Recombinant Acinetobacter sp. Bifunctional protein FolD 2 (folD2)

What is the biochemical classification of FolD2 in Acinetobacter species?

FolD2 is a bifunctional enzyme belonging to the N(5),N(10)-methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DHCH or FolD) family. This enzyme plays a critical role in folate cofactor biosynthesis, which is essential for bacterial growth and cellular development. The bifunctional nature of this protein enables it to catalyze sequential reactions in one-carbon metabolism pathways, making it an efficient metabolic component in Acinetobacter species. The enzyme contains distinct catalytic domains that work in concert to facilitate the conversion of folate derivatives in bacterial cells. High-resolution crystal structures have been determined for FolD from Acinetobacter baumannii, revealing important details about its molecular architecture and catalytic mechanisms .

How does FolD2 contribute to Acinetobacter metabolism?

FolD2 contributes significantly to one-carbon metabolism in Acinetobacter by catalyzing two sequential reactions in the folate pathway. First, as a dehydrogenase, it converts N(5),N(10)-methylenetetrahydrofolate to N(5),N(10)-methenyltetrahydrofolate while reducing NAD+ to NADH. Second, as a cyclohydrolase, it converts N(5),N(10)-methenyltetrahydrofolate to N(10)-formyltetrahydrofolate. These reactions are essential for synthesizing purines, thymidylate, and several amino acids, making FolD2 central to bacterial growth and survival. The bifunctional nature of this enzyme likely increases metabolic efficiency by channeling intermediates between the sequential reactions, minimizing the need for substrate diffusion between separate enzymes .

What distinguishes FolD2 from other proteins in Acinetobacter species?

FolD2 is distinguished by its bifunctional catalytic capabilities and its significance in folate metabolism. Unlike many other proteins in Acinetobacter that may have single functions, FolD2 performs multiple catalytic steps in a critical metabolic pathway. Crystallographic studies have revealed unique structural features that enable this dual functionality. Additionally, the importance of folate biosynthesis for bacterial survival has made FolD2 a potential antimicrobial target, distinguishing it from many other bacterial proteins. The specific role of FolD2 in Acinetobacter metabolism suggests potential links to the organism's pathogenicity and survival mechanisms, although direct evidence connecting FolD2 to virulence factors remains an area for further investigation .

What crystal structures have been determined for Acinetobacter FolD proteins?

High-resolution crystal structures of Acinetobacter baumannii FolD have been determined in complex with cofactors and inhibitors, revealing critical details about the enzyme's catalytic mechanism. These structures show the binding modes of both natural cofactors and synthetic inhibitors, providing atomic-level insights into the protein's function. The structural data demonstrate how the enzyme accommodates its substrates and how inhibitors can effectively block its activity. Particularly noteworthy is the revelation regarding the structure of the inhibitor LY374571, which differed from previously published descriptions. This discrepancy highlights the importance of crystallographic validation in inhibitor design studies. The structural information has proven valuable for understanding the molecular basis of catalysis and inhibition, guiding future research in antimicrobial development .

How can computational methods like AlphaFold2 enhance research on FolD2?

AlphaFold2 and similar computational tools can significantly advance FolD2 research by predicting protein structures with atomic-level accuracy when experimental structures are unavailable. These tools can predict how mutations might affect protein structure, identify potential binding sites for inhibitors, and model protein-protein interactions involving FolD2. For Acinetobacter proteins specifically, the predictive power of AlphaFold2 (achieving GDT_TS values exceeding 85% for medium and difficult proteins in CASP14) offers researchers the ability to generate high-quality structural models rapidly and cost-effectively . This computational approach could be particularly valuable for exploring structural variations between different Acinetobacter species' FolD proteins, understanding evolutionary relationships, and identifying species-specific features that might be exploited for targeted antimicrobial development. The integration of AlphaFold2 predictions with experimental validation represents a powerful combined approach for FolD2 characterization .

What ligand complexes with FolD have been characterized in Acinetobacter?

Crystallographic studies have characterized multiple ligand complexes with Acinetobacter baumannii FolD, including complexes with its natural cofactor and two potent inhibitors. These structural studies have provided critical insights into both the catalytic mechanism of the enzyme and potential inhibition strategies. One particularly significant finding from these studies was the discovery that the actual structure of the inhibitor LY374571 differed from what had been previously published in the literature. This discovery has important implications for understanding structure-activity relationships and for the rational design of more effective inhibitors targeting FolD. The detailed structural information obtained from these ligand complexes offers valuable guidance for structure-based drug design efforts aimed at developing novel antimicrobials targeting folate metabolism in Acinetobacter species .

