The unusual substrate specificity of Escherichia coli cardiolipin synthase C does not require the product of the transcriptionally engaged ymdB gene.

Polycistronic transcription and translation of ymdB-clsC have been thought to be required for full activity of ClsC. The authentic initiation codon of the clsC gene is present within the open reading frame of the upstream located ymdB gene. ClsC translated from authentic initiation codon drives cardiolipin (CL) synthesis without transcriptionally paired YmdB. YmdB is not necessary for the substrate specificity of ClsC utilizing phosphatidylethanolamine (PE) as a co-substrate.

Inositol-Exchange Activity in Human Primordial Placenta.

Human placenta is an intensively growing tissue. Phosphatidylinositol (PI) and its derivatives are part of the signaling pathway in the regulation of trophoblast cell differentiation. There are two different enzymes that take part in the direct PI synthesis: phosphatidylinositol synthase (PIS) and inositol exchange enzyme (IE). The presence of PIS is known in the human placenta, but IE activity has not been documented before. In our study, we describe the physiological properties of the two enzymes in vitro. PIS and IE were studied in different Mn2+ and Mg2+ concentrations that enabled us to separate the individual enzyme activities. Enzyme activity was measured by incorporation of 3[H]inositol in human primordial placenta tissue or microsomes. Optimal PIS activity was achieved between 0.5 and 2.0 mM Mn2+ concentration, but higher concentrations inhibit enzyme activity. In the presence of Mg2+, the enzyme activity increases continuously up to a concentration of 100 mM. PIS was inhibited by nucleoside di- and tri-phosphates. PI production increases between 0.1 and 10 mM Mn2+ concentration. The incorporation of [3H]inositol into PI increased by 57% when adding stabile GTP analog. The described novel pathway of inositol synthesis may provide an additional therapeutic approach of inositol supplementation before and during pregnancy.

Two natural compounds as potential inhibitors against the Helicobacter pylori and Acinetobacter baumannii IspD enzymes.

In a vast majority of bacteria, protozoa and plants, the methylerythritol phosphate (MEP) pathway is utilized for the synthesis of isopentenyl diphosphate (IDP) and dimethylallyl diphosphate (DMADP), which are precursors for isoprenoids. Isoprenoids, such as cholesterol and coenzyme Q, play a variety of crucial roles in physiological activities, including cell-membrane formation, protein degradation, cell apoptosis, and transcription regulation. In contrast, humans employ the mevalonate (MVA) pathway for the production of IDP and DMADP, rendering proteins in the MEP pathway appealing targets for antimicrobial agents. This pathway consists of seven consecutive enzymatic reactions, of which 4-diphosphocytidyl-2C-methyl-D-erythritol synthase (IspD) and 2C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (IspF) catalyze the third and fifth steps, respectively. In this study, we characterized the enzymatic activities and protein structures of Helicobacter pylori IspDF and Acinetobacter baumannii IspD. Then, using the direct interaction-based thermal shift assay, we conducted a compound screening of an approved drug library and identified 27 hit compounds potentially binding to AbIspD. Among them, two natural products, rosmarinic acid and tanshinone IIA sodium sulfonate, exhibited inhibitory activities against HpIspDF and AbIspD, by competing with one of the substrates, MEP. Moreover, tanshinone IIA sodium sulfonate also demonstrated certain antibacterial effects against H. pylori. In summary, we identified two IspD inhibitors from approved ingredients, broadening the scope for antibiotic discovery targeting the MEP pathway.

SGMS2 in primary osteoporosis with facial nerve palsy.

Pathogenic heterozygous variants in SGMS2 cause a rare monogenic form of osteoporosis known as calvarial doughnut lesions with bone fragility (CDL). The clinical presentations of SGMS2-related bone pathology range from childhood-onset osteoporosis with low bone mineral density and sclerotic doughnut-shaped lesions in the skull to a severe spondylometaphyseal dysplasia with neonatal fractures, long-bone deformities, and short stature. In addition, neurological manifestations occur in some patients. SGMS2 encodes sphingomyelin synthase 2 (SMS2), an enzyme involved in the production of sphingomyelin (SM). This review describes the biochemical structure of SM, SM metabolism, and their molecular actions in skeletal and neural tissue. We postulate how disrupted SM gradient can influence bone formation and how animal models may facilitate a better understanding of SGMS2-related osteoporosis.

