Functional chararacterization of the enzymes TabB and TabD involved in tabtoxin biosynthesis by Pseudomonas syringae.

Pseudomonas syringae pv. tabaci ATCC 11528 produces tabtoxin, a ß-lactam-containing dipeptide phytotoxin. Tabtoxinine-ß-lactam (TßL), one of tabtoxin's constituent amino acids, structurally mimics lysine, and many of the proteins encoded by the tabtoxin biosynthetic gene cluster are homologs of lysine biosynthetic enzymes, suggesting that the tabtoxin and lysine biosynthetic routes parallel one another. We cloned and expressed TabB and TabD, predicted homologs of tetrahydrodipicolinate (THDPA)-N-acyltransferase and N-acyl-THDPA aminotransferase, respectively, to determine their activities in vitro. We confirmed that TabB succinylates THDPA and that TabD is a PLP-dependent aminotransferase that utilizes glutamate as an amine donor. Surprisingly, we also found that though TabD could utilize the TabB product N-succinyl-THDPA as a substrate, THDPA itself was also recognized. These observations reveal that TabB functionally duplicates DapD, the THDPA-N-succinyltransferase involved in lysine biosynthesis, and reinforce the close relationship between the metabolic logics underpinning the respective biosynthetic pathways.

ORF7 from Amycolatopsis orientalis catalyzes decarboxylation of Nδ-methylarginine and amine oxidation of arginine: Biosynthetic implications.

A key step in the biosynthesis of the polyene polyketide ECO-0501 by Amycolatopsis orientalis ATCC 43491 is thought to involve oxidative decarboxylation of arginine or Nδ-methylarginine to the corresponding primary amide. This reaction is the centerpiece of a recently identified biosynthetic cassette that generates 4-guanidinobutyryl thioesters to serve as starter units for polyketide synthesis. We examined the reaction of ORF7, the predicted ECO-0501 biosynthetic decarboxylase, with arginine, and saw no evidence of decarboxylation. Instead, we observed exclusive amine oxidation to generate 2-oxoarginine, with a kcat/KM,Arg of 5.6×106M-1s-1, typical of values measured for physiological amino acid decarboxylases. In contrast, when ORF7 was incubated with Nδ-methylarginine, we observed exclusive decarboxylation to generate 4-(N1-methylguanidino)butyramide. These differing reactive pathways provide insight into the biosyntheses of guanidinobutyryl-derived polyketides and demonstrate the biosynthetic versatility of arginine-processing decarboxylases. In addition, it suggests that ORF7 may be an incisive model system for dissecting the determinants of flavoprotein-catalyzed oxidase and monooxygenase modes of reactivity.

A ketoreductase domain in the PksJ protein of the bacillaene assembly line carries out both alpha- and beta-ketone reduction during chain growth.

The polyketide signaling metabolites bacillaene and dihydrobacillaene are biosynthesized in Bacillus subtilis on an enzymatic assembly line with both nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS) modules acting along with catalytic domains servicing the assembly line in trans. These signaling metabolites possess the unusual starter unit alpha-hydroxyisocaproate (alpha-HIC). We show here that it arises from initial activation of alpha-ketoisocaproate (alpha-KIC) by the first adenylation domain of PksJ (a hybrid PKS/NRPS) and installation on the pantetheinyl arm of the adjacent thiolation (T) domain. The alpha-KIC unit is elongated to alpha-KIC-Gly by the second NRPS module in PksJ as demonstrated by mass spectrometric analysis. The third module of PksJ uses PKS logic and contains an embedded ketoreductase (KR) domain along with two adjacent T domains. We show that this KR domain reduces canonical 3-ketobutyryl chains but also the alpha-keto group of alpha-KIC-containing intermediates on the PksJ T-domain doublet. This KR activity accounts for the alpha-HIC moiety found in the dihydrobacillaene/bacillaene pair and represents an example of an assembly-line dual-function alpha- and beta-KR acting on disparate positions of a growing chain intermediate.

Polyunsaturated fatty-acid-like trans-enoyl reductases utilized in polyketide biosynthesis.

Polyketide biosynthesis is typically directed by cis-acting catalytic domains. In the case of the Bacillus subtilis secondary metabolite dihydrobacillaene, the cis-acting domains are not sufficient to generate the saturated C14'-C15' bond. In this communication, we identify PksE as a trans-acting enoyl reductase utilized in the biosynthesis of this portion of dihydrobacillaene. PksE is homologous to the enzymes predicted to serve as enoyl reductases in polyunsaturated fatty acid (PUFA) biosynthesis, and we confirmed this functional assignment in vitro. These results suggest a general enoyl reduction pathway in polyketide biosynthesis and a means by which PUFA-like biosynthetic machinery can modulate small-molecule function.

Incorporation of nonmethyl branches by isoprenoid-like logic: multiple beta-alkylation events in the biosynthesis of myxovirescin A1.