What expression systems yield optimal recombinant FolD2 production?

For optimal recombinant production of FolD2 from Acinetobacter species, researchers have successfully employed bacterial expression systems based on E. coli. When designing expression constructs, consideration should be given to codon optimization for E. coli usage patterns to enhance translation efficiency. The addition of affinity tags (such as His-tags) facilitates subsequent purification while typically maintaining enzymatic function. Expression conditions require careful optimization, with induction typically performed at lower temperatures (16-20°C) to promote proper folding of the bifunctional protein. Based on approaches used for similar bifunctional enzymes, IPTG concentrations around 0.2-0.5 mM and overnight induction periods often yield the best balance between protein quantity and quality. For structural studies requiring high purity and homogeneity, additional considerations for expression strain selection (such as BL21(DE3) derivatives) may improve outcomes by reducing proteolytic degradation and ensuring proper folding of this complex bifunctional enzyme .

What are the optimal methods for kinetic characterization of FolD2?

Kinetic characterization of FolD2 requires careful consideration of its bifunctional nature, with distinct approaches needed for each catalytic activity. For dehydrogenase activity, spectrophotometric assays tracking NADP+ reduction at 340 nm provide real-time monitoring of reaction progression. The cyclohydrolase activity can be measured through coupled assays or direct product detection using HPLC or other analytical methods. Researchers should determine key kinetic parameters (Km, kcat, kcat/Km) for both activities under consistent conditions to enable meaningful comparisons. Temperature and pH optimization studies are essential, as these parameters significantly affect enzymatic performance. When evaluating potential inhibitors, IC50 determinations followed by detailed inhibition kinetics (competitive, uncompetitive, or mixed-mode) provide crucial insights for drug development. The bifunctional nature of FolD2 necessitates careful data interpretation, as inhibition of one activity may indirectly affect the other through structural perturbations or substrate channeling disruption .

How can researchers effectively analyze inhibitor interactions with FolD2?

Effective analysis of inhibitor interactions with FolD2 requires a multi-faceted approach combining biochemical, structural, and computational methods. Enzymatic assays should quantify inhibition constants (Ki) and determine inhibition mechanisms for both catalytic activities. Isothermal titration calorimetry (ITC) provides thermodynamic parameters of binding, while thermal shift assays can rapidly screen potential inhibitors through changes in protein stability. For structural insights, X-ray crystallography of FolD2-inhibitor complexes reveals atomic-level binding details, as demonstrated with Acinetobacter baumannii FolD complexes that identified unexpected structural features of the inhibitor LY374571 . Surface plasmon resonance (SPR) offers kinetic binding parameters including association and dissociation rates. To complement experimental approaches, molecular docking and dynamics simulations can predict binding modes and guide rational optimization of inhibitor structures. This integrated approach ensures comprehensive characterization of inhibitor interactions, supporting structure-guided development of more potent and selective FolD2 inhibitors.

How does FolD2 potentially contribute to Acinetobacter pathogenicity?

FolD2 likely contributes to Acinetobacter pathogenicity primarily through its essential role in folate metabolism, which supports bacterial growth and survival during infection. While direct evidence specifically linking FolD2 to virulence mechanisms is limited, its function in providing one-carbon units for nucleotide synthesis, amino acid metabolism, and methylation reactions is critical for bacterial proliferation in host environments. The essential nature of folate metabolism makes FolD2 indirectly important for the expression of virulence factors and stress responses during infection. In Acinetobacter baumannii, various metabolic pathways interact with virulence mechanisms, suggesting that FolD2's role in central metabolism may influence pathogenicity through complex regulatory networks. For example, studies integrating genome-scale metabolic modeling with transcriptomics and metabolomics have revealed how metabolic adaptations in A. baumannii relate to antibiotic resistance and virulence, implying that folate metabolism enzymes like FolD2 may be part of these adaptive responses .

Is there evidence connecting FolD2 to biofilm formation in Acinetobacter?

While direct evidence specifically linking FolD2 to biofilm formation in Acinetobacter is not explicitly presented in the available literature, there are reasonable connections that warrant investigation. Biofilm formation is a critical virulence factor in Acinetobacter species, particularly in A. baumannii, where it contributes significantly to antimicrobial resistance and persistence on abiotic surfaces in healthcare settings . Research has identified various proteins that influence A. baumannii's ability to form biofilms and colonize surfaces. For instance, studies have shown that an RNase T2 family protein positively regulates A. baumannii's ability to colonize inanimate surfaces and affects motility, which is relevant to biofilm development . Given that FolD2 plays an essential role in folate metabolism, which supports bacterial growth and protein synthesis, it may indirectly contribute to biofilm formation by providing the metabolic foundation necessary for producing biofilm components. Future research specifically investigating the relationship between FolD2 expression and biofilm formation could provide valuable insights into this potential connection.

