[Rh(bpy)2(chrysi)]3+ is a novel, sterically bulky DNA intercalator that has been designed to bind specifically in the destabilized regions near DNA base mismatches and, upon photoactivation, to cleave the DNA backbone. Here the molecule is shown to be both a general and remarkably specific mismatch recognition agent. Specific DNA cleavage is observed at over 80% of mismatch sites in all the possible single base pair sequence contexts around the mispaired bases. Moreover, the complex is highly site-specific; it is shown to recognize and photocleave at a single base mismatch in a 2725 base pair linearized plasmid heteroduplex. Sterically demanding intercalators such as [Rh(bpy)2(chrysi)]3+ may have application both in mutation detection systems and as mismatch-specific chemotherapeutic agents.
Polyketides are a medicinally important class of natural products. The architecture of modular polyketide synthases (PKSs), composed of multiple covalently linked domains grouped into modules, provides an attractive framework for engineering novel polyketide-producing assemblies. However, impaired domain-domain interactions can compromise the efficiency of engineered polyketide biosynthesis. To facilitate the study of these domain-domain interactions, we have used nuclear magnetic resonance (NMR) spectroscopy to determine the first solution structure of an acyl carrier protein (ACP) domain from a modular PKS, 6-deoxyerythronolide B synthase (DEBS). The tertiary fold of this 10-kD domain is a three-helical bundle; an additional short helix in the second loop also contributes to the core helical packing. Superposition of residues 14-94 of the ensemble on the mean structure yields an average atomic RMSD of 0.64 6 0.09 Å for the backbone atoms (1.21 6 0.13 Å for all non-hydrogen atoms). The three major helices superimpose with a backbone RMSD of 0.48 6 0.10 Å (0.99 6 0.11 Å for non-hydrogen atoms). Based on this solution structure, homology models were constructed for five other DEBS ACP domains. Comparison of their steric and electrostatic surfaces at the putative interaction interface (centered on helix II) suggests a model for protein-protein recognition of ACP domains, consistent with the previously observed specificity. Site-directed mutagenesis experiments indicate that two of the identified residues influence the specificity of ACP recognition.Keywords: polyketide; acyl carrier protein; NMR; structure; protein interaction; electrostatic; homology modeling; mutagenesis Supplemental material: see www.proteinscience.org Reprint requests to: Chaitan Khosla, Department of Chemistry, Stanford University, MC5080, Stanford CA 94305, USA; e-mail: khosla@stanford.edu; fax: (650) 723-6538.Abbreviations ACP, acyl carrier protein; ArCP, aryl carrier protein; AT, acyltransferase; CoA, coenzyme A; DEBS, 6-deoxyerythronolide B synthase; DH, dehydratase; ER, enoyl reductase; ESI-MS, electrospray ionization mass spectrometry; FAS, fatty acid synthase; HSNAc, N-acetylcysteamine; HSQC, heteronuclear single-quantum coherence; IPTG, isopropyl-b-D-thiogalactopyranoside; kD, kilodalton; KR, ketoreductase; KS, ketosynthase; NDK-SNAc, the natural diketide substrate, 2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester; NMR, nuclear magnetic resonance; NOE, nuclear Overhauser effect; Ni-NTA, nickel-nitrilotriacetic acid; NRPS, nonribosomal peptide synthetase; PCP, peptidyl carrier protein; PKS, polyketide synthase; RMSD, root-mean-square deviation; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis; TCEP, tris-(2-carboxyethyl)phosphine hydrochloride; TE, thioesterase; TLC, thin layer chromatography.Article and publication are at
Cytochrome P450 (P450) 1A2 is the major enzyme involved in the metabolism of 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ) and other heterocyclic arylamines and their bioactivation to mutagens. Random mutant libraries of human P450 1A2, in which mutations were made throughout the entire open reading frame, were screened with Escherichia coli DJ3109pNM12, a strain designed to bioactivate MeIQ and detect mutagenicity of the products. Mutant clones with enhanced activity were confirmed using quantitative measurement of MeIQ N-hydroxylation. Three consecutive rounds of random mutagenesis and screening were performed and yielded a highly improved P450 1A2 mutant, SF513 (E225N/Q258H/G437D), with >10-fold increased MeIQ activation based on the E. coli genotoxicity assay and 12-fold enhanced catalytic efficiency (k(cat)/K(m)) in steady-state N-hydroxylation assays done with isolated membrane fractions. SF513 displayed selectively enhanced activity for MeIQ compared to other heterocyclic arylamines. The enhanced catalytic activity was not attributed to changes in any of several individual steps examined, including substrate binding, total NADPH oxidation, or H(2)O(2) formation. Homology modeling based on an X-ray structure of rabbit P450 2C5 suggested that the E225N and Q258H mutations are located in the F-helix and G-helix, respectively, and that the G437D mutation is in the "meander" region, apparently rather distant from the substrate. In summary, the approach generated a mutant enzyme with selectively elevated activity for a single substrate, even to the extent of a difference of a single methyl group, and several mutations had interacting roles in the development of the selected mutant protein.
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