Substrate Binding Specificity Research Articles

Molecularly Imprinted Polymers for Highly Specific Bioorthogonal Catalysis Inside Cells.

Transition metal catalysts (TMCs) mediated bioorthogonal catalysis expand the chemical possibilities within cells. Developing synthetic TMCs tools that emulate the efficiency and specificity of natural metalloenzymes is a rewarding yet challenging endeavor. Here, we highlight the potential of molecularly imprinted enzyme mimics (MIEs) containing a Cu center and specific substrate binding domain, for conducing dimethylpropargyloxycarbonyl (DmProc) cleavage reactions within cells. Our studies reveal that the Cu-MIEs act as highly specific guides, precisely catalyzing target substrates, even in glutathione (GSH)-rich cellular environments. By adapting templates similar to the target substrates, we evolved Cu-MIEs activity to a high level and provided a method to broaden its scope to other unique substrates. This system was applied to a thyroid hormone (T3)-responsive gene switch model, inducing firefly luciferase expression by T3 in cells. This approach verifies that MIEs effectively rescue DmProc-bearing T3 prodrugs and seamlessly integrating themself into cellular biocatalytic networks.

Molecularly Imprinted Polymers for Highly Specific Bioorthogonal Catalysis Inside Cells

AbstractTransition metal catalysts (TMCs) mediated bioorthogonal catalysis expand the chemical possibilities within cells. Developing synthetic TMCs tools that emulate the efficiency and specificity of natural metalloenzymes is a rewarding yet challenging endeavor. Here, we highlight the potential of molecularly imprinted enzyme mimics (MIEs) containing a Cu center and specific substrate binding domain, for conducing dimethylpropargyloxycarbonyl (DmProc) cleavage reactions within cells. Our studies reveal that the Cu‐MIEs act as highly specific guides, precisely catalyzing target substrates, even in glutathione (GSH)‐rich cellular environments. By adapting templates similar to the target substrates, we evolved Cu‐MIEs activity to a high level and provided a method to broaden its scope to other unique substrates. This system was applied to a thyroid hormone (T3)‐responsive gene switch model, inducing firefly luciferase expression by T3 in cells. This approach verifies that MIEs effectively rescue DmProc‐bearing T3 prodrugs and seamlessly integrating themself into cellular biocatalytic networks.

Biochemical and structural elucidation of the L-carnitine degradation pathway of the human pathogen Acinetobacter baumannii.

Acinetobacter baumannii is an opportunistic human pathogen which can use host-derived L-carnitine as sole carbon and energy source. Recently, an L-carnitine transporter (Aci1347) and a specific monooxygense (CntA/CntB) for the intracellular cleavage of L-carnitine have been characterized. Subsequent conversion of the resulting malic semialdehyde into the central metabolite L-malate was hypothesized. Alternatively, L-carnitine degradation via D-malate with subsequent oxidation into pyruvate was proposed. Here we describe the in vitro and in vivo reconstitution of the entire pathway, starting from the as yet uncharacterized gene products of the carnitine degradation gene operon. Using recombinantly purified enzymes, enantiomer-specific formation of D-malate by the NAD(P)+-dependent malic semialdehyde dehydrogenase (MSA-DH) is demonstrated. The solved X-ray crystal structure of tetrameric MSA-DH reveals the key catalytic residues Cys290 and Glu256, accessible through opposing substrate and cofactor funnels. Specific substrate binding is enabled by Arg166, Arg284 and Ser447 while dual cofactor specificity for NAD+ and NADP+ is mediated by Asn184. The subsequent conversion of the unusual D-malate reaction product by an uncharacterized NAD+-dependent malate dehydrogenase (MDH) is shown. Tetrameric MDH is a β-decarboxylating dehydrogenase that synthesizes pyruvate. MDH experiments with alternative substrates showed a high degree of substrate specificity. Finally, the entire A. baumannni pathway was heterologously reconstituted, allowing E. coli to grow on L-carnitine as a carbon and energy source. Overall, the metabolic conversion of L-carnitine via malic semialdehyde and D-malate into pyruvate, CO2 and trimethylamine was demonstrated. Trimethylamine is also an important gut microbiota-dependent metabolite that is associated with an increased risk of cardiovascular disease. The pathway reconstitution experiments allowed us to assess the TMA forming capacity of gut microbes which is related to human cardiovascular health.

The Role of Key Amino Acids of the Human Fe(II)/2OG-Dependent Dioxygenase ALKBH3 in Structural Dynamics and Repair Activity toward Methylated DNA.