How might FolD2 interact with other virulence factors in Acinetobacter infections?

The interaction between FolD2 and other virulence factors in Acinetobacter likely occurs through metabolic integration and regulatory networks, though specific mechanisms remain to be fully elucidated. FolD2's essential role in folate metabolism positions it as a provider of fundamental metabolic precursors that support the synthesis of numerous bacterial components, including virulence factors. In Acinetobacter baumannii, research integrating transcriptomics and metabolomics with genome-scale metabolic modeling has revealed complex relationships between metabolism and virulence . Such multiomics approaches have demonstrated how bacterial metabolic pathways respond to environmental challenges, suggesting that folate metabolism enzymes like FolD2 may interact with virulence factor expression through shared regulatory mechanisms. Additionally, surface colonization and biofilm formation, which are important for A. baumannii pathogenicity, involve numerous proteins whose expression may be metabolically linked to FolD2 function . For example, the discovery that an RNase T2 family protein modulates A. baumannii's surface colonization ability demonstrates how seemingly unrelated proteins can influence virulence traits, suggesting similar complex relationships may exist for FolD2.

What evidence supports FolD2 as a potential antimicrobial target?

FolD2 represents a promising antimicrobial target based on several compelling factors. First, its bifunctional role in the essential folate biosynthesis pathway makes it critical for bacterial survival, as evidenced by studies of FolD in Acinetobacter baumannii . Second, high-resolution crystal structures of A. baumannii FolD in complex with potent inhibitors demonstrate the feasibility of developing small molecules that can effectively block its activity . Third, the absence of this specific bifunctional enzyme in humans reduces the likelihood of host toxicity, providing a selective advantage for antimicrobial development. Research has successfully characterized the kinetic properties of FolD from A. baumannii and determined crystal structures of complexes with a cofactor and two potent inhibitors, revealing important details about the molecular basis of catalysis and inhibition . These structural and functional insights provide a solid foundation for structure-based drug design approaches targeting FolD2. The increasing prevalence of multidrug-resistant Acinetobacter infections further emphasizes the value of exploring novel targets like FolD2 for antimicrobial development.

What methodologies have proven effective for identifying FolD2 inhibitors?

Effective methodologies for identifying FolD2 inhibitors combine structural insights with biochemical screening approaches. High-resolution X-ray crystallography has been particularly valuable, having successfully determined structures of Acinetobacter baumannii FolD in complex with a cofactor and two potent inhibitors . These structural studies revealed unexpected details about inhibitor binding, including identification of a different structure for the inhibitor LY374571 than had been previously published . Enzyme kinetic assays measuring both dehydrogenase and cyclohydrolase activities provide quantitative assessment of inhibitor potency and mechanism. Structure-based virtual screening can efficiently identify potential inhibitors from large compound libraries based on complementarity to the FolD2 active site. Fragment-based drug discovery approaches, which identify small molecular fragments that bind to the target and then link or grow them into more potent inhibitors, have proven successful for similar enzymes. Thermal shift assays (differential scanning fluorimetry) offer a high-throughput method for initial inhibitor screening. These complementary approaches, when integrated with structural biology and medicinal chemistry expertise, create a robust pipeline for FolD2 inhibitor discovery and optimization.

How do FolD2 inhibition mechanisms compare with other antimicrobial approaches in Acinetobacter?

FolD2 inhibition represents a distinct antimicrobial strategy compared to conventional approaches used against Acinetobacter species. Unlike antibiotics that target cell wall synthesis, protein synthesis, or DNA replication, FolD2 inhibitors disrupt folate metabolism, an essential pathway relatively underexploited in current antimicrobial therapies against Acinetobacter. This mechanistic distinction may provide advantages against multidrug-resistant strains. Compared to fosfomycin, which faces resistance through the AbaF efflux pump in A. baumannii , properly designed FolD2 inhibitors might circumvent such efflux mechanisms if structurally dissimilar. While polymyxins (like colistin) target bacterial membranes and face resistance through lipopolysaccharide modifications , FolD2 inhibitors would face different resistance barriers. Crystal structures of A. baumannii FolD with potent inhibitors have revealed detailed binding mechanisms , enabling structure-guided optimization to enhance efficacy and reduce resistance development. The essentiality of folate metabolism for bacterial growth suggests that resistance to FolD2 inhibitors might require complex adaptations, potentially slowing resistance emergence compared to some existing antibiotics classes.