Sphingomyelin synthase-related protein SMSr is a phosphatidylethanolamine phospholipase C that promotes nonalcoholic fatty liver disease.

Sphingomyelin synthase (SMS)-related protein (SMSr) is a phosphatidylethanolamine phospholipase C (PE-PLC) that is conserved and ubiquitous in mammals. However, its biological function is still not clear. We previously observed that SMS1 deficiency-mediated glucosylceramide accumulation caused nonalcoholic fatty liver diseases (NAFLD), including nonalcoholic steatohepatitis (NASH) and liver fibrosis. Here, first, we evaluated high-fat diet/fructose-induced NAFLD in Smsr KO and WT mice. Second, we evaluated whether SMSr deficiency can reverse SMS1 deficiency-mediated NAFLD, using Sms1/Sms2 double and Sms1/Sms2/Smsr triple KO mice. We found that SMSr/PE-PLC deficiency attenuated high-fat diet/fructose-induced fatty liver and NASH, and attenuated glucosylceramide accumulation-induced NASH, fibrosis, and tumor formation. Further, we found that SMSr/PE-PLC deficiency reduced the expression of many inflammatory cytokines and fibrosis-related factors, and PE supplementation in vitro or in vivo mimicked the condition of SMSr/PE-PLC deficiency. Furthermore, we demonstrated that SMSr/PE-PLC deficiency or PE supplementation effectively prevented membrane-bound ß-catenin transfer to the nucleus, thereby preventing tumor-related gene expression. Finally, we observed that patients with NASH had higher SMSr protein levels in the liver, lower plasma PE levels, and lower plasma PE/phosphatidylcholine ratios, and that human plasma PE levels are negatively associated with tumor necrosis factor-α and transforming growth factor ß1 levels. In conclusion, SMSr/PE-PLC deficiency causes PE accumulation, which can attenuate fatty liver, NASH, and fibrosis. These results suggest that SMSr/PE-PLC inhibition therapy may mitigate NAFLD.

The formation of migrasomes is initiated by the assembly of sphingomyelin synthase 2 foci at the leading edge of migrating cells.

The migrasome is an organelle of migrating cells with diverse physiological functions. How migrasome formation is initiated is unknown. We found that sphingomyelin is enriched in migrasomes and identified sphingomyelin synthase 2 (SMS2) as an essential protein for migrasome biogenesis. SMS2 assembles into immobile foci that adhere on the basal membrane at the leading edge. When cells migrate away, the SMS2 foci 'move' out of cells and into retraction fibres, where they become migrasome formation sites and eventually grow into migrasomes. Mechanistically, SMS2 foci seed migrasomes by converting ceramide to sphingomyelin, which is essential for migrasome formation. Furthermore, CerS5, which is required for the synthesis of long-chain ceramide, and CERT, which transports ceramide from the endoplasmic reticulum to Golgi, are both required for migrasome formation. Our data reveal the essential role of ceramide and sphingomyelin in migrasome formation and suggest that SMS2 forms basal membrane-surface-connecting structures that pre-determine where migrasomes will grow.

Functional screening of lysosomal storage disorder genes identifies modifiers of alpha-synuclein neurotoxicity.

Heterozygous variants in the glucocerebrosidase (GBA) gene are common and potent risk factors for Parkinson's disease (PD). GBA also causes the autosomal recessive lysosomal storage disorder (LSD), Gaucher disease, and emerging evidence from human genetics implicates many other LSD genes in PD susceptibility. We have systemically tested 86 conserved fly homologs of 37 human LSD genes for requirements in the aging adult Drosophila brain and for potential genetic interactions with neurodegeneration caused by α-synuclein (αSyn), which forms Lewy body pathology in PD. Our screen identifies 15 genetic enhancers of αSyn-induced progressive locomotor dysfunction, including knockdown of fly homologs of GBA and other LSD genes with independent support as PD susceptibility factors from human genetics (SCARB2, SMPD1, CTSD, GNPTAB, SLC17A5). For several genes, results from multiple alleles suggest dose-sensitivity and context-dependent pleiotropy in the presence or absence of αSyn. Homologs of two genes causing cholesterol storage disorders, Npc1a / NPC1 and Lip4 / LIPA, were independently confirmed as loss-of-function enhancers of αSyn-induced retinal degeneration. The enzymes encoded by several modifier genes are upregulated in αSyn transgenic flies, based on unbiased proteomics, revealing a possible, albeit ineffective, compensatory response. Overall, our results reinforce the important role of lysosomal genes in brain health and PD pathogenesis, and implicate several metabolic pathways, including cholesterol homeostasis, in αSyn-mediated neurotoxicity.

