Aspergillus nidulans AmyG Functions as an Intracellular α-Amylase to Promote α-Glucan Synthesis.

α-Glucan is a major cell wall component and a virulence and adhesion factor for fungal cells. However, the biosynthetic pathway of α-glucan was still unclear. α-Glucan was shown to be composed mainly of 1,3-glycosidically linked glucose, with trace amounts of 1,4-glycosidically linked glucose. Besides the α-glucan synthetases, amylase-like proteins were also important for α-glucan synthesis. In our previous work, we showed that Aspergillus nidulans AmyG was an intracellular protein and was crucial for the proper formation of α-glucan. In the present study, we expressed and purified AmyG in an Escherichia coli system. Enzymatic characterization found that AmyG mainly functioned as an α-amylase that degraded starch into maltose. AmyG also showed weak glucanotransferase activity. Most intriguingly, supplementation with maltose in shaken liquid medium could restore the α-glucan content and the phenotypic defect of a ΔamyG strain. These data suggested that AmyG functions mainly as an intracellular α-amylase to provide maltose during α-glucan synthesis in A. nidulans. IMPORTANCE Short α-1,4-glucan was suggested as the primer structure for α-glucan synthesis. However, the exact structure and its source remain elusive. AmyG was essential to promote α-glucan synthesis and had a major impact on the structure of α-glucan in the cell wall. Data presented here revealed that AmyG belongs to the GH13_5 family and showed strong amylase function, digesting starch into maltose. Supplementation with maltose efficiently rescued the phenotypic defect and α-glucan deficiency in an ΔamyG strain but not in an ΔagsB strain. These results provide the first piece of evidence for the primer structure of α-glucan in fungal cells, although it might be specific to A. nidulans.

Heterologous Production of Unnatural Flavipucine Family Products Provides Insights into Flavipucines Biosynthesis.

Heterologous expression of the flavipucine biosynthetic gene cluster in Aspergillus nidulans led to the production of flavipucine (1) and dihydroisoflavipucine (3), as well as six unusual flavipucine related products containing three classes of heterocycles. This combined with gene inactivation, chemical complementation, and transcriptome analysis demonstrated unprecedented ways to form 2-pyridone and 2-pyrone structures by the oxidative rearrangements of pyrrolinone precursors as well as provided insights into the biosynthesis of this important class of natural products.

High-Titer Production of Olivetolic Acid and Analogs in Engineered Fungal Host Using a Nonplant Biosynthetic Pathway.

The microbial synthesis of cannabinoids and related molecules requires access to the intermediate olivetolic acid (OA). Whereas plant enzymes have been explored for E. coli and yeast biosynthesis, moderate yields and shunt product formation are major hurdles. Here, based on the chemical logic to form 2,4-dihydroxybenzoate-containing natural products, we discovered a set of fungal tandem polyketide synthases that can produce OA and the related octanoyl-primed derivative sphaerophorolcarboxylic acid in high titers using the model organism Aspergillus nidulans. This new set of enzymes will enable new synthetic biology strategies to access microbial cannabinoids.

Duplication and Functional Divergence of Branched-Chain Amino Acid Biosynthesis Genes in Aspergillus nidulans.

Fungi, bacteria, and plants, but not animals, synthesize the branched-chain amino acids: leucine, isoleucine, and valine. While branched-chain amino acid (BCAA) biosynthesis has been well characterized in the yeast Saccharomyces cerevisiae, it is incompletely understood in filamentous fungi. The three BCAAs share several early biosynthesis steps before divergence into specific pathways. In Aspergillus nidulans, the genes for the first two dedicated steps in leucine biosynthesis have been characterized, but the final two have not. We used sequence searches of the A. nidulans genome to identify two genes encoding ß-isopropylmalate dehydrogenase, which catalyzes the penultimate step of leucine biosynthesis, and six genes encoding BCAA aminotransferase, which catalyzes the final step in biosynthesis of all three BCAA. We have used combinations of gene knockouts to determine the relative contribution of each of these genes to BCAA biosynthesis. While both ß-isopropylmalate dehydrogenase genes act in leucine biosynthesis, the two most highly expressed BCAA aminotransferases are responsible for BCAA biosynthesis. We have also characterized the expression of leucine biosynthesis genes using reverse transcriptase-quantitative PCR and found regulation in response to leucine availability is mediated through the Zn(II)2Cys6 transcription factor LeuB. IMPORTANCE Branched-chain amino acid (BCAA) biosynthesis is important for pathogenic fungi to successfully cause disease in human and plant hosts. The enzymes for their production are absent from humans and, therefore, provide potential antifungal targets. While BCAA biosynthesis is well characterized in yeasts, it is poorly understood in filamentous fungal pathogens. Developing a thorough understanding of both the genes encoding the metabolic enzymes for BCAA biosynthesis and how their expression is regulated will inform target selection for antifungal drug development.

