Condensation-Incompetent Ketosynthase Inhibits trans-Acyltransferase Activity.

Nonelongating modules with condensation-incompetent ketosynthase (KS0) are frequently found in many trans-acyltransferase polyketide synthases ( trans-AT PKS). KS0 catalyzes translocation of carbon chain without decarboxylative condensation. Unlike typical elongating modules where malonylation of acyl carrier protein (ACP) precedes elongation, the malonylation of ACP downstream of KS0 is assumed to be prevented. In this study, the regulation mechanism(s) of ACP malonylation in a non-elongating module of difficidin biosynthase was investigated. In vitro reconstitution, protein mass spectrometry, and enzyme kinetics demonstrated that KS0 controls the pathway by inhibiting the trans-AT activity. Protein-protein interactions of the surrounding domains also contribute to the regulation. Enzyme kinetics further identified the DfnKS05 as an allosteric inhibitor of trans-AT. The principle and knowledge discovered from this study will enhance the understanding of this unusual PKS system.

Stereospecific Formation of E- and Z-Disubstituted Double Bonds by Dehydratase Domains from Modules 1 and 2 of the Fostriecin Polyketide Synthase.

The dehydratase domain FosDH1 from module 1 of the fostriecin polyketide synthase (PKS) catalyzed the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP1 (5) and (E)-2-butenoyl-FosACP1 (11), as established by a combination of direct LC-MS/MS and chiral GC-MS. FosDH1 did not act on either (3S)-3-hydroxybutyryl-FosACP1 (6) or (Z)-2-butenoyl-FosACP1 (12). FosKR2, the ketoreductase from module 2 of the fostriecin PKS that normally provides the natural substrate for FosDH2, was shown to catalyze the NADPH-dependent stereospecific reduction of 3-ketobutyryl-FosACP2 (23) to (3S)-3-hydroxybutyryl-FosACP2 (8). Consistent with this finding, FosDH2 catalyzed the interconversion of the corresponding triketide substrates (3R,4E)-3-hydroxy-4-hexenoyl-FosACP2 (18) and (2Z,4E)-2,4-hexadienoyl-FosACP2 (21). FosDH2 also catalyzed the stereospecific hydration of (Z)-2-butenoyl-FosACP2 (14) to (3S)-3-hydroxybutyryl-FosACP2 (8). Although incubation of FosDH2 with (3S)-3-hydroxybutyryl-FosACP2 (8) did not result in detectable accumulation of (Z)-2-butenoyl-FosACP2 (14), FosDH2 catalyzed the slow exchange of the 3-hydroxy group of 8 with [18O]-water. FosDH2 unexpectedly could also support the stereospecific interconversion of (3R)-3-hydroxybutyryl-FosACP2 (7) and (E)-2-butenoyl-FosACP2 (13).

Structure and stereospecificity of the dehydratase domain from the terminal module of the rifamycin polyketide synthase.

RifDH10, the dehydratase domain from the terminal module of the rifamycin polyketide synthase, catalyzes the stereospecific syn dehydration of the model substrate (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, resulting in the exclusive formation of (E)-2-methyl-2-pentenoyl-RifACP10. RifDH10 does not dehydrate any of the other three diastereomeric, RifACP10-bound, diketide thioester substrates. On the other hand, when EryACP6, from the sixth module of the erythromycin polyketide synthase, is substituted for RifACP10, RifDH10 stereospecifically dehydrates only (2R,3R)-2-methyl-3-hydroxypentanoyl-EryACP6 to give exclusively (E)-2-methyl-2-pentenoyl-EryACP6, with no detectable dehydration of any of the other three diastereomeric, EryACP6-bound, diketides. An identical alteration in substrate diastereospecificity was observed for the corresponding N-acetylcysteamine or pantetheine thioester analogues, regardless of acyl chain length or substitution pattern. Incubation of (2RS)-2-methyl-3-ketopentanoyl-RifACP10 with the didomain reductase-dehydratase RifKR10-RifDH10 yielded (E)-2-methyl-2-pentenoyl-RifACP10, the expected product of syn dehydration of (2S,3S)-2-methyl-3-hydroxypentanoyl-RifACP10, while incubation with the corresponding EryACP6-bound substrate, (2RS)-2-methyl-3-ketopentanoyl-EryACP6, gave only the reduction product (2S,3S)-2-methyl-3-hydroxypentanoyl-EryACP6 with no detectable dehydration. These results establish the intrinsic syn dehydration stereochemistry and substrate diastereoselectivity of RifDH10 and highlight the critical role of the natural RifACP10 domain in chaperoning the proper recognition and processing of the natural ACP-bound undecaketide substrate. The 1.82 Å resolution structure of RifDH10 reveals the atomic-resolution details of the active site and allows modeling of the syn dehydration of the (2S,3S)-2-methyl-3-hydroxyacyl-RifACP10 substrate. These results suggest that generation of the characteristic cis double bond of the rifamycins occurs after formation of the full-length RifACP10-bound acyclic trans-unsaturated undecaketide intermediate, most likely during the subsequent macrolactamization catalyzed by the amide synthase RifF.