Directed Evolution of the BpsA Carrier Protein Domain for Recognition by Non-cognate 4'-Phosphopantetheinyl Transferases to Enable Inhibitor Screening.

4'-Phosphopantetheinyl transferases (PPTases) play an essential role in activating the carrier protein domains of mega-synthases involved in primary and secondary metabolism and have been validated as promising drug targets in multiple pathogens. Monitoring phosphopantetheinylation of the non-ribosomal peptidase synthetase BpsA, which produces blue indigoidine pigment upon activation, is a useful strategy to screen chemical collections for inhibitors of a target PPTase. However, PPTases can exhibit carrier protein specificity and some medically important PPTases do not activate BpsA. Here, we describe how to conduct a directed evolution campaign to evolve the BpsA carrier protein domain for improved recognition by a candidate PPTase, as exemplified for the human Sfp-like PPTase. This method can be applied to other non-cognate PPTases for discovery of new drug candidates or chemical probes, or to enable development of next-generation biosensors that utilize BpsA as a reporter.

Effect of Total SMS Activity on LDL Catabolism in Mice.

BACKGROUND: Sphingomyelin (SM) and cholesterol are 2 key lipid partners on cell membranes and on lipoproteins. Many studies have indicated the influence of cholesterol on SM metabolism. This study examined the influence of SM biosynthesis on cholesterol metabolism. METHODS: Inducible global Sms1 KO (knockout)/global Sms2 KO mice were prepared to evaluate the effect of whole-body SM biosynthesis deficiency on lipoprotein metabolism. Tissue cholesterol, SM, ceramide, and glucosylceramide levels were measured. Triglyceride production rate and LDL (low-density lipoprotein) catabolism were measured. Lipid rafts were isolated and LDL receptor mass and function were evaluated. Also, the effects of exogenous sphingolipids on hepatocytes were investigated. RESULTS: We found that total SMS (SM synthase) depletion significantly reduced plasma SM levels. Also, the total deficiency significantly induced plasma cholesterol, apoB (apolipoprotein B), and apoE (apolipoprotein E) levels. Importantly, total SMS deficiency, but not SMS2 deficiency, dramatically decreased LDL receptors in the liver and attenuated LDL uptake through the receptor. Further, we found that total SMS deficiency greatly reduced LDL receptors in the lipid rafts, which contained significantly lower SM and significantly higher glucosylceramide, as well as cholesterol. Furthermore, we treated primary hepatocytes and Huh7 cells (a human hepatoma cell line) with SM, ceramide, or glucosylceramide, and we found that only SM could upregulate LDL receptor levels in a dose-dependent fashion. CONCLUSIONS: Whole-body SM biosynthesis plays an important role in LDL cholesterol catabolism. The total SMS deficiency, but not SMS2 deficiency, reduces LDL uptake and causes LDL cholesterol accumulation in the circulation. Given the fact that serum SM level is a risk factor for cardiovascular diseases, inhibiting SMS2 but not SMS1 should be the desirable approach.

LARP6 suppresses colorectal cancer progression through ZNF267/SGMS2-mediated imbalance of sphingomyelin synthesis.