Epoxidation Catalyzed by the Nonheme Iron(II)- and 2-Oxoglutarate-Dependent Oxygenase, AsqJ: Mechanistic Elucidation of Oxygen Atom Transfer by a Ferryl Intermediate.

Mechanisms of enzymatic epoxidation via oxygen atom transfer (OAT) to an olefin moiety is mainly derived from the studies on thiolate-heme containing epoxidases, such as cytochrome P450 epoxidases. The molecular basis of epoxidation catalyzed by nonheme-iron enzymes is much less explored. Herein, we present a detailed study on epoxidation catalyzed by the nonheme iron(II)- and 2-oxoglutarate-dependent (Fe/2OG) oxygenase, AsqJ. The native substrate and analogues with different para substituents ranging from electron-donating groups (e.g., methoxy) to electron-withdrawing groups (e.g., trifluoromethyl) were used to probe the mechanism. The results derived from transient-state enzyme kinetics, Mössbauer spectroscopy, reaction product analysis, X-ray crystallography, density functional theory calculations, and molecular dynamic simulations collectively revealed the following mechanistic insights: (1) The rapid O2 addition to the AsqJ Fe(II) center occurs with the iron-bound 2OG adopting an online-binding mode in which the C1 carboxylate group of 2OG is trans to the proximal histidine (His134) of the 2-His-1-carboxylate facial triad, instead of assuming the offline-binding mode with the C1 carboxylate group trans to the distal histidine (His211); (2) The decay rate constant of the ferryl intermediate is not strongly affected by the nature of the para substituents of the substrate during the OAT step, a reactivity behavior that is drastically different from nonheme Fe(IV)-oxo synthetic model complexes; (3) The OAT step most likely proceeds through a stepwise process with the initial formation of a C(benzylic)-O bond to generate an Fe-alkoxide species, which is observed in the AsqJ crystal structure. The subsequent C3-O bond formation completes the epoxide installation.

Prenylated quinolinone alkaloids and prenylated isoindolinone alkaloids from the fungus Aspergillus nidulans.

Two undescribed prenylated quinolinone alkaloids, aspoquinolones E and F, and three undescribed prenylated isoindolinone alkaloids aspernidines F-H, were isolated from the fungus Aspergillus nidulans. Their structures and configurations were elucidated based on spectroscopic analyses and ECD spectra. Aspoquinolones E and F possess a C10 moiety with an unusual 2,2,4-trimethyl-3oxa-bicyclo[3.1.0]hexane unit, and aspernidines F-H own a C15 side chain. These compounds were evaluated for cytotoxic activities against five human cancer cell lines, compounds 1 and 5 exhibited strong inhibitory activities against A-549 and SW-480 cells with IC50 values of 3.50 and 4.77 µM, respectively.

Catalytic mechanism of the PrhA (V150L/A232S) double mutant involved in the fungal meroterpenoid biosynthetic pathway: a QM/MM study.

PrhA from Penicillium brasilianum and AusE from Aspergillus nidulans are nonheme Fe(ii)/α-ketoglutarate-dependent oxygenases, which are involved in the fungal meroterpenoid biosynthetic pathways. Both enzymes use preaustinoid A1 as a common substrate to form divergent products through dynamic skeletal rearrangement. Importantly, structure-guided mutagenesis results in the successful interconversion of AusE and PrhA functions, for example, the PrhA(V150L/A232S) double mutant carried out the same catalysis as AusE. Here, on the basis of the crystal structure of the PrhA (V150L/A232S) double mutant in complex with Fe(ii), αKG and the substrate preaustinoid A1, computational models were constructed, and combined quantum mechanics/molecular mechanics (QM/MM) calculations were performed to illuminate the reaction mechanism at the atomistic level. According to our calculation results, the whole reaction occurs on the quintet state surface. All three steps, including desaturation, ring rearrangement and hydroxylation, require three hydrogen abstractions by FeIV[double bond, length as m-dash]O to trigger the reaction. Owing to the relative position of FeIV[double bond, length as m-dash]O to the hydrogen atoms in the substrate to be extracted, the three H-abstractions correspond to different energy barriers, which are 17.9, 23.6 and 21.8 kcal mol-1, respectively. For the ring rearrangement, as soon as the H5 is extracted, the skeletal rearrangement is very easy. However, in the hydroxylation of intermediate preaustinoid A3, the final O-rebound corresponds to a high barrier, which is mainly caused by the long distance between the Fe-OH and -CH2 radical. It is the relative orientation of the substrate to the highly reactive FeIV[double bond, length as m-dash]O that controls the catalytic chemistry of these enzymes. The reaction barriers are sensitive to the geometry of FeIV[double bond, length as m-dash]OHx (Hx is the hydrogen atom to be extracted). These results may provide useful information for understanding the mechanisms of AusE and PrhA as well as other nonheme Fe(ii)/α-ketoglutarate-dependent oxygenases.

