Biomedical subjects

D V Santi

Publications and source records attributed to D V Santi.

At least 19 recordsLinked to original sources

PubMed2003

Approaches to stabilization of inter-domain recombination in polyketide synthase gene expression plasmids.

Regions of extremely high sequence identity are recurrent in modular polyketide synthase (PKS) genes. Such sequences are potentially detrimental to the stability of PKS expression plasmids used in the combinatorial biosynthesis of polyketide metabolites. We present two different solutions for circumventing intra-plasmid recombination within the megalomicin PKS genes in Streptomyces coelicolor. In one example, a synthetic gene was used in which the codon usage was reengineered without affecting the primary amino acid sequence. The other approach utilized a heterologous subunit complementation strategy to replace one of the problematic regions. Both methods resulted in PKS complexes capable of 6-deoxyerythronolide B analogue biosynthesis in S. coelicolor CH999, permitting reproducible scale-up to at least 5-l stirred-tank fermentation and a comparison of diketide precursor incorporation efficiencies between the erythromycin and megalomicin PKSs.

Base Sequence↗

PubMed2001

An FKBP12 binding assay based upon biotinylated FKBP12.

A binding assay was developed for measuring the affinity of FKBP12 ligands. A biotinylation signal sequence was fused to the 5' end of the human FKBP12 gene, and the fusion protein was expressed in Escherichia coli with biotin ligase. The fusion protein was immobilized in avidin-coated multiwell plates, and varying concentrations of test ligands were allowed to compete with [3H]FK506 for FKBP12 sites on the plate. The assay provided Kd values for FK520, 32-hydroxyethyl indolyl FK520, and 18-ene, 20-oxa FK520 that are in agreement with previously reported values. The assay provides a convenient and rapid method for the assessment of FKBP12 binding by small molecules.

Bacterial Proteins↗

PubMed2000

m5C RNA and m5C DNA methyl transferases use different cysteine residues as catalysts.

A family of RNA m(5)C methyl transferases (MTases) containing over 55 members in eight subfamilies has been identified recently by an iterative search of the genomic sequence databases by using the known 16S rRNA m(5)C 967 MTase, Fmu, as an initial probe. The RNA m(5)C MTase family contained sequence motifs that were highly homologous to motifs in the DNA m(5)C MTases, including the ProCys sequence that contains the essential Cys catalyst of the functionally similar DNA-modifying enzymes; it was reasonable to assign the Cys nucleophile to be that in the conserved ProCys. The family also contained an additional conserved Cys residue that aligns with the nucleophilic catalyst in m(5)U54 tRNA MTase. Surprisingly, the mutant of the putative Cys catalyst in the ProCys sequence was active and formed a covalent complex with 5-fluorocytosine-containing RNA, whereas the mutant at the other conserved Cys was inactive and unable to form the complex. Thus, notwithstanding the highly homologous sequences and similar functions, the RNA m(5)C MTase uses a different Cys as a catalytic nucleophile than the DNA m(5)C MTases. The catalytic Cys seems to be determined, not by the target base that is modified, but by whether the substrate is DNA or RNA. The function of the conserved ProCys sequence in the RNA m(5)C MTases remains unknown.

Amino Acid Sequence↗

PubMed2000

An approach for obtaining perfect hybridization probes for unknown polyketide synthase genes: a search for the epothilone gene cluster.

An approach is described for obtaining 'perfect probes' for type I modular polyketide synthase (PKS) gene clusters that in turn enables the identification of all such gene clusters in a genome. The approach involves sequencing small fragments of a random genomic DNA library containing one or more modular PKS gene clusters, and identifying which fragments emanate from PKS genes. Knowing the approximate sizes of the genome and the target gene cluster, one can predict the the frequency that a PKS gene fragment will be present in the library sequenced. Computer simulations of the approach were applied to the known PKS and non-ribosomal peptide synthetase (NRPS) gene clusters in the Bacillus subtilus genome. The approach was then used to identify PKS gene fragments in a strain of Sorangium cellulosum that produces epothilone. In addition to identifying fragments of the epothilone gene cluster, we obtained 11 unique fragments from other PKS gene clusters; the results suggest that there may be six to eight PKS gene clusters in this organism. In addition, we identified four unique fragments of NRPS genes, demonstrating that the approach is also applicable for identification of these modular gene clusters.