BACKGROUND: With increasing incidence and mortality, colorectal cancer (CRC) seriously endangers human health. LARP6, a member of La-related protein (LARP) family, is a RNA binding protein and probably associates with CRC progression, but its specific roles and mechanisms in CRC still remain unknown. METHOD: Quantitative real-time PCR (qPCR), western blot, and immunohistochemistry were employed to examine LARP6 expression in CRC tissues. Using the stable LARP6 overexpression or interference CRC cell lines, the effect of LARP6 on CRC progression were evaluated. High-throughput RNA immunoprecipitation sequencing (RIP-seq) and a series of relevant experiments were conducted to explain how LARP6 functions. SPSS software was used for statistical analysis. RESULT: In this study, we found that LARP6 expression is downregulated in CRC and correlates with patients' overall survival and relapse-free survival. Furthermore, altered LARP6 expression influences CRC cells invasion and metastasis. Mechanically, we discovered that LARP6 bind ZNF267 mRNA and regulated its stability and translation. LARP6 inhibited expression of SGMS2, a downstream target of ZNF267, resulting in ceramide and sphingomyelin imbalance in CRC cells. Interestingly, LARP6 also enhances autophagy activity of CRC cells, and the effect was at least partially determined by the inhibition of SGMS2-mediated sphingomyelin synthesis. CONCLUSION: Our study showed how LARP6/ZNF267/SGMS2 axis influence CRC progression, which contributes to further understanding of the molecular mechanisms underlying CRC development.

Lysosomal enzyme trafficking factor LYSET enables nutritional usage of extracellular proteins.

Mammalian cells can generate amino acids through macropinocytosis and lysosomal breakdown of extracellular proteins, which is exploited by cancer cells to grow in nutrient-poor tumors. Through genetic screens in defined nutrient conditions, we characterized LYSET, a transmembrane protein (TMEM251) selectively required when cells consume extracellular proteins. LYSET was found to associate in the Golgi with GlcNAc-1-phosphotransferase, which targets catabolic enzymes to lysosomes through mannose-6-phosphate modification. Without LYSET, GlcNAc-1-phosphotransferase was unstable because of a hydrophilic transmembrane domain. Consequently, LYSET-deficient cells were depleted of lysosomal enzymes and impaired in turnover of macropinocytic and autophagic cargoes. Thus, LYSET represents a core component of the lysosomal enzyme trafficking pathway, underlies the pathomechanism for hereditary lysosomal storage disorders, and may represent a target to suppress metabolic adaptations in cancer.

GCAF(TMEM251) regulates lysosome biogenesis by activating the mannose-6-phosphate pathway.

The mannose-6-phosphate (M6P) biosynthetic pathway for lysosome biogenesis has been studied for decades and is considered a well-understood topic. However, whether this pathway is regulated remains an open question. In a genome-wide CRISPR/Cas9 knockout screen, we discover TMEM251 as the first regulator of the M6P modification. Deleting TMEM251 causes mistargeting of most lysosomal enzymes due to their loss of M6P modification and accumulation of numerous undigested materials. We further demonstrate that TMEM251 localizes to the Golgi and is required for the cleavage and activity of GNPT, the enzyme that catalyzes M6P modification. In zebrafish, TMEM251 deletion leads to severe developmental defects including heart edema and skeletal dysplasia, which phenocopies Mucolipidosis Type II. Our discovery provides a mechanism for the newly discovered human disease caused by TMEM251 mutations. We name TMEM251 as GNPTAB cleavage and activity factor (GCAF) and its related disease as Mucolipidosis Type V.

Sphingomyelin Synthase Family and Phospholipase Cs.

The sphingomyelin synthase (SMS) gene family has three members: SMS1 and SMS2 have SM synthase activity, while SMS-related protein (SMSr) has no SM synthase activity but has ceramide phosphorylethanolamine (CPE) synthase activity in vitro. Recently, we found that SMS family members are a group of phospholipase Cs (PLC). SMS1 and SMS2 are two phosphatidylcholine (PC)-PLCs and SMSr is a phosphatidylethanolamine (PE)-PLC. SMS family members not only influence SM levels but also influence the levels of diacylglycerol (DAG), PC, PE, and glycosphingolipids, thus influencing cell functions. In this chapter, we will discuss the recent progress in the research field of SMS family and will focus on its impact on metabolic diseases.

[Bridge between Total Synthesis of Bioactive Natural Products and Development of Drug Leads].

