T. Ashton Cropp

Biochemical Evidence for an Editing Role of Thioesterase II in the Biosynthesis of the Polyketide Pikromycin

The pikromycin biosynthetic gene cluster contains the pikAV gene encoding a type II thioesterase (TEII). TEII is not responsible for polyketide termination and cyclization, and its biosynthetic role has been unclear. During polyketide biosynthesis, extender units such as methylmalonyl acyl carrier protein (ACP) may prematurely decarboxylate to generate the corresponding acyl-ACP, which cannot be used as a substrate in the condensing reaction by the corresponding ketosynthase domain, rendering the polyketide synthase module inactive. It has been proposed that TEII may serve as an “editing” enzyme and reactivate these modules by removing acyl moieties attached to ACP domains. Using a purified recombinant TEII we have tested this hypothesis by using in vitro enzyme assays and a range of acyl-ACP, malonyl-ACP, and methylmalonyl-ACP substrates derived from either PikAIII or the loading didomain of DEBS1 (6-deoxyerythronolide B synthase; ATL-ACPL). The pikromycin TEII exhibited highK m values (>100 μm) with all substrates and no apparent ACP specificity, catalyzing cleavage of methylmalonyl-ACP from both ATL-ACPL(k cat/K m 3.3 ± 1.1m −1 s−1) and PikAIII (k cat/K m 2.9 ± 0.9m −1 s−1). The TEII exhibited some acyl-group specificity, catalyzing hydrolysis of propionyl (k cat/K m 15.8 ± 1.8m −1 s−1) and butyryl (k cat/K m 17.5 ± 2.1m −1 s−1) derivatives of ATL-ACPL faster than acetyl (k cat/K m 4.9 ± 0.7m −1 s−1), malonyl (k cat/K m 3.9 ± 0.5m −1 s−1), or methylmalonyl derivatives. PikAIV containing a TEI domain catalyzed cleavage of propionyl derivative of ATL-ACPL at a dramatically lower rate than TEII. These results provide the first unequivocal in vitro evidence that TEII can hydrolyze acyl-ACP thioesters and a model for the action of TEII in which the enzyme remains primarily dissociated from the polyketide synthase, preferentially removing aberrant acyl-ACP species with long half-lives. The lack of rigorous substrate specificity for TEII may explain the surprising observation that high level expression of the protein inStreptomyces venezuelae leads to significant (>50%) titer decreases.

Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin

The side chain of the antifungal antibiotic ansatrienin A from Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC)-derived moiety. This moiety is also observed in trace amounts of ω-cyclohexyl fatty acids (typically less than 1% of total fatty acids) produced by S. collinus. Coenzyme A-activated CHC (CHC-CoA) is derived from shikimic acid through a reductive pathway involving a minimum of nine catalytic steps. Five putative CHC-CoA biosynthetic genes in the ansatrienin biosynthetic gene cluster of S. collinus have been identified. Plasmid-based heterologous expression of these five genes in Streptomyces avermitilis or Streptomyces lividans allows for production of significant amounts of ω-cyclohexyl fatty acids (as high as 49% of total fatty acids). In the absence of the plasmid these organisms are dependent on exogenously supplied CHC for ω-cyclohexyl fatty acid production. Doramectin is a commercial antiparasitic avermectin analog produced by fermenting a bkd mutant of S. avermitilis in the presence of CHC. Introduction of the S. collinus CHC-CoA biosynthetic gene cassette into this organism resulted in an engineered strain able to produce doramectin without CHC supplementation. The CHC-CoA biosynthetic gene cluster represents an important genetic tool for precursor-directed biosynthesis of doramectin and has potential for directed biosynthesis in other important polyketide-producing organisms.

Nonenzymatic assembly of natural polyubiquitin chains of any linkage composition and isotopic labeling scheme.

Polymeric chains made of a small protein ubiquitin act as molecular signals regulating a variety of cellular processes controlling essentially all aspects of eukaryotic biology. Uncovering the mechanisms that allow differently linked polyubiquitin chains to serve as distinct molecular signals requires the ability to make these chains with the native connectivity, defined length, linkage composition, and in sufficient quantities. This, however, has been a major impediment in the ubiquitin field. Here, we present a robust, efficient, and widely accessible method for controlled iterative nonenzymatic assembly of polyubiquitin chains using recombinant ubiquitin monomers as the primary building blocks. This method uses silver-mediated condensation reaction between the C-terminal thioester of one ubiquitin and the ε-amine of a specific lysine on the other ubiquitin. We augment the nonenzymatic approaches developed recently by using removable orthogonal amine-protecting groups, Alloc and Boc. The use of bacterially expressed ubiquitins allows cost-effective isotopic enrichment of any individual monomer in the chain. We demonstrate that our method yields completely natural polyubiquitin chains (free of mutations and linked through native isopeptide bonds) of essentially any desired length, linkage composition, and isotopic labeling scheme, and in milligram quantities. Specifically, we successfully made Lys11-linked di-, tri-, and tetra-ubiquitins, Lys33-linked diubiquitin, and a mixed-linkage Lys33,Lys11-linked triubiquitin. We also demonstrate the ability to obtain, by high-resolution NMR, residue-specific information on ubiquitin units at any desired position in such chains. This method opens up essentially endless possibilities for rigorous structural and functional studies of polyubiquitin signals.