Novel peroxiredoxin-based sensor for sensitive detection of hydrogen peroxide.

A series of genetically encoded sensors have been developed to detect the important signaling molecule H2O2 in living cells. However, more responsive and sensitive biosensors need to be developed. To address these demands, we used E. coli as a platform to develop a novel fluorescent H2O2 sensor, which we refer to as TScGP. This sensor employs a circularly permuted YFP (cpYFP) and is based on a redox relay between peroxiredoxin (Prx) and thioredoxin (Trx). Structurally, cpYFP is sandwiched between a fungal PrxA and a C-terminal cysteine mutated TrxA that can form a stabilized disulfide bond between PrxA and TrxA in response to H2O2. We confirmed that TScGP can be used for detecting exogenous H2O2 in the range of 0.5-5 µM with high selectivity and rapidly detecting H2O2 within 30 s in E. coli. To demonstrate an application, cellular H2O2 production by menadione was detected directly by TScGP. Our results demonstrated that using Prx-Trx combination as a sensing moiety is another strategy in designing H2O2 sensor with high performance.

Comprehensive Analysis of Aspergillus nidulans PKA Phosphorylome Identifies a Novel Mode of CreA Regulation.

In filamentous fungi, an important kinase responsible for adaptation to changes in available nutrients is cyclic AMP (cAMP)-dependent protein kinase (protein kinase A [PKA]). This kinase has been well characterized at a molecular level, but its systemic action and direct/indirect targets are generally not well understood in filamentous fungi. In this work, we used a pkaA deletion strain (ΔpkaA) to identify Aspergillus nidulans proteins for which phosphorylation is dependent (either directly or indirectly) on PKA. A combination of phosphoproteomic and transcriptomic analyses revealed both direct and indirect targets of PKA and provided a global perspective on its function. One of these targets was the transcription factor CreA, the main repressor responsible for carbon catabolite repression (CCR). In the ΔpkaA strain, we identified a previously unreported phosphosite in CreA, S319, which (based on motif analysis) appears to be a direct target of Stk22 kinase (AN5728). Upon replacement of CreA S319 with an alanine (i.e., phosphonull mutant), the dynamics of CreA import to the nucleus are affected. Collectively, this work provides a global overview of PKA function while also providing novel insight regarding significance of a specific PKA-mediated phosphorylation event.IMPORTANCE The cyclic AMP (cAMP)-dependent protein kinase A (PKA) signaling pathway is well conserved across eukaryotes, and previous work has shown that it plays an important role in regulating development, growth, and virulence in a number of fungi. PKA is activated in response to extracellular nutrients and acts to regulate metabolism and growth. While a number of components in the PKA pathway have been defined in filamentous fungi, current understanding does not provide a global perspective on PKA function. Thus, this work is significant in that it comprehensively identifies proteins and functional pathways regulated by PKA in a model filamentous fungus. This information enhances our understanding of PKA action and may provide information on how to manipulate it for specific purposes.

Functional characterization of host toxic EcdB transcription factor protein of echinocandin B biosynthetic gene cluster.