Although natural products are rich sources for drug discovery, only a small percentage of natural products themselves have been approved for clinical use, thus it is necessary to modulate various properties, such as efficacy, toxicity, and metabolic stability. A question in natural product drug discovery is how to logically design natural product derivatives with desired biological properties. This review describes our recent studies regarding the medicinal chemistry of tunicamycin. Tunicamycin inhibits bacterial phospho-N-acetylmuramic acid (MurNAc)-pentapeptide translocase (MraY), which is an essential enzyme in bacteria and a good target for antibacterial drug discovery. The usefulness of tunicamycin as antibacterial agents is limited by off-target inhibition of human UDP-N-acetylglucosamine (GlcNAc): polyprenol phosphate translocase (GPT). We positioned the total synthesis of tunicamycin as a starting point for the research and have accomplished the synthesis of tunicamycin V by using the Achmatowicz reaction, [3,3] sigmatropic rearrangement of allyl cyanate, and stereoselective glycosylation as key reactions. Next, the minimum structural requirements for tunicamycin V for MraY inhibition were established by systematic structure-activity relationship studies with truncated analogs of tunicamycin V. Our collaborative study elucidated a crystal structure of human GPT in complex with tunicamycin. This structural information was then exploited to rationally design an MraY-specific inhibitor of tunicamycin V in which the GlcNAc moiety was modified to a MurNAc amide. The analog was identified as a highly selective MraYAA inhibitor.

HetI-Like Phosphopantetheinyl Transferase Posttranslationally Modifies Acyl Carrier Proteins in Xanthomonas spp.

In Xanthomonas spp., the biosynthesis of the yellow pigment xanthomonadin and fatty acids originates in the type II polyketide synthase (PKS II) and fatty acid synthase (FAS) pathways, respectively. The acyl carrier protein (ACP) is the central component of PKS II and FAS and requires posttranslational phosphopantetheinylation to initiate these pathways. In this study, for the first time, we demonstrate that the posttranslational modification of ACPs in X. campestris pv. campestris is performed by an essential 4'-phosphopantetheinyl transferase (PPTase), XcHetI (encoded by Xc_4132). X. campestris pv. campestris strain XchetI could not be deleted from the X. campestris pv. campestris genome unless another PPTase-encoding gene such as Escherichia coli acpS or Pseudomonas aeruginosa pcpS was present. Compared with wild-type strain X. campestris pv. campestris 8004 and mutant XchetI::PapcpS, strain XchetI::EcacpS failed to generate xanthomonadin pigments and displayed reduced pathogenicity for the host plant, Brassica oleracea. Further experiments showed that the expression of XchetI restored the growth of E. coli acpS mutant HT253 and, when a plasmid bearing XchetI was introduced into P. aeruginosa, pcpS, which encodes the sole PPTase in P. aeruginosa, could be deleted. In in vitro enzymatic assays, XcHetI catalyzed the transformation of 4'-phosphopantetheine from coenzyme A to two X. campestris pv. campestris apo-acyl carrier proteins, XcAcpP and XcAcpC. All of these findings indicate that XcHetI is a surfactin PPTase-like PPTase with a broad substrate preference. Moreover, the HetI-like PPTase is ubiquitously conserved in Xanthomonas spp., making it a potential new drug target for the prevention of plant diseases caused by Xanthomonas.[Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

A phosphopantetheinyl transferase gene restricted to Porphyromonas.

The phosphopantetheinyl transferases (PPTases) catalyze the post-translational modification of carrier proteins (CPs) from fatty acid synthases (FASs) in primary metabolism and from polyketide synthases (PKSs) and non-ribosomal polypeptide synthases (NRPSs) in secondary metabolism. Based on the conserved sequence motifs and substrate specificities, two types (AcpS-type and Sfp-type) of PPTases have been identified in prokaryotes. We present here that Porphyromonas gingivalis, the keystone pathogen in chronic periodontitis, harbors merely one PPTase, namely PptP. Complementation and gene deletion experiments clearly show that PptP can replace the function of Escherichia coli AcpS and is essential for the growth of P. gingivalis. Purified PptP transfers the 4-phosphopantetheine moiety of CoA to inactive apo-acyl carrier protein (ACP) to form holo-ACP, which functions as an active carrier of the acyl intermediates of fatty acid synthesis. Moreover, PptP exhibits broad substrate specificity, modifying all ACP substrates tested and catalyzing the transfer of coenzyme A (CoA) derivatives. The lack of sequence alignment with known PPTases together with phylogenetic analyses revealed PptP as a new class of PPTases. Identification of the new PPTase gene pptP exclusive in Porphyromonas species reveals a potential target for treating P. gingivalis infections.

