). hHsp90alpha9-236 bound to [3H]AAG with a K(d) value of 0.4+/-0.1 microM. [3H]AAG had similar affinities for full-length hHsp90alpha and for hHsp90alpha9-236 variants containing biotinylated N-terminal biotinylation signal sequences and N- or C-terminal His(6) tags. Geldanamycin, ADP, ATP, and radicicol-all known to bind to the ATP domain of Hsp90-competed with [3H]AAG for binding to hHsp90alpha9-236, showing K(d) values in good agreement with reported values.

Adenosine Diphosphate↗
PubMed2003

A model of structure and catalysis for ketoreductase domains in modular polyketide synthases.

A putative catalytic triad consisting of tyrosine, serine, and lysine residues was identified in the ketoreductase (KR) domains of modular polyketide synthases (PKSs) based on homology modeling to the short chain dehydrogenase/reductase (SDR) superfamily of enzymes. This was tested by constructing point mutations for each of these three amino acid residues in the KR domain of module 6 of the 6-deoxyerythronolide B synthase (DEBS) and determining the effect on ketoreduction. Experiments conducted in vitro with the truncated DEBS Module 6+TE (M6+TE) enzyme purified from Escherichia coli indicated that any of three mutations, Tyr --> Phe, Ser --> Ala, and Lys --> Glu, abolish KR activity in formation of the triketide lactone product from a diketide substrate. The same mutations were also introduced in module 6 of the full DEBS gene set and expressed in Streptomyces lividans for in vivo analysis. In this case, the Tyr --> Phe mutation appeared to completely eliminate KR6 activity, leading to the 3-keto derivative of 6-deoxyerythronolide B, whereas the other two mutations, Ser --> Ala and Lys --> Glu, result in a mixture of both reduced and unreduced compounds at the C-3 position. The results support a model analogous to SDRs in which the conserved tyrosine serves as a proton donating catalytic residue. In contrast to deletion of the entire KR6 domain of DEBS, which causes a loss in substrate specificity of the adjacent acyltransferase (AT) domain in module 6, these mutations do not affect the AT6 specificity and offer a potentially superior approach to KR inactivation for engineered biosynthesis of novel polyketides. The homology modeling studies also led to identification of amino acid residues predictive of the stereochemical nature of KR domains. Finally, a method is described for the rapid purification of engineered PKS modules that consists of a biotin recognition sequence C-terminal to the thioesterase domain and adsorption of the biotinylated module from crude extracts to immobilized streptavidin. Immobilized M6+TE obtained by this method was over 95% pure and as catalytically effective as M6+TE in solution.

Alcohol Oxidoreductases↗
PubMed2003

Crystal structure and molecular modeling of 17-DMAG in complex with human Hsp90.

Hsp90 is an attractive chemotherapeutic target because it chaperones the folding of proteins found in multiple signal transduction pathways. We describe the 1.75 A resolution crystal structure of human Hsp90 alpha (residues 9-236) complexed with 17-desmethoxy-17-N,N-dimethylaminoethylamino-geldanamycin (17-DMAG). The structure revealed an altered set of interactions between the 17-substituent and the protein compared to geldanamycin and the 17-dimethylaminoethyl moiety pointing into solvent, but otherwise was similar to that reported for the complex with geldanamycin. Targeted molecular dynamics simulations and energetic analysis indicate that geldanamycin undergoes two major conformational changes when it binds Hsp90, with the key step of the conversion being the trans to cis conformational change of the macrocycle amide bond. We speculate that 17-DMAG analogs constrained to a cis-amide in the ground state could provide a significant increase in affinity for Hsp90.

Benzoquinones↗
PubMed2003

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase.

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.

Conserved Sequence↗
PubMed2002

Metabolic engineering of a methylmalonyl-CoA mutase-epimerase pathway for complex polyketide biosynthesis in Escherichia coli.

