RESUMO
The proteins of trans-acyltransferase modular polyketide synthases (PKSs) self-organize into assembly lines, enabling the multienzyme biosynthesis of complex organic molecules. Docking domains comprised of â¼25 residues at the C- and N-termini of these polypeptides (CDDs and NDDs) help drive this association through the formation of four-helix bundles. Molecular connectors like these are desired in synthetic contexts, such as artificial biocatalytic systems and biomaterials, to orthogonally join proteins. Here, the ability of six CDD/NDD pairs to link non-PKS proteins is examined using green fluorescent protein (GFP) variants. As observed through size-exclusion chromatography and Förster resonance energy transfer (FRET), matched but not mismatched pairs of Venus+CDD and NDD+mTurquoise2 fusion proteins associate with low micromolar affinities.
Assuntos
Simulação de Acoplamento Molecular , Policetídeo Sintases/metabolismo , Sequência de Aminoácidos , Cromatografia em Gel , Transferência Ressonante de Energia de Fluorescência , Proteínas de Fluorescência Verde/química , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Mutagênese , Peptídeos/química , Peptídeos/metabolismo , Policetídeo Sintases/químicaRESUMO
The methyl substituents in products of trans-acyltransferase assembly lines are usually incorporated by S-adenosyl-methionine (SAM)-dependent methyltransferase (MT) domains. The gem-dimethyl moieties within the polyketide disorazol are installed through the iterative action of an MT in the third module of its assembly line. The 1.75-Å-resolution crystal structure of this MT helps elucidate how it catalyzes the addition of two methyl groups. Activity assays of point mutants on ß-ketoacyl chains linked to an acyl carrier protein and N-acetylcysteamine provide additional insights into the roles of active site residues. The replacement of an alanine with a phenylalanine at an apparent gatekeeping position resulted in more monomethylation than dimethylation. MTs may form an interface with ketoreductases (KRs) and even mediate the docking of trans-acyltransferase assembly line polypeptides through this association.
Assuntos
Metiltransferases/química , Policetídeo Sintases/química , Oxirredutases do Álcool/química , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Domínio Catalítico/genética , Cristalografia por Raios X , Metilação , Metiltransferases/genética , Metiltransferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Mutação , Myxococcales/enzimologia , Oxazóis/química , Oxazóis/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/metabolismo , Policetídeos/química , Policetídeos/metabolismo , Ligação Proteica , Domínios Proteicos , Alinhamento de SequênciaRESUMO
Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line.
Assuntos
Motivos de Aminoácidos/genética , Complexos Multiproteicos/química , Polienos/química , Policetídeo Sintases/química , Bacillus subtilis/química , Bacillus subtilis/enzimologia , Cristalografia por Raios X , Modelos Moleculares , Complexos Multiproteicos/genética , Polienos/metabolismo , Policetídeo Sintases/genética , Policetídeo Sintases/ultraestrutura , Estrutura Terciária de ProteínaRESUMO
Thiolases catalyze the formation of carbon-carbon bonds in diverse biosynthetic pathways. The promiscuous ß-ketoacyl thiolase B of Ralstonia eutropha (ReBktB) has been utilized in the in vivo conversion of Coenzyme A (CoA)-linked precursors such as acetyl-CoA and glycolyl-CoA into ß-hydroxy acids, including the pharmaceutically-important 3,4-dihydroxybutyric acid. Such thiolases could serve as powerful carbon-carbon bond-forming biocatalysts in vitro if handles less costly than CoA were employable. Here, thiolase activity is demonstrated toward substrates linked to the readily-available CoA mimic, N-acetylcysteamine (NAC). ReBktB was observed to catalyze the retro-Claisen condensation of several ß-ketoacyl-S-NAC substrates, with a preference for 3-oxopentanoyl-S-NAC over 3-oxobutanoyl-, 3-oxohexanoyl-, and 3-oxoheptanoyl-S-NAC. A 2.0 Å-resolution crystal structure, in which the asymmetric unit consists of four ReBktB tetramers, provides insight into acyl group specificity and how it may be engineered. By replacing an active site methionine with an alanine, a mutant possessing significant activity towards α-methyl substituted, NAC-linked substrates was engineered. The ability of ReBktB and its engineered mutants to utilize NAC-linked substrates will facilitate the in vitro biocatalytic synthesis of diketide chiral building blocks from feedstock molecules such as acetate and propionate.