Several polyketide secondary metabolites are predicted to undergo isoprenoid-like beta-alkylations during biosynthesis. One such secondary metabolite is myxovirescin A1, produced by Myxococcus xanthus. Myxovirescin is of special interest in that it appears to undergo two distinct beta-alkylations. Additionally, the myxovirescin biosynthetic gene cluster lacks tandem thiolation domains required in the synthesis of other beta-branched secondary metabolites. To probe the origins of the beta-branches in myxovirescin, we heterologously overexpressed the proteins predicted to be responsible for myxovirescin beta-alkylation and reconstituted their activities in vitro on model substrates. Our results confirm that myxovirescin undergoes two isoprenoid-like beta-alkylations during its biosynthesis, including an unprecedented beta-ethylation. The study of its biosynthesis should shed light on the scope and requirements for isoprenoid-like biosynthetic logic in a polyketide context.

Nucleic-acid-templated synthesis as a model system for ancient translation.

The translation of nucleic acids into synthetic structures with expanded functional potential has been the subject of considerable research, with applications including small-molecule and polymer evolution, reaction discovery and sensing. Here, we review properties of nucleic-acid-templated synthesis in the context of requirements for prebiotic translation. This analysis highlights the chemical possibilities of ancient translation systems, as well as challenges that these systems may have faced.

Changes in motion vs. bonding in positively vs. negatively cooperative interactions.

From a consideration of the interactions between non-covalent bonds, it is concluded that positively cooperative binding will occur with a benefit in enthalpy and a cost in entropy, and that negatively cooperative binding will occur with a cost in enthalpy and a benefit in entropy; experimental data support these conclusions.

Isoprenoid-like alkylations in polyketide biosynthesis.

Polyketides are secondary metabolites biosynthesized by the iterative Claisen condensation of malonate units. Despite utilizing only a small set of biochemical transformations, the polyketide biosynthetic machinery yields products of striking structural complexity and diversity. Recently, a new polyketide alkylation pathway was characterized that allows access to "beta-branched" structures. This Highlight will describe this alkylation sequence, with special emphasis on its parallels to isoprenoid biosynthesis from primary metabolism and the scope of structures accessible via this pathway.

Convergence of isoprene and polyketide biosynthetic machinery: isoprenyl-S-carrier proteins in the pksX pathway of Bacillus subtilis.

The pksX gene cluster from Bacillus subtilis is predicted to encode the biosynthesis of an as yet uncharacterized hybrid nonribosomal peptide/polyketide secondary metabolite. We used a combination of biochemical and mass spectrometric techniques to assign functional roles to the proteins AcpK, PksC, PksL, PksF, PksG, PksH, and PksI, and we conclude that they act to incorporate an acetate-derived beta-methyl branch on an acetoacetyl-S-carrier protein and ultimately generate a Delta(2)-isoprenyl-S-carrier protein. This work highlights the power of mass spectrometry to elucidate the functions of orphan biosynthetic enzymes, and it details a mechanism by which single-carbon beta-branches can be inserted into polyketide-like structures. This pathway represents a noncanonical route to the construction of prenyl units and serves as a prototype for the intersection of isoprenoid and polyketide biosynthetic manifolds in other natural product biosynthetic pathways.

Facile detection of acyl and peptidyl intermediates on thiotemplate carrier domains via phosphopantetheinyl elimination reactions during tandem mass spectrometry.

With the emergence of drug resistance and the genomic revolution, there has been a renewed interest in the genes that are responsible for the generation of bioactive natural products. Secondary metabolites of one major class are biosynthesized at one or more sites by ultralarge enzymes that carry covalent intermediates on phosphopantetheine arms. Because such intermediates are difficult to characterize in vitro, we have developed a new approach for streamlined detection of substrates, intermediates, and products attached to a phosphopantetheinyl arm of the carrier site. During vibrational activation of gas-phase carrier domains, facile elimination occurs in benchtop and Fourier-transform mass spectrometers alike. Phosphopantetheinyl ejections quickly reduce >100 kDa megaenzymes to <1000 Da ions for structural assignment of intermediates at <0.007 Da mass accuracy without proteolytic digestion. This "top down" approach quickly illuminated diverse acyl intermediates on the carrier domains of the nonribosomal peptide synthetases (NRPSs) or polyketide synthases (PKSs) found in the biosynthetic pathways of prodigiosin, pyoluteorin, mycosubtilin, nikkomycin, enterobactin, gramicidin, and several proteins from the orphan pksX gene cluster from Bacillus subtilis. By focusing on just those regions undergoing covalent chemistry, the method delivered clean proof for the reversible dehydration of hydroxymethylglutaryl-S-PksL via incorporation of 2H or 18O from the buffer. The facile nature of this revised assay will allow diverse laboratories to spearhead their NRPS-PKS projects with benchtop mass spectrometers.