Non-heme dioxygenases of the AlkB family hold a unique position among enzymes that repair alkyl lesions in nucleic acids. These enzymes activate the Fe(II) ion and molecular oxygen through the coupled decarboxylation of the 2-oxoglutarate co-substrate to subsequently oxidize the substrate. ALKBH3 is a human homolog of E. coli AlkB, which displays a specific activity toward N1-methyladenine and N3-methylcytosine bases in single-stranded DNA. Due to the lack of a DNA-bound structure of ALKBH3, the basis of its substrate specificity and structure-function relationships requires further exploration. Here we have combined biochemical and biophysical approaches with site-directed mutational analysis to elucidate the role of key amino acids in maintaining the secondary structure and catalytic activity of ALKBH3. Using stopped-flow fluorescence spectroscopy we have shown that conformational dynamics play a crucial role in the catalytic repair process catalyzed by ALKBH3. A transient kinetic mechanism, which comprises the steps of the specific substrate binding, eversion, and anchoring within the DNA-binding cleft, has been described quantitatively by rate and equilibrium constants. Through CD spectroscopy, we demonstrated that replacing side chains of Tyr143, Leu177, and His191 with alanine results in significant alterations in the secondary structure content of ALKBH3 and decreases the stability of mutant proteins. The bulky side chain of Tyr143 is critical for binding the methylated base and stabilizing its flipped-out conformation, while its hydroxyl group is likely involved in facilitating the product release. The removal of the Leu177 and His191 side chains substantially affects the secondary structure content and conformational flexibility, leading to the complete inactivation of the protein. The mutants lacking enzymatic activity exhibit a marked decrease in antiparallel β-strands, offset by an increase in the helical component.

Human lipocalins bind and export fatty acids through the secretory pathway of yeast cells.

The activation of fatty acids to their acyl-CoA derivatives is a crucial step for their integration into more complex lipids or their degradation via beta-oxidation. Yeast cells employ five distinct acyl-CoA synthases to facilitate this ATP-dependent activation of acyl chains. Notably, mutant cells that are deficient in two of these fatty acid-activating (FAA) enzymes, namely, Faa1 and Faa4, do not take up free fatty acids but rather export them out of the cell. This unique fatty acid export pathway depends on small, secreted pathogenesis-related yeast proteins (Pry). In this study, we investigate whether the expression of human fatty acid-binding proteins, including Albumin, fatty acid-binding protein 4 (Fabp4), and three distinct lipocalins (ApoD, Lcn1, and Obp2a), could promote fatty acid secretion in yeast. To optimize the expression and secretion of these proteins, we systematically examined various signal sequences in both low-copy and high-copy number plasmids. Our findings reveal that directing these fatty-acid binding proteins into the secretory pathway effectively promotes fatty acid secretion from a sensitized quadruple mutant model strain (faa1∆ faa4∆ pry1∆ pry3∆). Furthermore, the level of fatty acid secretion exhibited a positive correlation with the efficiency of protein secretion. Importantly, the expression of all human lipid-binding proteins rescued Pry-dependent fatty acid secretion, resulting in the secretion of both long-chain saturated and unsaturated fatty acids. These results not only affirm the in vitro binding capabilities of lipocalins to fatty acids but also present a novel avenue for enhancing the secretion of valuable lipidic compounds. Given the growing interest in utilizing yeast as a cellular factory for producing poorly soluble compounds and the potential of lipocalins as platforms for engineering substrate-binding specificity, our model is considered as a powerful tool for promoting the secretion of high-value lipid-based molecules.

UBR4 deficiency causes male sterility and testis abnormal in Drosophila.

It has been established that UBR4 encodes E3 ubiquitin ligase, which determines the specificity of substrate binding during protein ubiquitination and has been associated with various functions of the nervous system but not the reproductive system. Herein, we explored the role of UBR4 on fertility with a Drosophila model. Different Ubr4 knockdown flies were established using the UAS/GAL4 activating sequence system. Fertility, hatchability, and testis morphology were studied, and bioinformatics analyses were conducted. Our results indicated that UBR4 deficiency could induce male sterility and influent egg hatchability in Drosophila. We found that Ubr4 deficiency affected the testis during morphological analysis. Proteomics analysis indicated 188 upregulated proteins and 175 downregulated proteins in the testis of Ubr4 knockdown flies. Gene Ontology analysis revealed significant upregulation of CG11598 and Sfp65A, and downregulation of Pelota in Ubr4 knockdown flies. These proteins were involved in the biometabolic or reproductive process in Drosophila. These regulated proteins are important in testis generation and sperm storage promotion. Bioinformatics analysis verified that UBR4 was low expressed in cryptorchidism patients, which further supported the important role of UBR4 in male fertility. Overall, our findings suggest that UBR4 deficiency could promote male infertility and may be involved in the protein modification of UBR4 by upregulating Sfp65A and CG11598, whereas downregulating Pelota protein expression.

