Analysis and Engineering of Substrate Shuttling by the Acyl Carrier Protein (ACP) in Fatty Acid Synthases (FASs)

In the large enzyme complexes of natural biosynthetic pathways, molecules are assembled like in a factory. Carrier domains shuttle substrates and intermediates as covalently attached cargo within the enzyme complex between active sites. The physical confinement of the reaction increases reaction rates and hinders pathway branching. Alternating interactions of substrate-loaded carrier domains with different catalytic domains modulate the chemical environment. In this study, we aim at assessing the impact of domain-domain interactions (DDIs) on the reaction progress of a multienzyme type I fatty acid synthase (FAS) in quantitative terms. We modulate DDIs by single interface mutations, and read out the impact on substrate shuttling by recording fatty acid (FA) chain length product spectra and FAS activities. Our data show that even single interface point mutations can severely affect FA synthesis. With molecular dynamics simulations and modeling, we relate the mutation effects to specific alterations in the molecular interaction networks and domain-domain binding energetics. Some of the presented mutations induce the synthesis of short-chain FAs. These compounds are important commodity products and potent precursors for microbial biofuel production.

synthase (KS) for another elongation, or released by a transferase (MPT) or a thio-esterase (TE) (Fig. SI1). [6][7][8] In bacterial and fungal multi-enzyme (type I) FASs, chain length is controlled by the competitive substrate acceptance of the KS domain and the MPT domain, and as such is influenced by the interfaces ACP:KS and ACP:MPT. [9][10] We recently engineered the G2559S-M2600W-mutated Corynebacterium ammoniagenes FAS, hereafter termed FAS GSMW , to produce a bimodal spectrum of C 8 -and C 14 /C 16 -CoA in vitro. [11][12] The two mutations of FAS GSMW are located in the KS-binding channel, where they attenuate the loading of octanoyl in the KS binding channel and promote the off-loading of the acyl chain by the substrate-promiscuous MPT to produce C 8 -CoA. With its bimodal product spectrum, the FAS GSMW mutant is a well-suited reporter system for elaborating the impact of domain-domain interactions (DDIs) in substrate shuttling (Fig. 1C). We  In light of the need for microbial short-chain FA production, [12][13][14] we aimed at mutating the ACP:KS interface to shift the FAS GSMW product spectrum towards short-chain products. To modulate the ACP:KS interface, mutations were introduced on the surface of the KS domain only, since an intact ACP leaves other DDIs during synthesis unaffected ( Fig. 2A). Mutation sites on the KS domain surface, such as D2553, D2556, and N2557, were derived from a study analyzing the transient interaction of FabB, a KS homologous protein in E. coli, 15 and its ACP. A2696 was identified from a crosslinking study on FabF, another KS homologue in E. coli. 16 Additionally, structural data of the S. cerevisiae FAS, which was crystallized with ACP docked to the KS domain, [17][18] were included in our consideration. On this basis, we selected two novel interaction sites, N2621 and D2622 ( Fig.   2A and Table SI1). The selected residues were mutated to alanine and/or to residues with opposite charges, and the mutant protein analyzed in activity and product spectra (SI Material and Methods). CoA production, yielding up to 77% C 8 -CoA of all detected CoA esters. The constructs with the highest yields in short-chain acyl-CoA were most severely impaired in their activity; i.e., N2557E, N2621D, and D2622A showed only a fraction of the specific activity of the template construct ( Fig   2C). Correlation of selective short acyl-CoA production and poor activities implies that changes in the product spectrum require the efficient suppression of specific DDIs. D2553A, D2553N and D2556A showed effects opposite to what was anticipated (Fig. 2B), and increased the fraction of long chain acyl-CoA.
We used structure-based homology modeling to gain a deeper understanding of the relation between ACP:KS domain interactions. We constructed three-dimensional models for ACP:KS using the S. cerevisiae FAS crystallographic structure (pdb: 2uv8) as a template (Note SI2, Fig.   SI4-SI8). 17 High crystallographic B-factors and low sequence conservation in a loop between K160 and P167 of ACP suggested local structural disorder (Fig. 3A). To account for possible flexibility in this region, we constructed two different models (A and B). In model A, we fully retained the secondary structure of the template. In model B, we locally relaxed the secondary structure restriction to account for a variation in bacterial and fungal systems (Fig. 3B). The multiple sequence alignment indicates that target loop residues V1753 and R1754 correspond to template K161 and S162, respectively (S. cerevisiae FAS numbering (2uv8); Fig. SI5). In S. cerevisiae FAS, The calculated binding affinities correlate both with the measured FAS activities and the observed product spectra (Fig. 4A). Strong ACP:KS binding is associated with the production of long acyl chains and high FAS activity. The good correlation implies that the stability of the ACP:KS interaction indeed modulates C. ammoniagenes FAS function, which allows us to establish detailed relations between structure, energetics, and FAS product spectrum.
