Tolerance and specificity of recombinant 6-methylsalicyclic acid synthase

BACKGROUND: 6-Methylsalicylic acid synthase (MSAS), a fungal polyketide synthase from Penicillium patulum, is perhaps the simplest polyketide synthase that embodies several hallmarks of this family of multifunctional enzymes--a large multidomain protein, a high degree of specificity toward acetyl-CoA and malonyl-CoA substrates, chain length control, and regiospecific ketoreduction. MSAS has recently been functionally expressed in Escherichia coli and Saccharomyces cerevisiae, leading to the engineered biosynthesis of 6-methylsalicylic acid in these hosts. These developments have set the stage for detailed mechanistic studies of this model system. RESULTS: A three--step purification procedure was developed to obtain >95% pure MSAS from extracts of E. coli. As reported earlier for the enzyme isolated from P. patulum, the recombinant enzyme produced 6-methylsalicylic acid (a reduced tetraketide) in the presence of acetyl-CoA, malonyl-CoA, and NADPH, but triacetic acid lactone (an unreduced triketide) in the absence of NADPH. Consistent with this observation, point mutations in the highly conserved nucleotide-binding motif of the ketoreductase domain also led to production of triacetic acid lactone in vivo. The enzyme showed some tolerance toward nonnatural primer units including propionyl- and butyryl-CoA, but was incapable of incorporating extender units from (R, S)-methylmalonyl-CoA. Interestingly, MSAS readily accepted the N-acetylcysteamine (NAC) analog of malonyl-CoA as a substrate. CONCLUSIONS: NAC thioesters are simple, cost-effective analogs of CoA thioester substrates, and therefore provide a facile strategy for probing the molecular recognition features of polyketide synthases using unnatural building blocks. The ability to produce 4-hydroxy-6-methyl-2-pyrone in both E. coli and yeast illustrates the feasibility of metabolic engineering of these hosts to produce unnatural polyketides. Finally, the abundant source of recombinant MSAS described here provides an opportunity to study this fascinating model system using a combination of structural, mechanistic, and mutagenesis approaches.     

TAK1 is critical for IkappaB kinase-mediated activation of the NF-kappaB pathway

Cytokine treatment stimulates the IkappaB kinases, IKKalpha and IKKbeta, which phosphorylate the IkappaB proteins, leading to their degradation and activation of NF-kappaB regulated genes. A clear definition of the specific roles of IKKalpha and IKKbeta in activating the NF-kappaB pathway and the upstream kinases that regulate IKK activity remain to be elucidated. Here, we utilized small interfering RNAs (siRNAs) directed against IKKalpha, IKKbeta and the upstream regulatory kinase TAK1 in order to better define their roles in cytokine-induced activation of the NF-kappaB pathway. In contrast to previous results with mouse embryo fibroblasts lacking either IKKalpha or IKKbeta, which indicated that only IKKbeta is involved in cytokine-induced NF-kappaB activation, we found that both IKKalpha and IKKbeta were important in activating the NF-kappaB pathway. Furthermore, we found that the MAP3K TAK1, which has been implicated in IL-1-induced activation of the NF-kappaB pathway, was also critical for TNFalpha-induced activation of the NF-kappaB pathway. TNFalpha activation of the NF-kappaB pathway is associated with the inducible binding of TAK1 to TRAF2 and both IKKalpha and IKKbeta. This analysis further defines the distinct in vivo roles of IKKalpha, IKKbeta and TAK1 in cytokine-induced activation of the NF-kappaB pathway.     

Structure-based mutagenesis of the malonyl-CoA:acyl carrier protein transacylase from Streptomyces coelicolor

Malonyl-CoA:acyl carrier protein transacylase (MAT) provides acyl-ACP thioesters for the biosynthesis of aromatic polyketides, and thus is the primary gatekeeper of substrate specificity in type II PKS. A recent report described the X-ray crystal structure of the Streptomyces coelicolor MAT and suggested active site residues which may be important for substrate selectivity [Keatinge-Clay, A. T., et al. (2003) Structure 11, 147-154]. Mutants were made to test the proposed roles of these residues, and the enzymes were characterized kinetically with respect to native and non-native substrates. The activity of the MAT was observed to be greatly attenuated in many of the observed mutants; however, the K(m) for malonyl-CoA was only modestly affected. Our results suggest the MAT uses an active site that is rigorously ordered around the acyl-thioester moiety of the acyl-CoA to facilitate rapid and efficient transacylation to an ACP. Our results also suggest that the MAT does not discriminate against alpha-substituted acyl-CoA thioesters solely on the basis of substrate size.     

Catalysis,specificity, and ACP docking site of Streptomyces coelicolor malonyl-CoA:ACP transacylase

Malonyl-CoA:ACP transacylase (MAT), the fabD gene product of Streptomyces coelicolor A3(2), participates in both fatty acid and polyketide synthesis pathways, transferring malonyl groups that are used as extender units in chain growth from malonyl-CoA to pathway-specific acyl carrier proteins (ACPs). Here, the 2.0 A structure reveals an invariant arginine bound to an acetate that mimics the malonyl carboxylate and helps define the extender unit binding site. Catalysis may only occur when the oxyanion hole is formed through substrate binding, preventing hydrolysis of the acyl-enzyme intermediate. Macromolecular docking simulations with actinorhodin ACP suggest that the majority of the ACP docking surface is formed by a helical flap. These results should help to engineer polyketide synthases (PKSs) that produce novel polyketides.     

