Condensation-Incompetent Ketosynthase Inhibits trans-Acyltransferase Activity.

Nonelongating modules with condensation-incompetent ketosynthase (KS) are frequently found in many trans-acyltransferase polyketide synthases ( trans-AT PKS). KS 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 KS is assumed to be prevented.

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).

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.

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.

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.

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.

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).

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.