Antineoplastic Agents↗

PubMed2000

Effects of subunit occupancy on partitioning of an intermediate in thymidylate synthase mutants.

Experimental evidence for a 5-exocyclic methylene-dUMP intermediate in the thymidylate synthase reaction was recently obtained by demonstrating that tryptophan 82 mutants of the Lactobacillus casei enzyme produced 5-(2-hydroxyethyl)thiomethyl-dUMP (HETM-dUMP) (Barret, J. E., Maltby, D. A., Santi, D. V., and Schultz, P. G. (1998) J. Am. Chem. Soc. 120, 449-450). The unusual product was proposed to emanate from trapping of the intermediate with beta-mercaptoethanol in competition with hydride transfer from H(4)folate to form dTMP. Using mutants of the C-terminal residue of thymidylate synthase, we found that the ratio of HETM-dUMP to dTMP varies as a function of CH(2)H(4)folate concentration. This observation seemed inconsistent with the conclusion that both products arose from a common intermediate in which CH(2)H(4)folate was already bound to the enzyme. The enigma was resolved by a kinetic model that allowed for differential partitioning of the intermediate formed on each of the two subunits of the homodimeric enzyme in forming the two different products. With three C-terminal mutants of L. casei TS, HETM-dUMP formation was consistent with a model in which product formation occurs upon occupancy of the first completely bound subunit, the rate of which is unaffected by occupancy of the second subunit. With one analogous E. coli TS mutant, HETM-dUMP formation occurred upon occupancy of the first subunit, but was inhibited when both subunits were occupied. With all mutants, dTMP formation occurs from occupied forms of both subunits at different rates; here, binding of cofactor to the first subunit decreased affinity for the second, but the reaction occurred faster in the enzyme form with both subunits bound to dUMP and CH(2)H(4)folate. The model resolves the apparent enigma of the cofactor-dependent product distribution and supports the conclusion that the exocyclic methylene intermediate is common to both HETM-dUMP and dTMP formation.

Alanine↗

PubMed2000

Energetic contributions of four arginines to phosphate-binding in thymidylate synthase are more than additive and depend on optimization of "effective charge balance".

In thymidylate synthase, four conserved arginines provide two hydrogen bonds each to the oxygens of the phosphate group of the substrate, 2'-deoxyuridine-5'-monophosphate. Of these, R23, R178, and R179 are far removed from the site of methyl transfer and contribute to catalysis solely through binding and orientation of ligands. These arginines can be substituted by other residues, while still retaining more than 1% activity of the wild-type enzyme. We compared the kinetics and determined the crystal structures of dUMP complexes of three of the most active, uncharged single mutants of these arginines, R23I, R178T, and R179T, and of double mutants (R23I, R179T) and (R178T, R179T). The dramatically higher K(m) for R178T compared to the other two single mutants arises from the effects of R178 substitution on the orientation of dUMP; 10-15-fold increases in for R23I and R178T reflect the role of these residues in stabilizing the closed conformation of TS in ternary complexes. The free energy for productive dUMP binding, DeltaG(S), increases by at least 1 kcal/mol for each mutant, even when dUMP orientation and mobility in the crystal structure is the same as in wild-type enzyme. Thus, the four arginines do not contribute excess positive charge to the PO(4)(-2) binding site; rather, they ideally complement the charge and geometry of the phosphate moiety. More-than-additive increases in DeltaG(S) seen in the double mutants are consistent with quadratic increases in DeltaG(S) predicted for deviations from ideal electrostatic interactions and may also reflect cooperative binding of the arginines to the phosphate oxygens.

Arginine↗

PubMed2000

The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I.