The role of TMS 12 in the staphylococcal multidrug efflux protein QacA.

To elucidate the importance of a region in QacA predicted to be important in antimicrobial substrate recognition. A total of 38 amino acid residues within or flanking putative transmembrane helix segment (TMS) 12 of QacA were individually replaced with cysteine using site-directed mutagenesis. The impact of these mutations on protein expression, drug resistance, transport activity and interaction with sulphhydryl-binding compounds was determined. Accessibility analysis of cysteine-substituted mutants identified the extents of TMS 12, which allowed for refinement of the QacA topology model. Mutation of Gly-361, Gly-379 and Ser-387 in QacA resulted in reduced resistance to at least one bivalent substrate. Interaction with sulphhydryl-binding compounds in efflux and binding assays demonstrated the role of Gly-361 and Ser-387 in the binding and transport pathway of specific substrates. The highly conserved residue Gly-379 was found to be important for the transport of bivalent substrates, commensurate with the role of glycine residues in helical flexibility and interhelical interactions. TMS 12 and its external flanking loop is required for the structural and functional integrity of QacA and contains amino acids directly involved in the interaction with substrates.

Regulation of Cell Proliferation and Nrf2-Mediated Antioxidant Defense: Conservation of Keap1 Cysteines and Nrf2 Binding Site in the Context of the Evolution of KLHL Family.

Keap1 (Kelch-like ECH-associated protein 1) is one of the major negative regulators of the transcription factor Nrf2 (nuclear factor erythroid-2-related factor 2), which induces the expression of numerous proteins defending the cell against different stress conditions. Keap1 is generally negatively regulated by post-translational modification (mostly via its cysteine residues) and interaction with other proteins that compete with Nrf2 for binding. Cysteine residues in Keap1 have different effects on protein regulation, as basic residues (Lys, Arg, and His) in close proximity to them increase cysteine modification potential. In this paper, we present an evolutionary analysis of residues involved in both mechanisms of Keap1 regulation in the broader context of the KLHL protein family in vertebrates. We identified the typical domain structure of the KLHL protein family in several proteins outside of this family (namely in KBTBD proteins 2, 3, 4, 6, 7, 8, 12 and 14). We found several cysteines that are flanked by basic residues (namely, C14, C38, C151, C226, C241, C273, C288, C297, C319, and C613) and, therefore, may be considered more susceptible to regulatory modification. The Nrf2 binding site is completely conserved in Keap1 in vertebrates but is absent or located in nonaligned DA and BC loops of the Kelch domain within the KLHL family. The development of specific substrate binding regions could be an evolutionary factor of diversification in the KLHL protein family.

Mechanism of chorismate dehydratase MqnA, the first enzyme of the futalosine pathway, proceeds via substrate-assisted catalysis

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Details of the MqnA reaction mechanism remain unclear. Here, we present crystal structures of Streptomyces coelicolor MqnA and its active site mutants in complex with chorismate and the product 3-enolpyruvyl-benzoate, produced during heterologous expression in Escherichia coli. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with the enol pyruvyl group of chorismate acting as catalytic base. Surprisingly, structures of the mutant Asn17Asp with copurified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway.

Definition of the Acceptor Substrate Binding Specificity in Plant Xyloglucan Endotransglycosylases Using Computational Chemistry.

Xyloglucan endotransglycosylases (XETs) play key roles in the remodelling and reconstruction of plant cell walls. These enzymes catalyse homo-transglycosylation reactions with xyloglucan-derived donor and acceptor substrates and hetero-transglycosylation reactions with a variety of structurally diverse polysaccharides. In this work, we describe the basis of acceptor substrate binding specificity in non-specific Tropaeolum majus (TmXET6.3) and specific Populus tremula x tremuloides (PttXET16A) XETs, using molecular docking and molecular dynamics (MD) simulations combined with binding free energy calculations. The data indicate that the enzyme-donor (xyloglucan heptaoligosaccharide or XG-OS7)/acceptor complexes with the linear acceptors, where a backbone consisted of glucose (Glc) moieties linked via (1,4)- or (1,3)-β-glycosidic linkages, were bound stably in the active sites of TmXET6.3 and PttXET16A. Conversely, the acceptors with the (1,6)-β-linked Glc moieties were bound stably in TmXET6.3 but not in PttXET16A. When in the (1,4)-β-linked Glc containing acceptors, the saccharide moieties were replaced with mannose or xylose, they bound stably in TmXET6.3 but lacked stability in PttXET16A. MD simulations of the XET-donor/acceptor complexes with acceptors derived from (1,4;1,3)-β-glucans highlighted the importance of (1,3)-β-glycosidic linkages and side chain positions in the acceptor substrates. Our findings explain the differences in acceptor binding specificity between non-specific and specific XETs and associate theoretical to experimental data.

