PKS-NRPS Enzymology and Structural Biology: Considerations in Protein Production

Abstract

The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions. The size and complexity of these proteins present numerous difficulties in the process of going from gene to structure. Here, we review the problems that may be encountered at the various steps of this process and discuss some of the solutions devised in our and other labs for the cloning, production, purification, and structure solution of complex proteins using Escherichia coli as a heterologous host.

Keywords: Chaperones; Coexpression; Cyanobacteria; Nonribosomal peptide synthetase; Polyketide synthase; Protein crystallization; Protein folding; Protein production; Protein purification; Protein solubility.

Figure 1

Architectures of PKS modification domains: DH (red), MT (cyan), KR (light and dark purple KRs and KRc sub-domains), ER (yellow). A two-stranded β-ribbon (orange and green β-strands) is an integral part of the KR regardless of the domain context. The presence of other modifying domains such as ER or MT can complicate identification of the full KR (β-orange – KRs – β-green – KRc). a) DEBS1 KR (PDB 2FR0): A β-strand (orange) at the KRs N-terminus pairs with a β-strand (green) between the KRs and KRc sub-domains (Keatinge-Clay et al., 2006). b) SpnB KR-ER (PDB 3SLK): The ER domain is located after the green β-strand following KRs but prior to the KRc (Zheng, Gay, Demeler, White & Keatinge-Clay, 2012). c) MAS-like PKS DH-KR-ER (PDB 5BP4): The orange β-strand preceding KRs follows the DH domain (Herbst et al., 2016). d) Modeled CurJ MT-KR (MT PDB 5THY, KR homology model): CMT domains are inserted between the orange β-strand and KRs in cis-AT PKS (Skiba et al., 2016).

Figure 2

Lysis buffer screen used in developing a purification protocol for the MalA halogenase (Fraley et al., 2017). Cells from equal-volume aliquots of an E. coli culture were lysed by sonication into 13 different lysis solutions (Table 6), and quickly purified by Ni affinity chromatography, and analyzed by SDS-PAGE. Lanes in the gel are molecular weight markers (M: 97.4, 66.2, 45.0, 31.0, 21.5, 14.4 kDa) and lysis solutions 1-13. MalA molecular weight is 73 kDa.