Insights into a dual function amide oxidase/macrocyclase from lankacidin biosynthesis

Abstract

Acquisition of new catalytic activity is a relatively rare evolutionary event. A striking example appears in the pathway to the antibiotic lankacidin, as a monoamine oxidase (MAO) family member, LkcE, catalyzes both an unusual amide oxidation, and a subsequent intramolecular Mannich reaction to form the polyketide macrocycle. We report evidence here for the molecular basis for this dual activity. The reaction sequence involves several essential active site residues and a conformational change likely comprising an interdomain hinge movement. These features, which have not previously been described in the MAO family, both depend on a unique dimerization mode relative to all structurally characterized members. Taken together, these data add weight to the idea that designing new multifunctional enzymes may require changes in both architecture and catalytic machinery. Encouragingly, however, our data also show LkcE to bind alternative substrates, supporting its potential utility as a general cyclization catalyst in synthetic biology.

Fig. 1

Proposed biosynthetic pathway to lankacidin C. The gene cluster, which has been shown to be sufficient for lankacidin biosynthesis, encodes five assembly-line proteins, LkcA (a hybrid NRPS/PKS subunit), LkcB (a discrete DH), LkcC, LkcF, and LkcG, together containing a total of only four KS domains, although eight KS-mediated extension cycles are required. One possibility that agrees with phylogenetic analysis of KS substrate specificity is shown here, in which the assembly-line incorporates multiple copies of the proteins LkcB, LkcC and LkcF (see ref. for an alternate view). The starter unit may be either pyruvoyl-ACP or lactoyl-ACP, both derived from 1,3-bisphosphoglycerate. However, we and others have found that in an LkcE-deleted mutant, only the lactoyl form of the first free intermediate LC-KA05 accumulates (Supplementary Fig. 8), so we propose that lactoyl-ACP serves as the starter unit. The enzyme responsible for acetyl transfer at C-7 likewise remains to be determined, although a candidate is LkcH, which shows homology to isochorismatases. The object of this study, LkcE, catalyzes the critical macrocyclization reaction of LC-KA05 (1), resulting in lankacidinol A (2). The carbons implicated in the ring closure are indicated in red. Domain abbreviations: C, condensation; A, adenylation; PCP, peptidyl carrier protein; KS, ketosynthase; DH, dehydratase; KR, ketoreductase; MT, C-methyltransferase; ACP, acyl carrier protein; TE, thioesterase

Fig. 2

Structures of selected compounds investigated in this study. Compound LC-KA05 (1) was shown by NMR to exist almost exclusively in the enol form, and thus its derivatives 6 and 7 are also represented as enols. The gray boxes indicate where 6 and 7 differ from 1. The stereochemistry of the C-6−C-7 double bond in 7 and cyclized 7 (8) is unknown, as indicated

Fig. 3

Crystal structures of LkcE and its mutants. a Crystal structure of homodimeric, wild-type LkcE. The FAD-binding domain is shown in light blue and light purple for the monomers A and B, respectively, whereas the substrate-binding domain is shown in dark blue and purple. The FAD is colored in yellow. b View of the active site of wild-type LkcE in the presence of bound DATD (pink) with its 2F_o−F_c map contoured at 1σ. Binding occurs mainly via hydrophobic interactions and a single hydrogen bond with T397. c View of the active site of wild-type LkcE in the presence of bound EMAA (orange) with the 2F_o−F_c map contoured at 0.6σ. Comparison with b shows the analog to be binding in the same region of the active site. d View of the active site of the LkcE E64Q mutant in the presence of bound LC-KA05 (green), which adopts a linear conformation. The 2F_o−F_c map surrounding the substrate is contoured at 1σ

Fig. 4

Characterization of recombinant LkcE by SAXS. a Fit between the ab initio model computed with GASBOR (black line) (χ² = 1.44), the theoretical scattering curve calculated on the basis of the structure of LkcE_WT with CRYSOL (blue line) (χ² = 3.8) and the experimental SAXS data (red dots). Inset is the Guinier plot, which yields an R_g of 31.0 Å. A molecular weight (MW) of 99.3 kDa was calculated using the SAXS MoW program (homodimer calculated MW = 98.6 kDa). b The distance distribution function derived for LkcE calculated with GNOM, yielding a D_max of 91 Å. c Averaged ab initio envelope of LkcE calculated using GASBOR (gray mesh) with superimposition of the LkcE_WT crystal structure carried out using SUPCOMB (blue)

Fig. 5

Alternate homodimerization mode of LkcE relative to hMAO B and 6HDNO. a Homodimeric structure of LkcE. b Homodimerization mode of 6HDNO. The dimer is superimposed on one monomer of LkcE (in blue in a), in the same orientation. c Homodimerization mode of hMAO B. Again, the dimer has been superimposed on a monomer of LkcE. d Interaction surface between the two monomers in LkcE (yellow), hMAO B (red), and 6HDNO (green), relative to a monomer of LkcE. This analysis clearly shows that the mode of homodimerization of LkcE, as well as its overall quarternary organization, differ from these two homologs

Fig. 6

Kinetic analysis by coupled assay of LkcE and its mutants. Data for which errors are shown were obtained by experiments repeated in duplicate (a) (the error bars thus show the two data points) or triplicate (b) (the error bars indicate the standard deviation). a LkcE acting on its native substrate, LC-KA05 (1), as a 2:1 mixture with eliminated product 7. b LkcE acting on deacetylated (7-OH) substrate 6. c LkcE acting on 6 but with each concentration adjusted to contain the maximum amount of DMSO (that present at 100 μM 6 in b). As the data in b and c are essentially identical, the concentration of DMSO in this range had no effect on the rate. d LkcE R326Q acting on 6. e LkcE Y182F acting on 6. It must be noted that as we were limited in these assays for reasons of sensitivity to higher concentrations of substrate, it is possible that we missed an earlier sigmoidal dependence on concentration, indicative of cooperative behavior between the two LkcE monomers

Fig. 7

Proposed mechanism of the LkcE-catalyzed reaction. LC-KA05 binds into the active site, and undergoes FAD-catalyzed oxidation to yield the iminium ion. A conformational change then occurs to bring the δ-lactone in proximity to the iminium, with the new LC-KA05 conformation potentially stabilized by interaction with R326. The final step is an intramolecular Mannich reaction, involving general base catalysis by E64 to generate the nucleophilic C-2 enolate