Measurement of gene expression following cryogenic mu-CT scanning of human iliac crest biopsies

An important consideration in interpreting indices of gene expression in human bone is relating mRNA levels to functional endpoints such as bone architecture. In the present study, a method was developed for quantitative measurement of gene expression and bone morphology in the same specimen. Three-dimensional images of iliac crest bone biopsies from healthy premenopausal women were obtained using a novel high resolution cryogenic mu-CT scanner. RNA was isolated from the biopsies and mRNA levels were measured for genes related to bone metabolism. The gene expression profile and variability of expression within iliac crest biopsies of women was similar to human osteoblastic cell lines and rat long bones. mRNA for alkaline phosphatase, bone matrix proteins, and selected cytokines and cytokine receptors were consistently detected in biopsies. As previously shown in rat bone, there was a tight correlation between mRNA levels for type 1 collagen and osteonectin, a weaker correlation between type 1 collagen and osteocalcin and no correlation between bone matrix proteins and alkaline phosphatase. The relative abundance of the mRNA for the three most prevalent transforming growth factor-beta (TGF-beta) isoforms in bone (TGF-beta(1)> TGF-beta(3)> TGF-beta(2)) was the same as the known abundance of the corresponding TGF-beta peptides in bone matrix. The results demonstrate the feasibility of analyzing the three-dimensional architecture of a bone biopsy using cryogenic mu-CT imaging and then measuring expression of genes related to bone cell function within the same specimen following RNA extraction and analysis.     

Kinetic and structural analysis of a new group of Acyl-CoA carboxylases found in Streptomyces coelicolor A3(2)

Two acyl-CoA carboxylases from Streptomyces coelicolor have been successfully reconstituted from their purified components. Both complexes shared the same biotinylated alpha subunit, AccA2. The beta and the epsilon subunits were specific from each of the complexes; thus, for the propionyl-CoA carboxylase complex the beta and epsilon components are PccB and PccE, whereas for the acetyl-CoA carboxylase complex the components are AccB and AccE. The two complexes showed very low activity in the absence of the corresponding epsilon subunits; addition of PccE or AccE dramatically increased the specific activity of the enzymes. The kinetic properties of the two acyl-CoA carboxylases showed a clear difference in their substrate specificity. The acetyl-CoA carboxylase was able to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity. The propionyl-CoA carboxylase could not recognize acetyl-CoA as a substrate, whereas the specificity constant for propionyl-CoA was 2-fold higher than for butyryl-CoA. For both enzymes the epsilon subunits were found to specifically interact with their carboxyltransferase component forming a beta-epsilon subcomplex; this appears to facilitate the further interaction of these subunits with the alpha component. The epsilon subunit has been found genetically linked to several carboxyltransferases of different Streptomyces species; we propose that this subunit reflects a distinctive characteristic of a new group of acyl-CoA carboxylases.     

A collection of sequenced and mapped Ds transposon insertion sites in Arabidopsis thaliana

Insertional mutagenesis is a powerful tool for generating knockout mutations that facilitate associating biological functions with as yet uncharacterized open reading frames (ORFs) identified by genomic sequencing or represented in EST databases. We have generated a collection of Dissociation (Ds) transposon lines with insertions on all 5 Arabidopsis chromosomes. Here we report the insertion sites in 260 independent single-transposon lines, derived from four different Ds donor sites. We amplified and determined the genomic sequence flanking each transposon, then mapped its insertion site by identity of the flanking sequences to the corresponding sequence in the Arabidopsis genome database. This constitutes the largest collection of sequence-mapped Ds insertion sites unbiased by selection against the donor site. Insertion site clusters have been identified around three of the four donor sites on chromosomes 1 and 5, as well as near the nucleolus organizers on chromosomes 2 and 4. The distribution of insertions between ORFs and intergenic sequences is roughly proportional to the ratio of genic to intergenic sequence. Within ORFs, insertions cluster near the translational start codon, although we have not detected insertion site selectivity at the nucleotide sequence level. A searchable database of insertion site sequences for the 260 transposon insertion sites is available at http://sgio2.biotec.psu.edu/sr. This and other collections of Arabidopsis lines with sequence-identified transposon insertion sites are a valuable genetic resource for functional genomics studies because the transposon location is precisely known, the transposon can be remobilized to generate revertants, and the Ds insertion can be used to initiate further local mutagenesis.     

Engineering of molecular and cellular biocatalysts: selected contributions by James E. Bailey

James (Jay) E. Bailey was a pioneer in biotechnology and biochemical engineering. During his 30 years in academia he made seminal contributions to many fields of chemical engineering science, including catalysis and reaction engineering, bioprocess engineering, mathematical modeling of cellular processes, recombinant DNA technology, enzyme engineering, and metabolic engineering. This article celebrates some of his contributions to the engineering of molecular and cellular biocatalysts, and identifies the influence he had on current and future research in biotechnology.