Using Steady-State Kinetics to Quantitate Substrate Selectivity and Specificity: A Case Study with Two Human Transaminases.

We examined the ability of two human cytosolic transaminases, aspartate aminotransferase (GOT1) and alanine aminotransferase (GPT), to transform their preferred substrates whilst discriminating against similar metabolites. This offers an opportunity to survey our current understanding of enzyme selectivity and specificity in a biological context. Substrate selectivity can be quantitated based on the ratio of the kcat/KM values for two alternative substrates (the 'discrimination index'). After assessing the advantages, implications and limits of this index, we analyzed the reactions of GOT1 and GPT with alternative substrates that are metabolically available and show limited structural differences with respect to the preferred substrates. The transaminases' observed selectivities were remarkably high. In particular, GOT1 reacted ~106-fold less efficiently when the side-chain carboxylate of the 'physiological' substrates (aspartate and glutamate) was replaced by an amido group (asparagine and glutamine). This represents a current empirical limit of discrimination associated with this chemical difference. The structural basis of GOT1 selectivity was addressed through substrate docking simulations, which highlighted the importance of electrostatic interactions and proper substrate positioning in the active site. We briefly discuss the biological implications of these results and the possibility of using kcat/KM values to derive a global measure of enzyme specificity.

Structural insights into how GlcNAc-1-phosphotransferase directs lysosomal protein transport.

GlcNAc-1-phosphotransferase catalyzes the initial step in the formation of the mannose-6-phosphate tag that labels ∼60 lysosomal proteins for transport. Mutations in GlcNAc-1-phosphotransferase are known to cause lysosomal storage disorders such as mucolipidoses. However, the molecular mechanism of GlcNAc-1-phosphotransferase activity remains unclear. Mammalian GlcNAc-1-phosphotransferases are α2ß2γ2 hexamers in which the core catalytic α- and ß-subunits are derived from the GNPTAB (N-acetylglucosamine-1-phosphate transferase subunits alpha and beta) gene. Here, we present the cryo-electron microscopy structure of the Drosophila melanogaster GNPTAB homolog, DmGNPTAB. We identified four conserved regions located far apart in the sequence that fold into the catalytic domain, which exhibits structural similarity to that of the UDP-glucose glycoprotein glucosyltransferase. Comparison with UDP-glucose glycoprotein glucosyltransferase also revealed a putative donor substrate-binding site, and the functional requirements of critical residues in human GNPTAB were validated using GNPTAB-knockout cells. Finally, we show that DmGNPTAB forms a homodimer that is evolutionarily conserved and that perturbing the dimer interface undermines the maturation and activity of human GNPTAB. These results provide important insights into GlcNAc-1-phosphotransferase function and related diseases.

In Vitro and In Vivo Inhibition of the Mycobacterium tuberculosis Phosphopantetheinyl Transferase PptT by Amidinoureas.

A newly validated target for tuberculosis treatment is phosphopantetheinyl transferase, an essential enzyme that plays a critical role in the biosynthesis of cellular lipids and virulence factors in Mycobacterium tuberculosis. The structure-activity relationships of a recently disclosed inhibitor, amidinourea (AU) 8918 (1), were explored, focusing on the biochemical potency, determination of whole-cell on-target activity for active compounds, and profiling of selective active congeners. These studies show that the AU moiety in AU 8918 is largely optimized and that potency enhancements are obtained in analogues containing a para-substituted aromatic ring. Preliminary data reveal that while some analogues, including 1, have demonstrated cardiotoxicity (e.g., changes in cardiomyocyte beat rate, amplitude, and peak width) and inhibit Cav1.2 and Nav1.5 ion channels (although not hERG channels), inhibition of the ion channels is largely diminished for some of the para-substituted analogues, such as 5k (p-benzamide) and 5n (p-phenylsulfonamide).

Engineered Chimeras Unveil Swappable Modular Features of Fatty Acid and Polyketide Synthase Acyl Carrier Proteins.

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein, we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the Escherichia coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based cross-linking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step toward the strategic engineering of "un-natural" natural products.