Computational Evaluation of Designed Phosphatase from Conserved Sequence Scratch for Diverse Substrate Specificity

The ability to design efficient enzymes for a broad class of different reactions would be of tremendous practical interest in both science and industry. Computer-assisted designing is a novel approach to generating industrial enzymes for biotechnological applications. Objectives: The main aim of this study was to design an enzyme construct with diverse substrate-binding specificity based on the evolutionary conservation of archaeal vanadium-dependent phosphatases. Materials and methods: A rational 3D structural model of enzyme construct was developed from conserved sequence scratch encompassing a vanadium-binding site and functional domain. Substrate-binding specificity of the designed enzyme was computed with different myo-inositol polyphosphate analogous by a molecular docking program. Results: A designed enzyme has shown more substrate-binding specificity with 1D-myo-inositol 3, 4, 5, 6-tetrakisphosphate. Its catalytic function closely resembled myo-inositol polyphosphate-5-phosphatase and multiple inositol polyphosphate phosphatases. Moreover, the enzyme construct was energetically stable with a low degree of conformational changes upon substrate-binding. Conclusion: Substrate specificity and catalytic competence of designed enzymes were computationally evaluated for further biotechnological applications.

Plastic structures for diverse substrates: A revisit of human ABC transporters

ATP-binding cassette (ABC) superfamily is one of the largest groups of primary active transporters that could be found in all kingdoms of life from bacteria to humans. In humans, ABC transporters can selectively transport a wide spectrum of substrates across membranes, thus playing a pivotal role in multiple physiological processes. In addition, due to the ability of exporting clinic therapeutics, some ABC transporters were originally termed multidrug resistance proteins. Increasing investigations of human ABC transporters in recent years have provided abundant information for elucidating their structural features, based on the structures at distinct states in a transport cycle. This review focuses on the recent progress in human ABC structural analyses, substrate binding specificities, and translocation mechanisms. We dedicate to summarize the common features of human ABC transporters in different subfamilies, and to discuss the possibility to apply the fast-developing techniques, such as cryogenic electron microscopy, and artificial intelligence-assisted structure prediction, for future studies.

Transmembrane helices 5 and 12 control transport dynamics, substrate affinity, and specificity in the elevator-type UapA transporter.

An increasing number of solute transporters have been shown to function with the so-called sliding-elevator mechanism. Despite structural and functional differences, all elevator-type transporters use a common mechanism of substrate translocation via reversible movements of a mobile core domain (the elevator) hosting the substrate binding site along a rigid scaffold domain stably anchored in the plasma membrane via homodimerization. One of the best-studied elevator transporters is the UapA uric acid-xanthine/H+ symporter of the filamentous fungus Aspergillus nidulans. Here, we present a genetic analysis for deciphering the role of transmembrane segments (TMS) 5 and 12 in UapA transport function. We show that specific residues in both TMS5 and TMS12 control, negatively or positively, the dynamics of transport, but also substrate binding affinity and specificity. More specifically, mutations in TMS5 can lead not only to increased rate of transport but also to an inactive transporter due to high-affinity substrate-trapping, whereas mutations in TMS12 lead to apparently uncontrolled sliding and broadened specificity, leading in specific cases to UapA-mediated purine toxicity. Our findings shed new light on how elevator transporters function and how this knowledge can be applied to genetically modify their transport characteristics.

Quinolinate Synthase: An Example of the Roles of the Second and Outer Coordination Spheres in Enzyme Catalysis.

