The monoterpene cyclase limonene synthase transforms geranyl diphosphate to a monocyclic olefin and constitutes the simplest model for terpenoid cyclase catalysis. (-)-4S-Limonene synthase preprotein from spearmint bears a long plastidial targeting sequence. Difficulty expressing the full-length preprotein in Escherichia coli is encountered because of host codon usage, inclusion body formation, and the tight association of bacterial chaperones with the transit peptide. The purified preprotein is also kinetically impaired relative to the mixture of N-blocked native proteins produced in vivo by proteolytic processing in plastids. Therefore, the targeting sequence, that precedes a tandem pair of arginines (R58R59) which is highly conserved in the monoterpene synthases, was removed. Expression of this truncated protein, from a vector that encodes a tRNA for two rare arginine codons (pSBET), affords a soluble, tractable 'pseudomature' form of the enzyme that is catalytically more efficient than the native species. Truncation up to and including R58, or substitution of R59, yields enzymes that are incapable of converting the natural substrate geranyl diphosphate, via the enzymatically formed tertiary allylic isomer 3S-linalyl diphosphate, to (-)-limonene. However, these enzymes are able to cyclize exogenously supplied 3S-linalyl diphosphate to the olefinic product. This result indicates a role for the tandem arginines in the unique diphosphate migration step accompanying formation of the intermediate 3S-linalyl diphosphate and preceding the final cyclization reaction catalyzed by the monoterpene synthases. The structural basis for this coupled isomerization-cyclization reaction sequence can be inferred by homology modeling of (-)-4S-limonene synthase based on the three-dimensional structure of the sesquiterpene cyclase epi-aristolochene synthase [Starks, C. M., Back, K., Chappell, J., and Noel, J. P. (1997) Science 277, 1815-1820].

Nucleosome remodeling complexes comprise several large families of chromatin modifiers that integrate multiple epigenetic control signals to play key roles in cell type-specific transcription regulation. We previously isolated a methyl-binding domain protein 2 (MBD2)-containing nucleosome remodeling and deacetylation (NuRD) complex from primary erythroid cells and showed that MBD2 contributes to DNA methylation-dependent embryonic and fetal β-type globin gene silencing during development in vivo. Here we present structural and biophysical details of the coiledcoil interaction between MBD2 and p66α, a critical component of the MBD2-NuRD complex. We show that enforced expression of the isolated p66α coiled-coil domain relieves MBD2-mediated globin gene silencing and that the expressed peptide interacts only with a subset of components of the MBD2-NuRD complex that does not include native p66α or Mi-2. These results demonstrate the central importance of the coiled-coil interaction and suggest that MBD2-dependent DNA methylation-driven gene silencing can be disrupted by selectively targeting this coiled-coil complex.epigenetics | gene regulation D NA methylation involves the enzymatic addition of a methyl group at the C5 position of symmetrically opposed cytosine bases in a double-stranded cytosine-guanosine sequence (CpG). Regions of increased CpG content (CpG islands) often are found associated with promoters and, when methylated, silence expression of the associated gene (1, 2). Although most CpG islands are largely unmethylated in normal adult tissues, a subset of CpG islands is methylated in specific tissue subtypes, stages of differentiation, and development. Importantly, hypermethylation and silencing of tumor suppressor genes represents a pro-oncogenic change found in a wide range of malignancies (3). These observations have raised interest in DNA methylation as both an important genetic regulatory mechanism and a potential therapeutic target for either re-expression of developmentally silenced genes or reversing tumor suppressor gene silencing in cancer (4, 5).The methyl cytosine binding proteins include a family that specifically recognizes the methylated CpG sequence through an ∼60 amino acid methyl-binding domain (MBD). There are five members of the MBD family in mammals: methyl CpG-binding protein 2 (MeCP2), the first to be identified (6), and MBD1 through MBD4 (7). We and others have isolated and characterized an MBD2-containing nucleosome remodeling and deacetylation (NuRD) complex (referred to as "MBD2-NuRD") that binds methylated DNA and regulates transcription of the associated gene (8-10). The MBD2-NuRD complex comprises at least one homolog of six core proteins: MBD2, retinoblastoma protein-associated protein (RbAp46 or -48) Mi-2(α or β), p66(α or β), histone deacetylase (HDAC1 or 2), and metastasis associated (MTA1 or -2) (Fig. 1A). However, the specific interactions involved in the formation of the MBD2-NuRD complex have not been delineated clearly; information that is key to understanding (i) ...