The ecdB is a transcription factor, located in the echinocandin B biosynthetic gene cluster of Emericella rugulosa NRRL11440. Here, we validated the ecdB mRNA sequence for functional expression and to explore the role of EcdB protein in the echinocandin B regulation. The sequence alignment study revealed that the ecdB coding sequence was found 75 bp shorter than the reference mRNA sequence. This coding sequence encodes for EcdB protein and comprises three conserved domains; DNA binding domain (DBD), coiled-coil domain, and signature middle homology region. The full-length and DBD (truncated) DNA sequences were expressed in Escherichia coli BL21(DE3) under different tested conditions. The expression of EcdB protein was found to be toxic, which curbs the cell growth. In contrast to truncated protein (GST:EcdB1-54), the full-length (GST:EcdB) protein was expressed at very low titer and not detectable in SDS-PAGE under the varying isopropyl ß-d-1-thiogalactopyranoside (IPTG), temperature, and media conditions. However, GST:EcdB1-54 was successfully purified under standard conditions (0.5 mM IPTG at 0.5OD) with 33 kDa expected size. The functionality of GST:EcdB1-54 was attained by electrophoretic mobility shift assay study as a clear band shifting showed with ecdA promoter. Taken together, we conclude that EcdB interacts with the ecdA promoter that reflected to require for echinocandin B regulation.

Fungal hydrophobins render stones impermeable for water but keep them permeable for vapor.

The conservation of architectural heritage is a big challenge in times with increasing air pollution with aggressive gases. A second major threat to buildings is the combination of water and air contaminants which may be used by microorganisms for their metabolism. Hence, myriads of different bacteria and fungi populate stone surfaces and penetrate into the fine pores and cracks. Whereas epoxid-based paintings (or other paintings) may protect the coated surfaces from water and aggressive gases, these chemicals seal the stone surface and prevent also the evaporation of vapor from the inside of the buildings. Here, we tested a natural, fungal protein-based coating method. Fungi use small, amphiphilic proteins to turn their surfaces hydrophobic. We found that Aspergillus nidulans hydrophobin DewA and Trichoderma reesei HFBI confer hydrophobicity to stones but keep their pores open. The effect resembles "Gore-tex" fabric material.

Niduterpenoids A and B: Two Sesterterpenoids with a Highly Congested Hexacyclic 5/5/5/5/3/5 Ring System from the Fungus Aspergillus nidulans.

Niduterpenoids A (1) and B (2), two sesterterpenoids with a highly congested hexacyclic 5/5/5/5/3/5 carbon skeleton but no unsaturated functional group, were isolated from Aspergillus nidulans. Their structures were determined by a combination of spectroscopic data and single-crystal X-ray diffraction analyses. Compounds 1 and 2 present the first examples of sesterterpenoids with a hexacyclic carbon ring system. Compound 1 showed no cytotoxicity but abolished 17-estradiol-induced cell proliferation (IC50 = 11.42 ± 0.85 µM).

SUMOlock reveals a more complete Aspergillus nidulans SUMOylome.

SUMOylation, covalent attachment of the small ubiquitin-like modifier protein SUMO to proteins, regulates protein interactions and activity and plays a crucial role in the regulation of many key cellular processes. Understanding the roles of SUMO in these processes ultimately requires identification of the proteins that are SUMOylated in the organism under study. The filamentous fungus Aspergillus nidulans serves as an excellent model for many aspects of fungal biology, and it would be of great value to determine the proteins that are SUMOylated in this organism (i.e. its SUMOylome). We have developed a new and effective approach for identifying SUMOylated proteins in this organism in which we lock proteins in their SUMOylated state, affinity purify SUMOylated proteins using the high affinity S-tag, and identify them using sensitive Orbitrap mass spectroscopy. This approach allows us to distinguish proteins that are SUMOylated from proteins that are binding partners of SUMOylated proteins or are bound non-covalently to SUMO. This approach has allowed us to identify 149 proteins that are SUMOylated in A. nidulans. Of these, 67 are predicted to be involved in transcription and particularly in the regulation of transcription, 21 are predicted to be involved in RNA processing and 16 are predicted to function in DNA replication or repair.

Crystal structure of LepI, a multifunctional SAM-dependent enzyme which catalyzes pericyclic reactions in leporin biosynthesis.

LepI is a novel multifunctional enzyme that catalyzes stereoselective dehydration, Diels-Alder reaction, and retro-Claisen rearrangement. Here we report the crystal structure of LepI in complex with its co-factor S-adenosyl methionine (SAM). LepI forms a tetramer via the N-terminal helical domain and binds to a SAM molecule in the C-terminal catalytic domain. The binding modes of various LepI substrates are investigated by docking simulations, which suggest that the substrates are bound via both hydrophobic and hydrophilic forces, as well as cation-π interactions with the positively charged SAM. The reaction starts with a dehydration step in which H133 possibly deprotonates the pyridone hydroxyl group of the substrate, while D296 might protonate an alkyl-chain hydroxyl group. Subsequent pericyclization may be facilitated by the correct fold of the substrate's alkyl chain and a thermodynamic driving force towards σ-bonds at the expense of π-bonds. These results provide structural insights into LepI catalysis and are important in understanding the mechanism of enzymatic pericyclization.