Mutants N2557E and N2621D are most effective in producing C 8 -CoA and display the highest (i.e., least favorable) relative binding energies. This can be explained by mutations introducing negative charges at the DDI that affect an ionic interaction network across residues 2557, 2621, 2622, and 2694 (KS) and 1774 (ACP) (Fig. 4B and C). In the unmodified interface, the polar residue R1774 is located at distances of 6.0 Å and 5.4 Å from N2557 and D2622, respectively. By contrast, the mutant E2557 sequesters R1774 in a salt-bridge interaction. The resulting charge imbalance around D2622 weakens the polar interaction network and the interface stability (Fig. 4B). Similarly, the change in charge of N2621D disrupts the polar interaction between N2621 and D2694 (Fig.   4C). Both mutations lower FAS enzymatic activity. By contrast, mutations N2557A and N2621A do not alter the charge balance of the interface and have no effect on FAS function ( Fig. 4B and C). and target (C. ammoniagenes) ACP:KS complexes. S. cerevisiae FAS template shown in gray and C. ammoniagenes FAS in magenta (ACP) and blue/white (KS 2 ). The ACP loop between K1752 and G1759 of ACP is highlighted in green. Models A and B differ in interactions between the interface residues R1774 and D2553.
A2696 is located at the interface periphery and its mutations do not trigger the modulation of FAS function (Note SI3 and Fig. SI9). Mutants D2553A, D2553N and D2556A revert the FAS GSMW spectrum to long chain production and display the highest (i.e., most favorable) relative binding energies. Interestingly, the energy shift of D2553A is only captured when combining the two alternative configurations of model A (the mutant) and model B (the wild-type). In such a scheme, the polarity of position 2553 (KS) influences the orientation of R1754 (ACP) that forms a salt-bridge with D2553 and is repelled by A2553 (Note SI4 and Fig. SI10). Mutant D2553N, despite a possible change in charge, has a minor impact on R1754 orientation (Note SI4 and Fig. SI10). In D2556A, the negative charge deletion leads to a minor but effective structural reorganization of the polar network at the interface (Note SI5 and Fig. SI11). In case of the charged-to-neutral mutation D2622A at the center of a network of ionic interactions, the limited sampling of motions in the computation of binding affinities is likely insufficient to capture a major reorganization of the interface, providing an explanation for this single outlier (Note SI6 and Fig. SI12).
As a further challenge to modeling substrate shuttling, binding energies are not the only determinant of DDI kinetics. Effects of accessibility and confinement will impact the frequency and duration of productive interaction between ACP and KS. As a first step towards capturing substrate shuttling kinetics, we set up coarse-grained simulations to count frequencies of competitive binding events of ACP to wildtype and surface mutated KS (Note SI7 and Fig. SI13). 19 The kinetic approach did, however, not lead to conclusive data, reflecting both the challenges in simulating binding kinetics in a confined environment and the paucity of experimental information required for model generation. perturb substrate shuttling in FASs. In silico modeling demonstrates that mutations are particularly invasive when inducing charge imbalances at the interfaces, which agrees with earlier finding on the importance of electrostatic complementarity of ACP:catalytic domains interfaces. 5,17,20 Engineering of DDIs proved to be powerful in modulating the chain length spectrum of C.
ammoniagenes FAS, and some mutations, i.e., N2557E, N2621D and N2622A, produced C 8 -CoA with high selectivity. However, the strong decrease in activity of the mutated FASs questions a true biotechnological relevance of DDI engineering in FASs, and rather illustrates an inherent "lowresolution" problem of the approach. While engineering of substrate binding channels 12 or the thioesterase-mediated hydrolyzation of acyl-ACP of certain chain length 13, [21][22] hijacks FA synthesis at a specific acyl-ACP chain length, ACP surface mutations interfere already in initial FA cycles, causing the overall drop in activity.
The observed sensitivity of substrate shuttling is also interesting for the related PKSs. Similar to FASs, engineering of substrate shuttling may enable modulation of product synthesis in iterative polyketide synthases (PKSs). 23 In modular PKSs, ACPs are involved in the substrate shuttling within one multienzyme complex (module), but also in the translocation of the substrate to a downstream module. The strong influence by even single mutations implies that a successful assembly of such modules for the design of new chimeric biosynthetic pathways will strongly rely on the effective adaptation of interfaces across the borders of the chimeric assembly lines. 24

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