The activation energy barrier of biochemical reactions is normally lowered by an enzyme catalyst, which directly helps the weakening of the bond(s) to be broken. In many metalloenzymes, this is a first coordination sphere effect. Besides having a direct catalytic action, enzymes can fix their reactive groups and substrates so that they are optimally positioned and also modify the water activity in the system. They can either activate substrates prior to their reaction or bind preactivated substrates, thereby drastically reducing local entropic effects. The latter type is well represented by some bisubstrate reactions, where they have been defined as "entropic traps". These can be described as "second coordination sphere" processes, but enzymes can also control the reactivity beyond this point through local conformational changes belonging to an "outer coordinate sphere" that can be modulated by substrate binding. We have chosen the [4Fe-4S] cluster-dependent enzyme quinolinate synthase to illustrate each one of these processes. In addition, this very old metalloenzyme shows low in vitro substrate binding specificity, atypical reactivity that produces dead-end products, and a unique modulation of its active site volume.

Identification of molecular basis that underlie enzymatic specificity of AzoRo from Rhodococcus opacus 1CP: A potential NADH:quinone oxidoreductase

Azo dyes are important to various industries such as textile industries. However, these dyes are known to comprise toxic, mutagenic, and carcinogenic representatives. Several approaches have already been employed to mitigate the problem such as the use of enzymes. Azoreductases have been well-studied in its capability to reduce azo dyes. AzoRo from Rhodococcus opacus 1CP has been found to be accepting only methyl red as a substrate, surmising that the enzyme may have a narrow active site. To determine the active site configuration of AzoRo at atomic level and identify the key residues involved in substrate binding and enzyme specificity, we have determined the crystal structure of holo-AzoRo and employed a rational design approach to generate AzoRo variants. The results reported here show that AzoRo has a different configuration of the active site when compared with other bacterial NAD(P)H azoreductases, having other key residues playing a role in the substrate binding and restricting the enzyme activity towards different azo dyes. Moreover, it was observed that AzoRo has only about 50% coupling yield to methyl red and p-benzoquinone – giving rise to the possibility that NADH oxidation still occurs even during catalysis. Results also showed that AzoRo is more active and more efficient towards quinones (about four times higher than methyl red).

An intermolecular salt bridge linking substrate binding and P1 substrate specificity switch of arterivirus 3C-like proteases

Equine arteritis virus (EAV) and porcine reproductive and respiratory syndrome virus (PRRSV) represent two members of the family Arteriviridae and pose a major threat to the equine- and swine-breeding industries throughout the world. Previously, we and others demonstrated that PRRSV 3C-like protease (3CLpro) had very high glutamic acid (Glu)-specificity at the P1 position (P1-Glu). Comparably, EAV 3CLpro exhibited recognition of both Glu and glutamine (Gln) at the P1 position. However, the underlying mechanisms of the P1 substrate specificity shift of arterivirus 3CLpro remain unclear. We systematically screened the specific amino acids in the S1 subsite of arterivirus 3CLpro using a cyclized luciferase-based biosensor and identified Gly116, His133 and Ser136 (using PRRSV 3CLpro numbering) are important for recognition of P1-Glu, whereas Ser136 is nonessential for recognition of P1-Gln. Molecular dynamics simulations and biochemical experiments highlighted that the PRRSV 3CLpro and EAV 3CLpro formed distinct S1 subsites for the P1 substrate specificity switch. Mechanistically, a specific intermolecular salt bridge between PRRSV 3CLpro and substrate P1-Glu (Lys138/P1-Glu) are invaluable for high Glu-specificity at the P1 position, and the exchange of K138T (salt bridge interruption, from PRRSV to EAV) shifted the specificity of PRRSV 3CLpro toward P1-Gln. In turn, the T139K exchange of EAV 3CLpro showed a noticeable shift in substrate specificity, such that substrates containing P1-Glu are likely to be recognized more efficiently. These findings identify an evolutionarily accessible mechanism for disrupting or reorganizing salt bridge with only a single mutation of arterivirus 3CLpro to trigger a substrate specificity switch.

Host manipulation by bacterial type III and type IV secretion system effector proteases.

Proteases are powerful enzymes, which cleave peptide bonds, leading most of the time to irreversible fragmentation or degradation of their substrates. Therefore they control many critical cell fate decisions in eukaryotes. Bacterial pathogens exploit this power and deliver protease effectors through specialised secretion systems into host cells. Research over the past years revealed that the functions of protease effectors during infection are diverse, reflecting the lifestyles and adaptations to specific hosts; however, only a small number of peptidase families seem to have given rise to most of these protease virulence factors by the evolution of different substrate-binding specificities, intracellular activation and subcellular targeting mechanisms. Here, we review our current knowledge about the enzymology and function of protease effectors, which Gram-negative bacterial pathogens translocate via type III and IV secretion systems to irreversibly manipulate host processes. We highlight emerging concepts such as signalling by protease cleavage products and effector-triggered immunity, which host cells employ to detect and defend themselves against a protease attack. TAKE AWAY: Proteases irreversibly cleave proteins to control critical cell fate decisions. Gram-negative bacteria use type III and IV secretion systems to inject effectors. Protease effectors are integral weapons for the manipulation of host processes. Effectors evolved from few peptidase families to target diverse substrates. Effector-triggered immunity upon proteolytic attack emerges as host defence.