Pseudouridine synthases catalyze the isomerization of specific uridines to pseudouridine in a variety of RNAs, yet the basis for recognition of the RNA sites or how they catalyze this reaction is unknown. The crystal structure of pseudouridine synthase I from Escherichia coli, which, for example, modifies positions 38, 39 and/or 40 in tRNA, reveals a dimeric protein that contains two positively charged, RNA-binding clefts along the surface of the protein. Each cleft contains a highly conserved aspartic acid located at its center. The structural domains have a topological similarity to those of other RNA-binding proteins, though the mode of interaction with tRNA appears to be unique. The structure suggests that a dimeric enzyme is required for binding transfer RNA and subsequent pseudouridine formation.

Amino Acid Sequence↗

PubMed2000

Mechanistic studies of RNA modifying enzymes. RNA pseudouridine synthase and m5cytosine methyl transferase.

RNA has numerous post-transcriptional modifications, but relatively little is known about the enzymes that catalyze such modifications or about the functions of the modified residues. Our laboratory has been engaged in studies of the structure and function of enzymes that catalyze the conversion of Urd residues in RNA to pseudouridine (psi Synthase), and Cyd to 5-methylcytidine methyl transferase (RNA m5C Mtase). The presentation will summarize recent results from our laboratory.

Amino Acid Motifs↗

PubMed2000

Replacement set mutagenesis of the four phosphate-binding arginine residues of thymidylate synthase.

Arginines R23, R178, R179 and R218 in thymidylate synthase (TS, EC 2. 1.1.45) are hydrogen bond donors to the phosphate moiety of the substrate, dUMP. In order to investigate how these arginines contribute to enzyme function, we prepared complete replacement sets of mutants at each of the four sites in Lactobacillus casei TS. Mutations of R23 increase K:(m) for dUMP 2-20-fold, increase K:(m) for cofactor 8-40-fold and decrease k(cat) 9-20-fold, reflecting the direct role of the R23 side chain in binding and orienting the cofactor in ternary complexes of the enzyme. Mutations of R178 increase K:(m) for dUMP 40-2000-fold, increase K:(m) for cofactor 3-20-fold and do not significantly affect k(cat). These results are consistent with the fact that this residue is an integral part of the dUMP-binding wall and contributes to the orientation and ordering of several other dUMP binding residues. Kinetic parameters for all R179 mutations except R179P were not significantly different from wild-type values, reflecting the fact that this external arginine does not directly contact the cofactor or other ligand-binding residues. R218 is essential for the structure of the catalytic site and all mutations of this arginine except R218K were inactive.

Arginine↗

PubMed1999

The mechanism of pseudouridine synthase I as deduced from its interaction with 5-fluorouracil-tRNA.

tRNA pseudouridine synthase I (PsiSI) catalyzes the conversion of uridine to Psi at positions 38, 39, and/or 40 in the anticodon loop of tRNAs. PsiSI forms a covalent adduct with 5-fluorouracil (FUra)-tRNA (tRNA(Phe) containing FUra in place of Ura) to form a putative analog of a steady-state intermediate in the normal reaction pathway. Previously, we proposed that a conserved aspartate of the enzyme serves as a nucleophilic catalyst in both the normal enzyme reaction and in the formation of a covalent complex with FUra-tRNA. The covalent adduct between FUra-tRNA and PsiSI was isolated and disrupted by hydrolysis and the FUra-tRNA was recovered. The target FU39 of the recovered FUra-tRNA was modified by the addition of water across the 5,6-double bond of the pyrimidine base to form 5,6-dihydro-6-hydroxy-5-fluorouridine. We deduced that the conserved aspartate of the enzyme adds to the 6-position of the target FUra to form a stable covalent adduct, which can undergo O-acyl hydrolytic cleavage to form the observed product. Assuming that an analogous covalent complex is formed in the normal reaction, we have deduced a complete mechanism for PsiS.

Fluorouracil↗

PubMed1999

A multiplasmid approach to preparing large libraries of polyketides.