The Oct and Sox transcription factors control many different aspects of neural development and embryogenesis, often binding to adjacent sites on DNA, and interacting with one another through their DNA binding domains to regulate transcription synergistically. Oct proteins contain two DNA binding domains (POU S and POU HD ) connected by a flexible linker, which interact with DNA in a bipartite manner. Residual dipolar coupling measurements on the binary Oct1⅐DNA complex reveal that the two domains are characterized by distinct alignment tensors in both phage pf1 and polyethylene glycol/hexanol liquid crystalline media. We show that this difference is due to a fast microscopic dissociation/association process involving alternative binding modes for the weaker binding POU S domain in the binary complex. Upon binding of Sox2 to an adjacent site in the Hoxb1 regulatory element, all components of the ternary Oct1⅐Sox2⅐DNA complex share a single alignment tensor. Thus ternary complex formation increases the site-specific affinity of Oct1 for DNA by effectively locking the POU S domain in a single orientation on the DNA. The solution NMR structure of the ternary 42 kDa Oct1⅐Sox2⅐Hoxb1-DNA complex, determined by novel procedures based on orientational restraints from dipolar couplings and conjoined rigid body/torsion angle dynamics, reveals that Sox2 and POU S interact through a predominantly hydrophobic interface, surrounded by a ring of electrostatic interactions. These observations suggest a mechanism of combinatorial control involving direct protein-protein interactions on the DNA whereby Oct1 in conjunction with a co-interacting transcription factor provide cell-specific transcription regulation.Transcription regulation in eukaryotes involves the formation of protein-DNA complexes that can interact with and modulate the downstream transcriptional machinery (1, 2). Unlike prokaryotes that often use a single protein for this function, eukaryotes generally employ complexes of multiple proteins in what has been termed combinatorial control (2, 3). This mechanism effectively integrates many different signaling pathways to provide a more complex regulatory network based on a finite number of transcription factors. Biological and structural studies of these complexes indicate that combinatorial control is achieved by employing transcription factors with adaptable DNA and protein binding surfaces. This adaptability allows different combinations of these factors to interact on specific promoter elements to drive synergistic transcription regulation (2, 3).Transcription regulation by the Oct and Sox families of transcription factors reflects many of the principles of combinatorial control. Different members of each family have been shown to interact on different promoter elements to regulate transcription during embryogenesis and neural development (4, 5). The Sox family is characterized by an HMG-box DNA binding domain that binds in the minor groove, bends DNA (50 -90°) and specifically recognizes variations of the consensus...

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.

The epigenetic code of DNA methylation is interpreted chiefly by methyl cytosine binding domain (MBD) proteins which in turn recruit multiprotein co-repressor complexes. We previously isolated one such complex, MBD2-NuRD, from primary erythroid cells and have shown it contributes to embryonic/fetal β-type globin gene silencing during development. This complex has been implicated in silencing tumor suppressor genes in a variety of human tumor cell types. Here we present structural details of chicken MBD2 bound to a methylated DNA sequence from the ρ-globin promoter to which it binds in vivo and mediates developmental transcriptional silencing in normal erythroid cells. While previous studies have failed to show sequence specificity for MBD2 outside of the symmetric mCpG, we find that this domain binds in a single orientation on the ρ-globin target DNA sequence. Further, we show that the orientation and affinity depends on guanine immediately following the mCpG dinucleotide. Dynamic analyses show that DNA binding stabilizes the central β-sheet, while the N- and C-terminal regions of the protein maintain mobility. Taken together, these data lead to a model in which DNA binding stabilizes the MBD2 structure and that binding orientation and affinity is influenced by the DNA sequence surrounding the central mCpG.