Aspergillus nidulans protein kinase C forms a complex with the formin SepA that is involved in apical growth and septation.

The Aspergillus nidulans orthologue of Protein kinase C (PkcA) and the A. nidulans formin SepA participate in polarized growth. PkcA localizes to growing hyphal apices and septation sites, and amino acid sequences within PkcA that are required for PkcA to localize to these sites of cell wall synthesis have been identified. SepA is associated with the contractile actomyosin ring (CAR), and it localizes at hyphal tips in association with the Spitzenkörper (SPK) and as an apical dome. A mutation in the sepA gene (sepA1) renders A. nidulans aseptate at elevated temperature. Progress towards understanding the spatiotemporal relationship between PkcA and SepA during polarized growth is presented here. Fluorescent chimeras of PkcA and SepA strongly overlapped in some hyphal tips in a dome pattern, while other tips displayed SepA SPK and PkcA dome localization within the same tip. At septation sites PkcA and SepA consistently colocalized through late stages of CAR constriction. Bimolecular fluorescence complementation experimental results provide evidence that SepA and PkcA are both present in complexes at both hyphal tip domes and at cortical rings. A Gal4-based yeast two-hybrid analysis confirmed the physical interaction between SepA and PkcA, and indicted that the FH2 domain of SepA is involved in its physical interaction with PkcA. A functional interaction between PkcA and SepA was shown through complementation of the pkcA calC2 mutant's hypersensitivity to cell wall perturbing agents by overexpressed sepA and by the ability of the sepA1 mutation to block PkcA's ability to form cortical rings. Taken together these results suggest that a PkcA/SepA complex is involved in polarized growth. Through experiments using the actin disrupter latrunculin B, evidence is presented suggesting that actin plays a role in the PkcA/SepA complex.

COPI localizes to the early Golgi in Aspergillus nidulans.

Coatomer-I (COPI) is a heteromeric protein coat that facilitates the budding of membranous carriers mediating Golgi-to-ER and intra-Golgi transport. While the structural features of COPI have been thoroughly investigated, its physiological role is insufficiently understood. Here we exploit the amenability of A. nidulans for studying intracellular traffic, taking up previous studies by Breakspear et al. (2007) with the α-COP/CopA subunit of COPI. Endogenously tagged α-COP/CopA largely localizes to SedVSed5 syntaxin-containing early Golgi cisterna, and acute inactivation of ER-to-Golgi traffic delocalizes COPI to a haze, consistent with the cisternal maturation model. In contrast, the Golgi localization of COPI is independent of the TGN regulators HypBSec7 and HypATrs120, implying that COPI budding predominates at the SedVSed5 early Golgi, with lesser contribution of the TGN. This finding agrees with the proposed role of COPI-mediated intra-Golgi retrograde traffic in driving cisternal maturation, which predicts that the capacity of the TGN to generate COPI carriers is low. The COPI early Golgi compartments intimately associates with Sec13-containing ER exit sites. Characterization of the heat-sensitive copA1ts (sodVIC1) mutation showed that it results in a single residue substitution in the ε-COP-binding Carboxyl-Terminal-Domain of α-COP that likely destabilizes its folding. However, we show that Golgi disorganization by copA1ts necessitates >150 min-long incubation at 42 °C. This weak subcellular phenotype makes it unsuitable for inactivating COPI traffic acutely for microscopy studies, and explains the aneuploidy-stabilizing role of the mutation at subrestrictive temperatures.

Discovery and Elucidation of the Biosynthesis of Aspernidgulenes: Novel Polyenes from Aspergillus Nidulans by Using Serial Promoter Replacement.