Stereochemistry of reductions catalyzed by methyl-epimerizing ketoreductase domains of polyketide synthases.

Ketoreductase (KR) domains from modular polyketide synthases (PKSs) catalyze the reduction of 2-methyl-3-ketoacyl acyl carrier protein (ACP) substrates and in certain cases epimerization of the 2-methyl group as well. The structural and mechanistic basis of epimerization is poorly understood, and only a small number of such KRs been studied. In this work, we studied three recombinant KR domains with putative epimerase activity: NysKR1 from module 1 of the nystatin PKS, whose stereospecificity can be predicted from both the protein sequence and the product structure; RifKR7 from module 7 of the rifamycin PKS, whose stereospecificity cannot be predicted from the protein sequence; and RifKR10 from module 10 of the rifamycin PKS, whose specificity is unclear from both the sequence and the structure. Each KR was individually incubated with NADPH and (2R)- or (2RS)-2-methyl-3-ketopentanoyl-ACP generated enzymatically in situ or via chemoenzymatic synthesis, respectively. Chiral GC-MS analysis revealed that each KR stereospecifically produced the corresponding (2S,3S)-2-methyl-3-hydroxypentanoyl-ACP in which the 2-methyl substituent had undergone KR-catalyzed epimerization. Thus, our results have led to the identification of a prototypical set of KR domains that generate (2S,3S)-2-methyl-3-hydroxyacyl products in the course of polyketide biosynthesis.

Stereospecificity of the dehydratase domain of the erythromycin polyketide synthase.

The dehydratase (DH) domain of module 4 of the 6-deoxyerythronolide B synthase (DEBS) has been shown to catalyze an exclusive syn elimination/syn addition of water. Incubation of recombinant DH4 with chemoenzymatically prepared anti-(2R,3R)-2-methyl-3-hydroxypentanoyl-ACP (2a-ACP) gave the dehydration product 3-ACP. Similarly, incubation of DH4 with synthetic 3-ACP resulted in the reverse enzyme-catalyzed hydration reaction, giving an ∼3:1 equilbrium mixture of 2a-ACP and 3-ACP. Incubation of a mixture of propionyl-SNAC (4), methylmalonyl-CoA, and NADPH with the DEBS ß-ketoacyl synthase-acyl transferase [KS6][AT6] didomain, DEBS ACP6, and the ketoreductase domain from tylactone synthase module 1 (TYLS KR1) generated in situ anti-2a-ACP that underwent DH4-catalyzed syn dehydration to give 3-ACP. DH4 did not dehydrate syn-(2S,3R)-2b-ACP, syn-(2R,3S)-2c-ACP, or anti-(2S,3S)-2d-ACP generated in situ by DEBS KR1, DEBS KR6, or the rifamycin synthase KR7 (RIFS KR7), respectively. Similarly, incubation of a mixture of (2S,3R)-2-methyl-3-hydroxypentanoyl-N-acetylcysteamine thioester (2b-SNAC), methylmalonyl-CoA, and NADPH with DEBS [KS6][AT6], DEBS ACP6, and TYLS KR1 gave anti-(2R,3R)-6-ACP that underwent syn dehydration catalyzed by DEBS DH4 to give (4R,5R)-(E)-2,4-dimethyl-5-hydroxy-hept-2-enoyl-ACP (7-ACP). The structure and stereochemistry of 7 were established by GC-MS and LC-MS comparison of the derived methyl ester 7-Me to a synthetic sample of 7-Me.

