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Crystal Structure and Biochemical Studies of the Trans-Acting Polyketide Enoyl Reductase Lovc from Lovastatin Biosynthesis

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Crystal Structure and Biochemical Studies of the Trans-Acting Polyketide Enoyl Reductase Lovc from Lovastatin Biosynthesis Crystal structure and biochemical studies of the trans-acting polyketide enoyl reductase LovC from lovastatin biosynthesis Brian D. Amesa, Chi Nguyena, Joel Brueggera, Peter Smitha, Wei Xub, Suzanne Mab, Emily Wonga, Steven Wonga, Xinkai Xieb, Jesse W.-H. Lic, John C. Vederasc, Yi Tangb,1, and Shiou-Chuan Tsaia,1 aDepartments of Molecular Biology and Biochemistry, Chemistry, and Pharmaceutical Sciences, University of California, Irvine, CA 92697; bDepartment of Chemical and Biomolecular Engineering, University of California, Los Angeles, CA 90095; and cDepartment of Chemistry, University of Alberta, Edmonton, AB, Canada T6G 2G2 Edited by Gregory A. Petsko, Brandeis University, Waltham, MA, and approved May 1, 2012 (received for review August 10, 2011) Lovastatin is an important statin prescribed for the treatment and bassiana (14). These trans-acting ERs are highly homologous (38– prevention of cardiovascular diseases. Biosynthesis of lovastatin 80% sequence identity; Fig. S1A) and are observed to interact with fi – uses an iterative type I polyketide synthase (PKS). LovC is a trans- megasynthases through speci cproteinprotein interactions (4). fi acting enoyl reductase (ER) that specifically reduces three out of However, how LovC speci cally interacts with LovB and why it eight possible polyketide intermediates during lovastatin biosyn- reduces only at the tetraketide, pentaketide, and heptaketide stages is not well understood. thesis. Such trans-acting ERs have been reported across a variety of Herein, we report the crystal structure of LovC, which repre- other fungal PKS enzymes as a strategy in nature to diversify poly- fi sents the structure of a polyketide ER from an iterative reducing ketides. How LovC achieves such speci city is unknown. The 1.9-Å Type I PKS. LovC is a 39.5-kDa protein (363 residues) and, sur- structure of LovC reveals that LovC possesses a medium-chain de- prisingly, is shown to be a unique monomeric member of the me- hydrogenase/reductase (MDR) fold with a unique monomeric as- dium-chain dehydrogenase/reductase (MDR) superfamily (15). sembly. Two LovC cocrystal structures and enzymological studies Two cocrystal structures are presented: the LovC–crotonoyl help elucidate the molecular basis of LovC specificity, define stereo- CoA complex and the LovC-NADP+ binary complex. To study the chemistry, and identify active-site residues. Sequence alignment trans-acting ERs and their unique substrate specificity, an in vitro indicates a general applicability to trans-acting ERs of fungal PKSs, assay is essential, and we report the successful development of as well as their potential application to directing biosynthesis. LovC enzyme assays that may be applied to other trans-acting ERs. We elucidated the structural basis of LovC substrate specificity and found that LovC mutations can alter its substrate specificity. The therosclerosis is the current leading cause of death for adults in results can be leveraged with our previous work using trans-acting Athe western world (1). Statins inhibit cholesterol biosynthesis ERs to biosynthesize new statin analogs such as the compactin and are the most widely prescribed drugs for the prevention and precursor (5), and the chemical diversity can potentially be further treatment of atherosclerosis (2). Lovastatin (compound 1) is the fi expanded by using other trans-acting ERs working in conjugation rst statin approved by the Food and Drug Administration and is with the megasynthases for directing biosynthesis. the direct precursor for manufacture of simvastatin, the second most-prescribed drug worldwide (1). The high impact of statins to Results and Discussion human health, including their possible use for cancer and neuro- degenerative diseases (3), has prompted vigorous efforts toward Overall Structure: LovC Is a Unique Monomeric ER and MDR Member. synthesis of lovastatin (1). However, the structural complexity of Unlike previously reported ER domains [Protein Data Bank lovastatin has prevented its commercial production by total chem- (PDB) ID codes 1GUF and 1ZSY], LovC exists as a monomer as ical synthesis (1). In nature, lovastatin is biosynthesized by the detected from the crystal structure (Fig. 2) and size-exclusion fungus Aspergillus terreus (4) using a gene cluster that contains two chromatography (Fig. 3). The monomeric state of LovC is highly polyketide synthases (PKSs), LovB and LovF (Fig. 1A)(5).Based unique in the MDR superfamily (15), the members of which are on past biochemical studies of lovastatin biosynthesis, our previous functionally dimeric or tetrameric, except for the monomeric bioengineering efforts led to rational generation of lovastatin ana- mannitol dehydrogenase (16). The MDR superfamily includes the zinc-containing alcohol dehydrogenases (ADHs), leukotriene logs with enhanced activities and reduced side effects (6). Despite dehydrogenases, quinone oxidoreductases, the