Structure and Mechanism of the trans-Acting Acyltransferase from the Disorazole Synthase

Abstract

The 1.51 Å resolution X-ray crystal structure of the trans-acyltransferase (AT) from the “AT-less” disorazole synthase (DSZS), and its acetate complex at 1.35 Å resolution, are reported. Separately, comprehensive alanine scanning mutagenesis of one of its acyl carrier protein substrates (ACP1 from DSZS) led to the identification of a conserved Asp45 residue on the ACP, which contributes to the substrate specificity of this unusual enzyme. Together, these experimental findings were used to derive a model for the selective association of the DSZS AT and its ACP substrate. Towards the goal of structurally characterizing the AT-ACP interface, a strategy was developed for covalently cross-linking active site Ser→Cys mutant of the DSZS AT to its ACP substrate, and for purifying the resulting AT-ACP complex to homogeneity. The S86C DSZS AT mutant was found to be functional, albeit with a 200-fold lower transacylation efficiency than its wild-type counterpart. Our findings provide new insights as well as new opportunities for high-resolution analysis of an important protein-protein interface in polyketide synthases.

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

“…2A). The overall architecture of VinK is similar to those of other structurally characterized ATs including FabD [Protein Data Bank (PDB) ID code 2G2Z; 25% sequence identity; 3.0-Å root mean square deviation (rmsd) for Cα atoms], which is a discrete malonyl-CoA:ACP transacylase (MCAT) of Escherichia coli fatty acid synthase (FAS) (14), malonyl-CoA-specific trans-acting AT (PDB ID code 3RGI; 21% sequence identity; 2.6 Å rmsd for Cα atoms) from disorazole synthase (DSZS) (15), and ZmaA AT (PDB ID code 4QBU; 18% sequence identity; 3.6 Å rmsd for Cα atoms), which recognizes hydroxymalonyl-ACP as a substrate (4). VinK has two conserved catalytic residues, Ser-106 and His-216, at the interface between the large domain and small domain as observed in other ATs ( Fig.…”

“…Thus, the exact AT-ACP contact interface remains elusive. Wong et al described the preparation of a covalent DSZS AT-ACP complex using cross-linkers such as 1,3-dibromopropanone and BMOE (15). Their method seems to be useful to capture ACP for the crystallization of the AT-ACP complex.…”

Use of transmission electron microscopy to identify nanocrystals of challenging protein targets

“…S1, along with the expression system, protein yield, and maximal concentration (while maintaining monodispersity) that were used to set up trays. The expression and purification of PTHR1, CD3Delta, and RPBII, as well as complex formation with RPBII, were performed as described (21), whereas DSZS AT expression and purification are described separately (22). The tPTHR was purified following the same protocol as described for full-length PTHR1.…”

Acyl hydrolases from trans-AT polyketide synthases target acetyl units on acyl carrier proteins

“…Analysis of the malonyl-complexed models for PedC and PedD revealed a strikingly obvious difference in the active site. The PedD and FabD structures both harbour an Arg residue at the back of the binding channel, which has been predicted to form a stabilising bidentate salt-bridge with the carboxyl region of the malonyl unit [28][29][30][31] . Furthermore, mutation of this Arg residue to a either Ala or Lys was found to abolish malonyl-specific transfer to the ACP in the fatty acid system 32 .…”