A three-plasmid system for heterologous expression of 6-deoxyerythronolide B synthase (DEBS) has been developed to facilitate combinatorial biosynthesis of polyketides made by type I modular polyketide synthases (PKSs). The eryA PKS genes encoding the three DEBS subunits were individually cloned into three compatible Streptomyces vectors carrying mutually selectable antibiotic resistance markers. A strain of Streptomyces lividans transformed with all three plasmids produced 6-deoxyerythronolide B at a level similar to that of a strain transformed with a single plasmid containing all three genes. The utility of this system in combinatorial biosynthesis was demonstrated through production of a library of modified polyketide macrolactones by using versions of each plasmid constructed to contain defined mutations. Combinations of these vector sets were introduced into S. lividans, resulting in strains producing a wide range of 6-deoxyerythronolide B analogs. This method can be extended to any modular PKS and has the potential to produce thousands of novel natural products, including ones derived from further modification of the PKS products by tailoring enzymes.

Anti-Bacterial Agents↗

PubMed1999

Exposition of a family of RNA m(5)C methyltransferases from searching genomic and proteomic sequences.

The Escherichia coli fmu gene product has recently been determined to be the 16S rRNA m(5)C 967 methyltransferase. As such, Fmu represents the first protein identified as an S -adenosyl-L-methionine (AdoMet)- dependent RNA m(5)C methyltransferase whose amino acid sequence is known. Using the amino acid sequence of Fmu as an initial probe in an iterative search of completed DNA sequence databases, 27 homologous ORF products were identified as probable RNA m(5)C methyltransferases. Further analysis of sequences in undeposited genomic sequencing data and EST databases yielded more than 30 additional homologs. These putative RNA m(5)C methyltransferases are grouped into eight subfamilies, some of which are predicted to consist of direct genetic counterparts, or orthologs. The enzymes proposed to be RNA m(5)C methyltransferases have sequence motifs closely related to signature sequences found in the well-studied DNA m(5)C methyltransferases and other AdoMet-dependent methyltransferases. Structure-function correlates in the known AdoMet methyltransferases support the assignment of this family as RNA m(5)C methyltransferases.

Amino Acid Sequence↗

PubMed1999

Phthalein derivatives as a new tool for selectivity in thymidylate synthase inhibition.

A new set of phthalein derivatives stemming from the lead compound, phenolphthalein, were designed to specifically complement structural features of a bacterial form of thymidylate synthase (Lactobacillus casei, LcTS) versus the human TS (hTS) enzyme. The new compounds were screened for their activity and their specificity against TS enzymes from different species, namely, L. casei (LcTS), Pneumocystis carinii (PcTS), Cryptococcus neoformans (CnTS), and human thymidylate synthase (hTS). Apparent inhibition constants (Ki) for all the compounds against LcTS were determined, and inhibition factors (IF, ratio between the initial rates of the enzymatic reaction in the presence and absence of each inhibitor) against each of the four TS species were measured. A strong correlation was found between the two activity parameters, IF and Ki, and therefore the simpler IF was used as a screening factor in order to accelerate biological evaluation. Compounds 5b, 5c, 5ba, and 6bc showed substantial inhibition of LcTS while remaining largely inactive against hTS, illustrating for the first time remarkable species specificity among TSs. Due to sequence homology between the enzymes, several compounds also showed high activity and specificity for CnTS. In particular, 3-hydroxy-3-(3-chloro-4-hydroxyphenyl)-6-nitro-1H, 3H-naphtho[1,8-c,d]pyran-1-one (6bc) showed an IF < 0.04 for CnTS (Ki = 0.45 microM) while remaining inactive in the hTS assay at the maximum solubility concentration of the compound (200 microM). In cell culture assays most of the compounds were found to be noncytotoxic to human cell lines but were cytotoxic against several species of Gram-positive bacteria. These results are consistent with the enzymatic assays. Intriguingly, several compounds also had selective activity against Cr. neoformans in cell culture assay. In general, the most active and selective compounds against the Gram-positive bacteria were those designed and found in the enzyme assay to be specific for LcTS versus hTS. The original lead compound was least selective against most of the cell lines tested. To our knowledge these compounds are the first TS inhibitors selective for bacterial TS with respect to hTS.

