Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase

Abstract

Polyketide metabolites produced by modular type I polyketide synthases (PKS) acquire their chemical diversity through the variety of catalytic domains within modules of the pathway. Methyltransferases are among the least characterized of the catalytic domains common to PKS systems. We determined the domain boundaries and characterized the activity of a PKS C-methyltransferase (C-MT) from the curacin A biosynthetic pathway. The C-MT catalyzes S-adenosylmethionine-dependent methyl transfer to the α-position of β-ketoacyl substrates linked to acyl carrier protein (ACP) or a small-molecule analog but does not act on β-hydroxyacyl substrates or malonyl-ACP. Key catalytic residues conserved in both bacterial and fungal PKS C-MTs were identified in a 2 Å crystal structure and validated biochemically. Analysis of the structure and the sequences bordering the C-MT provides insight into the positioning of this domain within complete PKS modules.

Figure 1

Introduction of α-methyl by CurJ C-MT. a) CurJ composition. Curacin A contains a single methyl branch derived from the C-MT in module CurJ, which contains ketosynthase (KS), acyltransferase (AT), dehydratase (DH), C-methyltransferase (CMT), ketoreductase (KR), and acyl carrier protein (ACP) domains. b) Potential routes for C-methylation in PKSs. Route 1-Methylation occurs on the β- ketoacyl intermediate after the KS condensation reaction. Route 2-Methylation occurs on malonyl-ACP prior to KS condensation.

Figure 2

LC-MS analysis of C-MT activity. Ion counts are shown in red for reactions via Route 1 in a) acetoacetyl-ACP (1) and b) β-ketopentanoyl-NAC (5), via Route 2 in c) malonyl-ACP (3), and via Route 3 in d) β-hydroxypentanoyl-NAC (7) and e) D,L-β-hydroxybutyryl-ACP (4). Blue traces are no-enzyme control reactions; green traces are no-substrate control reactions. See Supplementary Table 1 for expected masses and Supplementary Figure 3 for mass spectra of total ion chromatographs of acyl-NACs. f) Relative methylation activities of wild type CurJ C-MT, JamJ C-MT, and CurJ C-MT site-directed mutants with acetoacetyl-ACP (1) in an MS assay based on phosphopantetheine ejection (Supplementary Figure 5). NEC = no enzyme control.

Figure 3

CurJ C-MT structure. a) CurJ C-MT colored as a rainbow from blue (N-terminus) to red (C-terminus), shown in stereo. SAH is shown in sticks with atomic colors (C, gray; O, red; N, blue; S, yellow). b) CurJ C-MT colored by structure region. Helical seatbelt, blue; lid, dark blue; lid-to-core junction green; core, cyan; core insertion, orange; SAH, sticks with gray C. The transparent gray surface represents the substrate tunnel, which is lined with hydrophobic residues (Ile35, Phe157, Leu168, Val174, Ala307, Trp313, Val314, Phe318, Leu338). The CurJ C-MT substrate and phosphopantetheine (sticks with magenta C) were modeled into the active site. The views in A and B are from opposite sides of the C-MT.

Figure 4

PKS and mFAS modifying regions. The β-ribbon that is both an essential part of the KR domain and also an inter-domain linker is orange (1^st strand) and green (2^nd strand). a) Model of CurJ C-MT and KR domains, arranged as in mFAS. The model is based on superpositions of the CurJ C-MT core with the mFAS Ψ-MT core and of a PKS KR with the mFAS KR^, . b) mFAS with DH, Ψ-MT, ER and KR domains. c) MAS-like PKS with DH, ER and KR domains. d) DEBS1 module 1 modifying region with KR domain only. DH (red), ER (yellow), KR_C (purple) and KR_S (light blue) domains are represented as surfaces, the CurJ C-MT as a ribbon colored as in Fig. 3b, the mFAS Ψ-MT as a gray ribbon, and other domains as circles. Below each structure is a schematic of domain and β-ribbon strand arrangement colored as in the structure image. e) Sequence alignment and domain context of β-ribbon strand 1 in CurJ, DEBS module 1, phoslactomycin module 1, spinosyn module 2, MAS-like PKS and mFAS.

Figure 5

CurJ C-MT active site. Key amino acids, modeled substrate (magenta C), and SAH (white C) are shown in sticks. The MT core is represented as a transparent surface with ribbon. The junction and core insertion are shown as ribbons. C-MT structural regions are colored as in Fig. 3b.

Figure 6

Movement surrounding the CurJ C-MT active site. Structural regions are colored as in Fig. 3b. a) Closely related SeMet and wild type CurJ C-MT crystal forms (light and dark colors) are different in the lid-to-core junction and SAH homocysteine position. b) The position of Thr208 in MT motif I differs in the two C-MT chains in the SeMet crystal form.