Site-Specific Incorporation of Fluorotyrosines into Proteins in Escherichia coli by Photochemical Disguise

Fluorinated analogues of tyrosine can be used to manipulate the electronic environments of protein active sites. The ability to selectively mutate tyrosine residues to fluorotyrosines is limited, however, and can currently only be achieved through the total synthesis of proteins. As a general solution to this problem, we genetically encoded the unnatural amino acids o-nitrobenzyl-2-fluorotyrosine, -3-fluorotyrosine, and -2,6-difluorotyrosine in Escherichia coli. These amino acids are disguised from recognition by the endogenous protein biosynthetic machinery, effectively preventing global incorporation of fluorotyrosine into proteins.

Controlled enzymatic synthesis of natural-linkage, defined-length polyubiquitin chains using lysines with removable protecting groups

E2 enzymes catalyze the ATP-dependent polymerization of polyubiquitin chains which function as molecular signals in the regulation of numerous cellular processes. Here we present a method that uses genetically encoded unnatural amino acids to halt and re-start ubiquitin polymerization providing access to natural-linkage, precision-length ubiquitin chains that can be used for biochemical, structural, and dynamics studies.

Reprogramming the amino-acid substrate specificity of orthogonal aminoacyl-tRNA synthetases to expand the genetic code of eukaryotic cells.

The genetic code of living organisms has been expanded to allow the site-specific incorporation of unnatural amino acids into proteins in response to the amber stop codon UAG. Numerous amino acids have been incorporated including photo-crosslinkers, chemical handles, heavy atoms and post-translational modifications, and this has created new methods for studying biology and developing protein therapeutics and other biotechnological applications. Here we describe a protocol for reprogramming the amino-acid substrate specificity of aminoacyl-tRNA synthetase enzymes that are orthogonal in eukaryotic cells. The resulting aminoacyl-tRNA synthetases aminoacylate an amber suppressor tRNA with a desired unnatural amino acid, but no natural amino acids, in eukaryotic cells. To achieve this change of enzyme specificity, a library of orthogonal aminoacyl-tRNA synthetase is generated and genetic selections are performed on the library in Saccharomyces cerevisiae. The entire protocol, including characterization of the evolved aminoacyl-tRNA synthetase in S. cerevisiae, can be completed in approximately 1 month.

Linkage via K27 Bestows Ubiquitin Chains with Unique Properties among Polyubiquitins

Polyubiquitination, a critical protein post-translational modification, signals for a diverse set of cellular events via the different isopeptide linkages formed between the C terminus of one ubiquitin (Ub) and the ɛ-amine of K6, K11, K27, K29, K33, K48, or K63 of a second Ub. We assembled di-ubiquitins (Ub2) comprising every lysine linkage and examined them biochemically and structurally. Of these, K27-Ub2 is unique as it is not cleaved by most deubiquitinases. As this remains the only structurally uncharacterized lysine linkage, we comprehensively examined the structures and dynamics of K27-Ub2 using nuclear magnetic resonance, small-angle neutron scattering, and in silico ensemble modeling. Our structural data provide insights into the functional properties of K27-Ub2, in particular that K27-Ub2 may be specifically recognized by K48-selective receptor UBA2 domain from proteasomal shuttle protein hHR23a. Binding studies and mutagenesis confirmed this prediction, further highlighting structural/recognition versatility of polyubiquitins and the potential power of determining function from elucidation of conformational ensembles.

Recent developments in the production of novel polyketides by combinatorial biosynthesis.

Polyketides are a class of structurally diverse natural products which possess a wide range of biological activities (Hopwood, 1997). These compounds are used throughout medicine and agriculture as antimicrobials, immunosuppressants, antiparasitics, and anticancer agents. While structurally diverse, poIyketides are assembled by a common mechanism of decarboxylative condensations of simple malonate derivatives by polyketide synthases (PKSs) in a manner very similar to fatty acid biosynthesis. After assembly by the PKS, tailoring enzymes such as glycosyltransferases, hydroxylases, or methyltransferases can then further modify the polyketide product. These post-PKS modifications are almost always necessary in order for the molecule to be bioactive. It has been shown that, frequently, bacteria (primarily the actinomycetes) possess both multifunctional (type!) and multicomponent (type H) PKSs. The first type I PKS to be sequenced and characterized was 6-deoxyerythronolide B synthase (DEBS) from Saccharopolyspora etythraea, which produces the polyketide macrolactone ring of erythromycin (Figure 8.1) (Cortes et al., 1990; Donadio a al., 1991; Caffrey et al., 1992). This modular PKS contains a loading module responsible for selecting a primer unit (in this case, propionyl CoA) and six extension modules. It was shown

Condensed E. coli Cultures for Highly Efficient Production of Proteins Containing Unnatural Amino Acids

Current biosynthetic methods for producing proteins containing site-specifically incorporated unnatural amino acids are inefficient because the majority of the amino acid goes unused. Here we present a universal approach to improve the efficiency of such processes using condensed Escherichia coli cultures.

Photochemical control of FlAsH labeling of proteins

Spatiotemporal control of protein fluorescence is a powerful tool in tracking protein movements within cells. Here we report an approach to using genetically encoded photo-caged amino acids to control labeling protein tetracysteine tags with biarsenical fluorescein dyes (FlAsH).