Through serial promoter exchanges, we isolated several novel polyenes, the aspernidgulenes, from Aspergillus nidulans and uncovered their succinct biosynthetic pathway involving only four enzymes. An enoyl reductase (ER)-less highly reducing polyketide synthase (HR-PKS) putatively produces a 5,6-dihydro-α-pyrone polyene, which undergoes bisepoxidation, epoxide ring opening, cyclization, and hydrolytic cleavage by three tailoring enzymes to generate aspernidgulene A1 and A2. Our findings demonstrate the prowess of fungal-tailoring enzymes to transform a polyketide scaffold concisely and efficiently into complex structures. Moreover, comparison with citreoviridin and aurovertin biosynthesis suggests that methylation of the α-pyrone hydroxy group by methyltransferase (CtvB or AurB) is the branching point at which the biosynthesis of these two classes of compounds diverge. Therefore, scanning for the presence or absence of the gatekeeping α-pyrone methyltransferase gene in homologous clusters might be a potential way to classify the product bioinformatically as belonging to methylated α-pyrone polyenes or polyenes containing rings derived from the cyclization of the unmethylated 5,6-dihydro-α-pyrone, such as 2,3-dimethyl-γ-lactone and oxabicyclo[2.2.1]heptane.

Catabolism of phenylacetic acid in Penicillium rubens. Proteome-wide analysis in response to the benzylpenicillin side chain precursor.

Biosynthesis of benzylpenicillin in filamentous fungi (e.g. Penicillium chrysogenum - renamed as Penicillium rubens- and Aspergillus nidulans) depends on the addition of CoA-activated forms of phenylacetic acid to isopenicillin N. Phenylacetic acid is also detoxified by means of the homogentisate pathway, which begins with the hydroxylation of phenylacetic acid to 2-hydroxyphenylacetate in a reaction catalysed by the pahA-encoded phenylacetate hydroxylase. This catabolic step has been tested in three different penicillin-producing strains of P. rubens (P. notatum, P. chrysogenum NRRL 1951 and P. chrysogenum Wisconsin 54-1255) in the presence of sucrose and lactose as non-repressing carbon sources. P. chrysogenum Wisconsin 54-1255 was able to accumulate 2-hydroxyphenylacetate at late culture times. Analysis of the P. rubens genome showed the presence of several PahA homologs, but only Pc16g01770 was transcribed under penicillin production conditions. Gene knock-down experiments indicated that the protein encoded by Pc16g01770 seems to have residual activity in phenylacetic acid degradation, this catabolic activity having no effect on benzylpenicillin biosynthesis. Proteome-wide analysis of the Wisconsin 54-1255 strain in response to phenylacetic acid revealed that this molecule has a positive effect on some proteins directly related to the benzylpenicillin biosynthetic pathway, the synthesis of amino acid precursors and other important metabolic processes. SIGNIFICANCE: The adaptive response of Penicillium rubens to benzylpenicillin production conditions remains to be fully elucidated. This article provides important information about the molecular mechanisms interconnected with phenylacetate (benzylpenicillin side chain precursor) utilization and penicillin biosynthesis, and will contribute to the understanding of the complex physiology and adaptation mechanisms triggered by P. rubens (P. chrysogenum Wisconsin 54-1255) under benzylpenicillin production conditions.

Enzymatic one-step ring contraction for quinolone biosynthesis.

The 6,6-quinolone scaffolds on which viridicatin-type fungal alkaloids are built are frequently found in metabolites that display useful biological activities. Here we report in vitro and computational analyses leading to the discovery of a hemocyanin-like protein AsqI from the Aspergillus nidulans aspoquinolone biosynthetic pathway that forms viridicatins via a conversion of the cyclopenin-type 6,7-bicyclic system into the viridicatin-type 6,6-bicyclic core through elimination of carbon dioxide and methylamine through methyl isocyanate.

Allosteric regulation alters carrier domain translocation in pyruvate carboxylase.

Pyruvate carboxylase (PC) catalyzes the ATP-dependent carboxylation of pyruvate to oxaloacetate. The reaction occurs in two separate catalytic domains, coupled by the long-range translocation of a biotinylated carrier domain (BCCP). Here, we use a series of hybrid PC enzymes to examine multiple BCCP translocation pathways in PC. These studies reveal that the BCCP domain of PC adopts a wide range of translocation pathways during catalysis. Furthermore, the allosteric activator, acetyl CoA, promotes one specific intermolecular carrier domain translocation pathway. These results provide a basis for the ordered thermodynamic state and the enhanced carboxyl group transfer efficiency in the presence of acetyl CoA, and reveal that the allosteric effector regulates enzyme activity by altering carrier domain movement. Given the similarities with enzymes involved in the modular synthesis of natural products, the allosteric regulation of carrier domain movements in PC is likely to be broadly applicable to multiple important enzyme systems.