Distributive and directional behavior of lantibiotic synthetases revealed by high-resolution tandem mass spectrometry.

The lantibiotic synthetases LctM and HalM2 are bifunctional enzymes that catalyze both the dehydration of serine and threonine residues and the Michael-type additions of cysteine residues to the resulting dehydroamino acids in their substrate peptides. Using Fourier transform mass spectrometry to analyze these activities in vitro, the dehydration is shown to take place by a distributive mechanism, with build-up of intermediates observed in electrospray mass spectra. The cyclization activity of HalM2 was monitored through alkylation of free cysteines in intermediates, providing access to the regioselectivity of lanthionine ring formation using high-resolution tandem mass spectrometry. HalM2 is shown to catalyze the cyclization process in a largely N- to C-terminal directional fashion, forming a total of four lanthionine rings in its HalA2 substrate. These studies advance a model for lantibiotic production where substrate binding via an N-terminal leader results in dehydration and cyclization on similar time scales and with a high, though not strict, propensity for N-to-C directionality.

Lacticin 481 synthetase as a general serine/threonine kinase.

Methods that introduce posttranslational modifications in a general, mild, and non-sequence-specific manner using biologically produced peptides have great utility for investigation of the functions of these modifications. In this study, the substrate promiscuity of a lantibiotic synthetase was exploited for the preparation of phosphopeptides, glycopeptides, and peptides containing analogs of methylated or acetylated lysine residues. Peptides attached to the C-terminus of the leader peptide of the lacticin 481 precursor peptide were phosphorylated on serine residues in a wide variety of sequence contexts by the R399M and T405A mutants of lacticin 481 synthetase (LctM). Serine residues located as many as 30 amino acids C-terminal to the leader peptide were phosphorylated. Wild-type LctM was shown to dehydrate these peptides to generate dehydroalanine-containing products that can be conveniently modified with external nucleophiles including thiosaccharides, 2-(dimethylamino)ethanethiol, and N-acetyl cysteamine, resulting in mimics of O-linked glycopeptides and acetylated and methylated lysines.

Mechanistic investigations of the dehydration reaction of lacticin 481 synthetase using site-directed mutagenesis.

Lantibiotic synthetases catalyze the dehydration of Ser and Thr residues in their peptide substrates to dehydroalanine (Dha) and dehydrobutyrine (Dhb), respectively, followed by the conjugate addition of Cys residues to the Dha and Dhb residues to generate the thioether cross-links lanthionine and methyllanthionine, respectively. In this study ten conserved residues were mutated in the dehydratase domain of the best characterized family member, lacticin 481 synthetase (LctM). Mutation of His244 and Tyr408 did not affect dehydration activity with the LctA substrate whereas mutation of Asn247, Glu261, and Glu446 considerably slowed down dehydration and resulted in incomplete conversion. Mutation of Lys159 slowed down both steps of the net dehydration: phosphorylation of Ser/Thr residues and the subsequent phosphate elimination step to form the dehydro amino acids. Mutation of Arg399 to Met or Leu resulted in mutants that had phosphorylation activity but displayed greatly decreased phosphate elimination activity. The Arg399Lys mutant retained both activities, however. Similarly, the Thr405Ala mutant phosphorylated the LctA substrate but had compromised elimination activity. Finally, mutation of Asp242 or Asp259 to Asn led to mutant enzymes that lacked detectable dehydration activity. Whereas the Asp242Asn mutant retained phosphate elimination activity, the Asp259Asn mutant was not able to eliminate phosphate from a phosphorylated substrate peptide. A model is presented that accounts for the observed phenotypes of these mutant enzymes.