YASH and GHSXG motifs of ZmaA-AT compared to a methylmalonyl-CoA specific AT.

<p>The substrate binding-pocket amino acid residues (290–300 and 194) of ZmaA-AT (blue, with white span for disordered 293–295) are superimposed on those of AT from the DEB PKS module 3 (wheat). Bulky F193 is found next to the active site S192 in ZmaA-AT, instead of the glutamine residue found in methylmalonyl-CoA specific ATs. The catalytic H297 is positioned similarly to other ATs, despite its proposed steric hindrance to extender units with (2<i>R</i>) conformations. Despite high mobility for the substrate pocket lid YASH motif, we conclude based on the positions of well-ordered flanking residues that they must wander within the substrate binding pocket of ZmaA-AT, which holds co-crystallized formate (spheres). The red box, with its marked corner, can be compared to the same box in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110965#pone-0110965-g002" target="_blank">Figure 2</a> in order to orient the reader.</p

Overall structure of ZmaA-AT.

<p>The N-terminal KS-AT linker (green), α/β-hydrolase large subdomain (blue), small subdomain (gray), and post-AT linker (red) make up the complete asymmetric unit. The active site of ZmaA-AT (inside solid red box) is bounded on the left by the substrate pocket lid (containing the YASH motif, which in ZmaA-AT is GAAH) and on the top by the RVDVVQ motif (yellow) and is occupied by formate (spheres); cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110965#pone-0110965-g003" target="_blank">Figure 3</a>. Residues E293-G294-A295 are not observed and are indicated with a dashed line. The proposed substrate ACP binding surface M286-E293 contains the methionine residues of the RXR motif (MCM in ZmaAT) (dotted red box) which correspond to the inchoate β-strand of the ferredoxin fold in the smaller subdomain of other ATs; cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110965#pone-0110965-g004" target="_blank">Figure 4</a>.</p

The RXR Motifs of Select Acyltransferases Control Extender Unit Specificity.

<p>The RXR Motifs of Select Acyltransferases Control Extender Unit Specificity.</p

Proposed movement of the substrate pocket lid induced by ZmaD binding.

<p>(A) Based on the crystal structure of ZmaA-AT, the substrate pocket lid (blue) is shown in the closed position, restricting the entry of the extender unit, in the absence of substrate carrier protein. (B) Model structure of ZmaA-AT bound to substrate carrier protein, ZmaD (blue spheroid). The binding of the substrate carrier protein to the RXR motif (M286-C-M288 in ZmaA-AT; gray sticks) in the small subdomain of ZmaA-AT is proposed to cause the formation of the β-strand (red), resulting in the opening of the substrate pocket lid (blue).</p

Transacylase assay of ZmaA-AT Distinguishes ACP from Acyl Unit Recognition.

<p>SDS-PAGE of reaction mixtures and corresponding phosphorimage. Lane 1: Molecular mass markers (Prestained Broad-range, Biorad). Lane 2: ZmaA-AT. Lane 3: ZmaD (ACP). Lane 4: Sfp (4′-phosphopantetheinyl transferase). Lane 5: ZmaA-AT, Sfp, and *Malonyl-CoA. Lane 6: ZmaA-AT, Sfp, ZmaD, and *Malonyl-CoA. Lane 7: ZmaA-AT, Sfp, and *(2-<i>RS</i>)-methylmalonyl-CoA. Lane 8: ZmaA-AT, Sfp, ZmaD, and *(2-<i>RS</i>)-methylmalonyl-CoA.</p

Proposed AT-Domain Interaction with ACP Substrate Carrier.

<p>Approximate protein contact potential calculated using PyMOL vacuum electrostatics function. The colors represent potentials ranging from −70 mV (red) to +70 mV (blue). (A) Proposed AT/ACP interface of ZmaA-AT. FabD was aligned to the structure of ZmaA-AT to show the relative position of CoA (dots, FabD not shown). (B) AT/CoA interface of <i>E. coli</i> FabD (PBD ID: 2G2Z, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110965#s2" target="_blank">Methods</a><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110965#pone.0110965-Oefner1" target="_blank">[18]</a>). CoA is shown as spheres.</p

Biosynthesis of ZMA.

<p>(A) Biosynthetic pathway of hydroxymalonyl-ACP. The final FAD dependent oxidation step catalyzed by ZmaE may proceed through an endiol intermediate (red), resulting in the loss of stereospecificity at C2 of the final product, hydroxymalonyl-ACP. (B) ZMA PKS/NRPS. Nine extender units are utilized to form the precursor of metabolite A (green), zwittermicin A (red), and metabolite B (blue). Hydroxymalonyl-ACP is recognized by ZmaA (dotted line). Each circle represents a catalytic domain of the PKS/NRPS: C, condensation; A, adenylation; PCP, peptide carrier protein; E, epimerization; KS, ketosynthase; AT, acyltransferase; KR, ketoreductase; ACP, acyl carrier protein; Pr, protease; TE, thioesterase. (C) Natural prodrug activation. ZmaL is proposed to catalyze the cleavage of the ZMA precursor molecule from ZmaB-bound alanine, which is further condensed to leucine and methionine to form metabolite B (blue). ZmaM is proposed to catalyze the separation of metabolite A (green) from ZMA (red).</p

The GHSXG and the YASH Motifs of Select Acyltransferases are Responsible for ACP <i>vs</i> CoA Discrimination.

<p>The GHSXG and the YASH Motifs of Select Acyltransferases are Responsible for ACP <i>vs</i> CoA Discrimination.</p

Data Collection and Refinement Statistics.

<p>*Highest resolution shells in parentheses.</p>a<p><i>R</i>_sym(I) = ∑_hkl∑_i |I_i(<i>hkl</i>) – <<i>I</i>(<i>hkl</i>)>|/∑_hkl∑_i I_i(<i>hkl</i>) where <i>I</i>(<i>i</i>) is the intensity of the <i>i</i>th observation of the <i>hkl</i> reflection and <<i>I</i>(<i>hkl</i>)> is the mean intensity from multiple measurements of the <i>h</i>, <i>k</i>, <i>l</i> reflection.</p>b<p><i>R</i>_cryst(<i>F</i>) = ∑<i>_hkl</i>|<i>F</i>_obs(<i>hkl</i>)–<i>F</i>_calc(<i>hkl</i>)|/∑_hkl<i>F</i>_obs(<i>hkl</i>), where <i>F</i>_obs(<i>hkl</i>) and <i>F</i>_calc(<i>hkl</i>) are the observed and calculated structure factor amplitudes for the <i>h</i>, <i>k</i>, <i>l</i> reflection.</p>c<p><i>R</i>_free is <i>R</i>_cryst calculated for a randomly selected test set of reflections (5%) not included in the refinement.</p><p>Data Collection and Refinement Statistics.</p

Possible difference in substrate entry angles between DEB PKS AT-5 and ZmaA-AT.

<p>(A) In DEB PKS-AT5, Q643 has been proposed to orient the incoming (2<i>S</i>)-methylmalonyl-CoA so that Y742 makes a hydrophobic interaction with the methyl-group and H745 sterically hinders the entry of (2<i>R</i>)-methylmalonyl-CoA <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110965#pone.0110965-Tang2" target="_blank">[26]</a>. (B) In ZmaA-AT, F193 is not positioned to orient the incoming substrate, which may allow hydroxymalonyl-ACP with (2<i>R</i>)-stereochemistry to enter the substrate pocket unhindered.</p