eukaryotic the above advances, many questions about lovastatin biosynthesis ERs, and membrane-sensing proteins (15). Because none of the remain unanswered. Understanding lovastatin biosynthesis repre- MDR proteins of known structures shares significant sequence sents an opportunity to leverage our knowledge for future efforts in homology to LovC, we could not solve the LovC structure by directing biosynthesis of statin analogs. molecular replacement. Subsequently, the LovC structure was During lovastatin biosynthesis, the iterative type I PKS LovB solved by multiwavelength anomalous dispersion (MAD) tech- builds the nonaketide main framework with the aid of the trans- nique using selenomethionine-derived protein (Table S1). The acting enoyl reductase (ER) LovC (4, 7). A second type I iterative PKS, LovF, constructs the 2-methylbutyryl side chain. LovB and LovF are both large multidomain megasynthases that are highly homologous in sequence and domain arrangements (Fig. 1A). Author contributions: B.D.A., P.S., J.C.V., Y.T., and S.-C.T. designed research; B.D.A., C.N., When coupled with LovC, LovB iteratively catalyzes more than 30 P.S., W.X., E.W., S.W., and J.W.-H.L. performed research; B.D.A., S.M., X.X., and J.W.-H.L. contributed new reagents/analytic tools; B.D.A., J.B., and S.-C.T. analyzed data; and B.D.A., precisely synchronized reactions to yield a 19-carbon intermediate C.N., and S.-C.T. wrote the paper. dihydromonacolin L (DmL) (compound 2) (Fig. 1A). In compari- fl son, LovF only catalyzes one round of Claisen condensation to The authors declare no con ict of interest. produce a 2-methylbutyryl intermediate, which is attached to the C8 This article is a PNAS Direct Submission. hydroxyl of monacolin J (compound 3) to produce lovastatin. LovC Freely available online through the PNAS open access option. specifically interacts with only LovB, but not LovF, and accepts only Data deposition: The atomic coordinates and structure factors reported in this paper have three out of eight possible LovB intermediates as its substrates (Fig. been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes 3B6Z, 3B70, and 1A; tetra-, penta-, and heptaketides). Similar trans-acting ERs have 3GQV). also been reported in other fungi such as MokE in Monascus pilosus 1To whom correspondence may be addressed. E-mail: [email protected] or [email protected]. (8), MlcG in Penicillium citrinum (9), ApdC in Aspergilus nidulans This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (10–12), cytochalasans (12, 13), and tenellin_ORF3 in Beauveria 1073/pnas.1113029109/-/DCSupplemental. 11144–11149 | PNAS | July 10, 2012 | vol. 109 | no. 28 www.pnas.org/cgi/doi/10.1073/pnas.1113029109 Downloaded by guest on September 27, 2021 Fig. 1. (A) Proposed lovastatin biosynthetic pathway with 1-3 shown in their acidic forms. (B) Three putative substrates (tetra-, penta- and hepta-ketides) of LovC. (C) In vitro substrates. NADPH (compound 4) and NADH (compound 5) are discussed in the text. LovC structure contains two domains: the catalytic domain (res- The principal difference between LovC and these homologs lies idues 1–134, 283–363) and cofactor-binding domain (Rossmann in two transition regions between the catalytic and cofactor- fold; residues 135–282) (Fig. 2A). The structural comparison binding domains, consisting of α2-αA-loop and α3-loop-α4. LovC between LovC and selected MDR proteins (18–23% sequence has an additional 10 or more residues in each loop (xL1 and xL2; identity) shows 1.6–2.4 Å rms deviation (RMSD) between the Fig. 2A and Fig. S1B). The extended lengths of these two loops aligned Cα coordinates (Figs. S1B and S2). All are dimers except are a unique conserved feature of trans-acting ERs associated LovC. The least conserved region is located between α2 and βA. with fungal PKSs (Fig. S1A), and, as discussed below, the Fig. 2. (A) Overall fold and quaternary structure of LovC. Two extended loops, xL1 and xL2, are unique to the LovC structure. (B) Comparison of LovC-NADP+ (blue) vs. LovC-CO7 (yellow) structures showing the major change at Y296. (C) Comparison of both models (rendered as electrostatic surface) reveals that the BIOCHEMISTRY protein surface has significant changes. (D) Zoomed view shows that the active site does not change. Ames et al. PNAS | July 10, 2012 | vol. 109 | no. 28 | 11145 Downloaded by guest on September 27, 2021 Fig. 3. Gel-filtration interaction study indicative of LovB-LovC complex formation. Inset, SDS/ PAGE confirmed interaction: M, marker; 1, puri- fied LovB (335 kDa, doublet resulting from the loss of an N-terminal KS-containing fragment); 2, purified LovC (40 kDa); F9, fraction 9 from the combined run. extended loops may influence the oligomeric state and protein– (E)-2-butenoyl-acyl-N-acetylcysteamine [(E)-2-butenoyl-NAC] protein interactions of LovC during lovastatin biosynthesis. (compound 8), (2E,4E)-hexadienoyl-NAC (compound 9), and The LovC catalytic domain has three β-sheets: the anti-parallel (E)-2-octenoyl-NAC (compound 10) to mimic the α,β unsaturated Beta1 (β1-β2), the anti-parallel Beta2 (β5-β4-β6), and the six- di-, tri, and tetraketide intermediates, respectively, and (E)-2- stranded Beta3 (β7-β5-β4-β3-β9-β8) (Fig.
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