How can genome-scale metabolic modeling enhance understanding of FolD2 function?

Genome-scale metabolic modeling provides a powerful framework for understanding FolD2's role within the broader context of Acinetobacter metabolism. This approach has been successfully applied to A. baumannii, with researchers integrating transcriptomics and metabolomics data with genome-scale metabolic models to elucidate cellular responses to various conditions . For FolD2 specifically, these models can quantitatively predict how alterations in FolD2 activity propagate through interconnected metabolic networks, identifying unexpected dependencies and potential synthetic lethal interactions. Flux balance analysis can determine how FolD2 inhibition redirects metabolic fluxes and identifies potential metabolic vulnerabilities that could be exploited for combination therapies. In silico gene knockout simulations of folD2 can predict growth phenotypes under various nutritional conditions, generating testable hypotheses about the enzyme's essentiality in different environments. Integration of experimental omics data with these models enables contextualization of FolD2 function within condition-specific metabolic states, such as during antibiotic exposure or host infection. This systems-level understanding extends beyond traditional biochemical characterization to reveal emergent properties of FolD2's role in bacterial physiology and pathogenicity.

What considerations should guide the design of FolD2 mutational studies?

Designing effective FolD2 mutational studies requires careful consideration of the protein's bifunctional nature and structural features. Researchers should target conserved residues identified through sequence alignments across Acinetobacter species while referencing available crystal structures of A. baumannii FolD complexed with ligands . Site-directed mutagenesis should separately target residues in the dehydrogenase and cyclohydrolase domains to dissect their individual contributions and potential interdependence. Conservative mutations (maintaining similar physicochemical properties) can reveal subtle functional requirements, while non-conservative changes can establish essential structural features. Including mutations at the interface between domains may provide insights into substrate channeling and allosteric communication. Experimental characterization should employ kinetic assays for both enzymatic activities, thermal stability measurements to assess structural integrity, and crystallography where possible to confirm structural consequences. In vivo complementation studies in bacterial systems can connect biochemical effects to physiological relevance. For advanced insights, hydrogen-deuterium exchange mass spectrometry before and after mutation can map conformational changes across the protein. These comprehensive approaches enable correlation between structure, function, and biological significance of FolD2's catalytic mechanisms.

How might multiomics approaches advance FolD2 research in Acinetobacter?

Multiomics approaches offer unprecedented opportunities to contextualize FolD2 function within the complex cellular environment of Acinetobacter species. Integration of transcriptomics, proteomics, and metabolomics can reveal how FolD2 expression and activity respond to environmental changes, antibiotic exposures, or host interactions. This approach has been successfully applied to A. baumannii, where researchers integrated genome-scale metabolic modeling with transcriptomics and metabolomics data to elucidate cellular responses to colistin treatment . For FolD2 research specifically, transcriptomics can identify co-expressed genes, suggesting functional relationships or shared regulatory mechanisms. Proteomics can quantify FolD2 abundance across conditions and identify post-translational modifications that might regulate its activity. Metabolomics can trace flux through folate-dependent pathways, directly measuring the metabolic consequences of FolD2 inhibition or mutation. Interactomics approaches, including protein-protein interaction studies, can identify binding partners that might influence FolD2 function or localization. When combined with genome-scale metabolic models, these multiomics datasets enable predictive modeling of how perturbations to FolD2 propagate throughout cellular networks, generating testable hypotheses about its broader functional significance in Acinetobacter physiology and pathogenicity.

What metabolic pathways interact with FolD2 in Acinetobacter metabolism?

FolD2 functions as a crucial node connecting multiple metabolic pathways in Acinetobacter species. The enzyme participates directly in folate-mediated one-carbon metabolism, which interfaces with numerous other pathways through the provision of one-carbon units for biosynthetic reactions. Genome-scale metabolic modeling approaches, similar to those applied in A. baumannii studies integrating transcriptomics and metabolomics , can elucidate these interconnections. FolD2 activity directly impacts purine biosynthesis by providing formyl groups for specific steps in the pathway. It also influences pyrimidine metabolism through its effects on thymidylate synthesis. Amino acid metabolism is affected through relationships with serine, glycine, and methionine synthesis pathways, which both provide substrates for and utilize products of folate metabolism. The enzyme's activity influences S-adenosylmethionine levels, thereby affecting numerous methylation reactions throughout bacterial metabolism. Additionally, folate derivatives participate in specific steps of bacterial cell wall component synthesis. These interconnections position FolD2 as a critical metabolic hub, explaining why its inhibition can have pleiotropic effects on bacterial physiology and potentially virulence factor production.