A barrier to heterologous production of complex polyketides in Escherichia coli is the lack of (2S)-methylmalonyl-CoA, a common extender substrate for the biosynthesis of complex polyketides by modular polyketide synthases. One biosynthetic route to (2S)-methylmalonyl-CoA involves the sequential actions of two enzymes, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase, which convert succinyl-CoA to (2R)- and then to (2S)-methylmalonyl-CoA. As reported [McKie, N., et al. (1990) Biochem. J. 269, 293-298; Haller, T., et al. (2000) Biochemistry 39, 4622-4629], when genes encoding coenzyme B(12)-dependent methylmalonyl-CoA mutases were expressed in E. coli, the inactive apo-enzyme was produced. However, when cells harboring the mutase genes from Propionibacterium shermanii or E. coli were treated with the B12 precursor hydroxocobalamin, active holo-enzyme was isolated, and (2R)-methylmalonyl-CoA represented approximately 10% of the intracellular CoA pool. When the E. coli BAP1 cell line [Pfeifer, B. A., et al. (2001) Science 291, 1790-1792] harboring plasmids that expressed P. shermanii methylmalonyl-CoA mutase, Streptomyces coelicolor methylmalonyl-CoA epimerase, and the polyketide synthase DEBS (6-deoxyerythronolide B synthase) was fed propionate and hydroxocobalamin, the polyketide 6-deoxyerythronolide B (6-dEB) was produced. Isotopic labeling studies using [(13)C]propionate showed that the starter unit for polyketide synthesis was derived exclusively from exogenous propionate, while the extender units stemmed from methylmalonyl-CoA via the mutase-epimerase pathway. Thus, the introduction of an engineered mutase-epimerase pathway in E. coli enabled the uncoupling of carbon sources used to produce starter and extender units of polyketides.

Cloning, Molecular↗
PubMed2002

Stable expression of a synthetic gene for the human motilin receptor: use in an aequorin-based receptor activation assay.

A synthetic gene for the human motilin receptor containing 33 unique restriction sites was designed and stably coexpressed in HEK293 cells with the bioluminescent Ca(2+) indicator protein aequorin. The dose-dependent response of the receptor to motilin was demonstrated using transient transfections, and a stable cell line was selected. [(125)I]Motilin binding was used to estimate receptor expression level for the stable cell line, and titration of a membrane preparation indicated a K(d) value of 0.8 nM. The same cell line was used to evaluate a panel of erythromycin-derived agonists and provided EC(50) values for receptor activation that agree closely with data obtained in contractility assays. The peptide antagonist ANQ11125 (Phe3Leu13 motilin 1-14) inhibited motilin induced response with a K(i) value of 10 nM. The system is well-suited for the screening of compound libraries and receptor mutagenesis studies.

Aequorin↗
PubMed2002

Characterization of the 23 S ribosomal RNA m5U1939 methyltransferase from Escherichia coli.

An Escherichia coli open reading frame, ygcA, was identified as a putative 23 S ribosomal RNA 5-methyluridine methyltransferase (Gustafsson, C., Reid, R., Greene, P. J., and Santi, D. V. (1996) Nucleic Acids Res. 24, 3756-3762). We have cloned, expressed, and purified the 50-kDa protein encoded by ygcA. The purified enzyme catalyzed the AdoMet-dependent methylation of 23 S rRNA but did not act upon 16 S rRNA or tRNA. A high performance liquid chromatography-based nucleoside analysis identified the reaction product as 5-methyluridine. The enzyme specifically methylated U1939 as determined by a nuclease protection assay and by methylation assays using site-specific mutants of 23 S rRNA. A 40-nucleotide 23 S rRNA fragment (nucleotide 1930--1969) also served as an efficient substrate for the enzyme. The apparent K(m) values for the 40-mer RNA oligonucleotide and AdoMet were 3 and 26 microm, respectively, and the apparent k(cat) was 0.06 s(-1). The enzyme contains two equivalents of iron/monomer and has a sequence motif similar to a motif found in iron-sulfur proteins. We propose to name this gene rumA and accordingly name the protein product as RumA for RNA uridine methyltransferase.

Bacterial Proteins↗