Anti-Bacterial Agents↗

PubMed1999

Identification of the 16S rRNA m5C967 methyltransferase from Escherichia coli.

The fmu gene product has been proposed to be an RNA methyltransferase [Koonin, E. V. (1994) Nucleic Acids Res. 22, 2476-2478]. Fmu has been cloned and expressed, and the encoded 47 kDa protein has been purified and characterized. The enzyme catalyzed specific methylation of C967 of unmodified 16S rRNA transcripts. A 16mer stem-loop structure containing C967 (nt 960-975) was also a good substrate for the enzyme in vitro. Methylation of C967 was confirmed by several methods including analysis of RNase T1 digests and nearest-neighbor analysis. Fmu did not catalyze methylation of transcripts of 23S rRNA. E. coli cells that contained kanr-disrupted fmu produced 16S rRNA that could be specifically methylated by Fmu in vitro at C967 but not C1407. Further, fmu disruption did not significantly alter the growth rate of E. coli in rich or minimal media. We propose renaming this ORF "rrmB" and the enzyme "RrmB" for rRNA methyltransferase.

5-Methylcytosine↗

PubMed1999

Effect of amino acid substitutions at the subunit interface on the stability and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the Dimer interface of thymidylate synthase.

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.

Arginine↗

PubMed1999

Structure-based design of inhibitors specific for bacterial thymidylate synthase.

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.

Amino Acid Substitution↗

PubMed1999

Tyrosine 146 of thymidylate synthase assists proton abstraction from the 5-position of 2'-deoxyuridine 5'-monophosphate.

Tyr 146 of TS has been proposed to assist in the removal of the proton from the 5-carbon of the pyrimidine in a steady-state intermediate [Hyatt, D. C., Maley, F., and Montfort, W. R. (1997) Biochemistry 36, 4585-4594]. We prepared a replacement set of mutations at position 146 of L. casei TS. The kcat and kcat/Km values of 15 mutants studied were significantly lower than wild-type TS. There was no effect on the Km of dUMP, and only moderate effects on the Km of the cofactor. We concluded that Y146 is not directly involved in substrate binding, but contributes significantly to catalysis. We also examined the Y146 mutants as catalysts for cofactor-independent dehalogenation of BrdUMP, a reaction which simulates early steps of the normal pathway up to and including enzyme-nucleotide covalent adduct formation. Many mutants had activity comparable to the wild-type enzyme, and we concluded that the effects of Tyr 146 mutations occur after the initial covalent adduct is formed. A covalent steady-state intermediate-containing enzyme, dUMP, and cofactor accumulated with Tyr 146 mutants, and could be isolated by SDS-PAGE. The complex was kinetically competent as an intermediate in dTMP formation. Using Y146D and F, it was shown that removal of the C-5 proton from the covalent intermediate was defective. We conclude that in the wild-type enzyme Tyr 146 assists in proton removal from the covalent intermediate. Mutants containing fluorinated tyrosines at position 146 showed an inverse linear correlation of activity versus acidity, again indicating that the basicity of the phenolic oxygen plays an important catalytic role. Speculations of how the poorly basic phenol group might assist proton removal are made in which Tyr 146 acts as a proton conduit to N5 of the cofactor or as a cohort of a water molecule serving as the direct general base catalyst.

Binding Sites↗

PubMed1999

Disulfide engineering at the dimer interface of Lactobacillus casei thymidylate synthase: crystal structure of the T155C/E188C/C244T mutant.

The crystal structure of a covalently cross-linked Lactobacillus casei thymidylate synthase has been determined at 2.8 A resolution. The sites for mutation to achieve the bis-disulfide linked dimer were identified using the disulfide modeling program MODIP. The mutant so obtained was found to be remarkably thermostable. This increase in stability has been reasoned to be entirely a consequence of the covalent gluing between the two subunits.

Crystallography, X-Ray↗