TY - JOUR
T1 - Chemo- and Regioselective Peptide Cyclization Triggered by the N-Terminal Fatty Acid Chain Length
T2 - The Recombinant Cyclase of the Calcium-Dependent Antibiotic from Streptomyces coelicolor
AU - Grünewald, Jan
AU - Sieber, Stephan A.
AU - Marahiel, Mohamed A.
PY - 2004/3/16
Y1 - 2004/3/16
N2 - Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr2 and Ser1 residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser1 or Thr2 by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only L-configured Ser1 and Thr2 for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C2 to C6 as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.
AB - Here we report the first biochemical characterization of a recombinant nonribosomal peptide cyclase of a streptomycete, the model actinomycete Streptomyces coelicolor A3(2). This bacterium produces the calcium-dependent antibiotic (CDA), which is a branched cyclic macrolactone belonging to the group of acidic lipopeptides. The recombinant CDA3 cyclase from CDA synthetase efficiently catalyzes ring formation of linear peptidyl thioester substrates based on a sequence analogous to natural CDA. Four leaving groups were attached to the C-terminus of the undecapeptide: coenzyme A (CoA), phosphopantetheine, N-acetylcysteamine (SNAC), and thiophenol. The best rates for cyclization were determined for the thiophenol substrate, revealing that chemical reactivity is more important than cofactor recognition. The cyclase catalyzes the formation of two regioisomeric macrolactones, which arise from simultaneous nucleophilic attack of the two adjacent Thr2 and Ser1 residues onto the C-terminus of the acyl-enzyme intermediate. This relaxed regioselectivity has not been observed for any other recombinant NRPS or PKS cyclases so far. Substitution of either Ser1 or Thr2 by alanine led to selective formation of a decapeptide or undecapeptide lactone ring. In contrast to that, CDA3 cyclase strictly retains stereoselectivity for both nucleophiles, accepting only L-configured Ser1 and Thr2 for cyclization. Further, our studies provide evidence for the crucial role of N-terminal fatty acyl groups of lipopeptides in controlling the regio- and chemoselectivity of enzyme-catalyzed macrocyclization. Elongation of the fatty acyl group of our thioester substrate from C2 to C6 as in CDA turned the relaxed regioselectivity into a strict regioselectivity, yielding solely the decapeptide lactone ring with a significantly improved cyclization-to-hydrolysis ratio.
UR - http://www.scopus.com/inward/record.url?scp=12144287553&partnerID=8YFLogxK
U2 - 10.1021/bi036140d
DO - 10.1021/bi036140d
M3 - Article
C2 - 15005627
AN - SCOPUS:12144287553
SN - 0006-2960
VL - 43
SP - 2915
EP - 2925
JO - Biochemistry
JF - Biochemistry
IS - 10
ER -