Characterization of Acetylcholinesterase from Various Sources: A Mini Re

In this review, various aspects of acetylcholinesterase (AChE) are reviewed. Acetylcholinesterase (AChE, EC3.1.1.7) is an important neurotransmitter because it hydrolyses the neurotransmitter acetylcholine (ACh), it is also the target site of organophosphorus and carbamate insecticides. The discovery of the first neurotransmitter ACh was soon followed by the discovery of its hydrolysing enzyme, AChE. The role of AChE in terminating AChE-mediated neurotransmission made it the focus of intense research for much of the past century. The role of butyrylcholinesterase (BChE) is not known but it is assumed to participate in the growth and development and to scavenge the cholinergic toxins, it is also reported to have an auxiliary role in synaptic transmission. Acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) are the major groups of cholinesterase differentiated by their substrate preferences. Substrate binding specificity and catalytic efficiency are tangled in enzyme catalysis. The fact that an enzyme may exist in more than one molecular form has been accepted for a number of years.

Insights into Functional Dimerization of CYP121A1 of Mycobacterium tuberculosis

Tuberculosis is caused by the pathogenic bacterium Mycobacterium tuberculosis (Mtb) and remains the leading cause of death by infection world-wide. Out of twenty cytochrome P450 enzymes encoded by the Mtb genome, cytochrome P450 121A1 (CYP121A1) is essential, and therefore remains a target of drug design efforts. CYP121A1 mediates a phenol coupling reaction of the tyrosine dipeptide cyclo-L-Tyr-L-Tyr (cYY). In this work, a structure and function investigation of dimerization was performed as an overlooked feature of CYP121A1 function. This investigation showed that CYP121A1 dimers form via intermolecular contacts on the distal surface and are mediated by a network of solvent-exposed hydrophobic residues. Dimerization of CYP121A1 contributes to specific substrate binding in vitro, with 75% loss of function when dimers are disrupted. 19F labeling and nuclear magnetic resonance of the enzyme FG-loop was also combined with protein docking to develop a working model of a functional CYP121A1 dimer. Preliminary crystallographic data of a CYP121A1 homodimer confirm the distal-to-distal orientation of the dimers. These findings suggest a novel role for CYP dimer formation as a contributing factor in determining substrate selectivity, while also providing important mechanistic insight regarding a relevant drug target in the development of novel anti-tuberculosis agents.

Adrenodoxin and Substrate Binding Modulate the Vitamin‐D Metabolizing Human Cytochrome P450 24A1

Vitamin-D3 insufficiency is associated with rickets, cardiovascular disease, and chronic kidney disease, and therefore impacts nearly 50% of the U.S. population. In humans, vitamin-D3 is metabolized by a series of cytochrome P450 enzymes, including inactivation by the mitochondrial cytochrome P450 enzyme CYP24A1. CYP24A1 in humans first hydroxylates either the carbon-23 or carbon-24 position of 1,25-(OH)2D3. The human isoform of CYP24A1 is not easily expressed and purified recombinantly. Therefore, most current structural studies are based on the rat isoform, which does not display carbon-23 regioselective hydroxylation. As a way to study the human isoform, we have initiated use of a cleavable fusion construct of human CYP24A1 with its reduction-oxidation protein partner adrenodoxin. Cleaved and isolated human CYP24A1 provides a platform for interrogating the structural effects of an Adx-CYP24A1 complex on substrate, analog, and inhibitor binding. Notably, we observed that the presence Adx weakens the interaction between human CYP24A1 1.5-fold, while also increasing the population of the high spin shift that results from displacement of a water ligand. This effect is not seen with 1-(OH)D3, a vitamin-D3 analog, suggesting that specific substrate binding relates allosterically to Adx recognition. The path of this allostery is investigated by specific point mutations. These findings provide mechanistic insight into the interrelated nature of step I (substrate binding) and step II (redox partner binding) of a multi-functional mitochondrial cytochrome P450 enzyme.