Zinc-binding Domain Research Articles

Essential and multifunctional mpox virus E5 helicase-primase in double and single hexamer.

An outbreak of mpox virus in May 2022 has spread over 110 nonpandemic regions in the world, posing a great threat to global health. Mpox virus E5, a helicase-primase, plays an essential role in DNA replication, but the molecular mechanisms are elusive. Here, we report seven structures of mpox virus E5 in a double hexamer (DH) and six in single hexamer in different conformations, indicating a rotation mechanism for helicase and a coupling action for primase. The DH is formed through the interface of zinc-binding domains, and the central channel density indicates potential double-stranded DNA (dsDNA), which helps to identify dsDNA binding residues Arg249, Lys286, Lys315, and Lys317. Our work is important not only for understanding poxviral DNA replication but also for the development of novel therapeutics for serious poxviral infections including smallpox virus and mpox virus.

EIF2β zinc-binding domain interacts with the eIF2γ subunit through the guanine nucleotide binding interface to promote Met-tRNAiMet binding.

The heterotrimeric eIF2 complex consists of a core eIF2γ subunit to which binds eIF2α and eIF2β subunits and plays an important role in delivering the Met-tRNAiMet to the 40S ribosome and start codon selection. The intricacies of eIF2β-γ interaction in promoting Met-tRNAiMet binding are not clearly understood. Previously, the zinc-binding domain (ZBD) eIF2βS264Y mutation was reported to cause Met-tRNAiMet binding defect due to the intrinsic GTPase activity. We showed that the eIF2βS264Y mutation has eIF2β-γ interaction defect. Consistently, the eIF2βT238A intragenic suppressor mutation restored the eIF2β-γ and Met-tRNAiMet binding. The eIF2β-ZBD residues Asn252Asp and Arg253Ala mutation caused Met-tRNAiMet binding defect that was partially rescued by the eIF2βT238A mutation, suggesting the eIF2β-ZBD modulates Met-tRNAiMet binding. The suppressor mutation rescued the translation initiation fidelity defect of the eIF2γN135D SW-I mutation and eIF2βF217A/Q221A double mutation in the HTH domain. The eIF2βT238A suppressor mutation could not rescue the eIF2β binding defect of the eIF2γV281K mutation; however, combining the eIF2βS264Y mutation with the eIF2γV281K mutation was lethal. In addition to the previously known interaction of eIF2β with the eIF2γ subunit via its α1-helix, the eIF2β-ZBD also interacts with the eIF2γ subunit via guanine nucleotide-binding interface; thus, the eIF2β-γ interacts via two distinct binding sites.

Enhancing anti-cancer capacity: Novel class I/II HDAC inhibitors modulate EMT, cell cycle, and apoptosis pathways

Cancer has been a leading cause of death over the last few decades in western countries as well as in Taiwan. However, traditional therapies are limited by the adverse effects of chemotherapy and radiotherapy, and tumor recurrence may occur. Therefore, it is critical to develop novel therapeutic drugs. In the field of HDAC inhibitor development, apart from the hydroxamic acid moiety, 2-aminobenzamide also functions as a zinc-binding domain, which is shown in well-known HDAC inhibitors such as Entinostat and Chidamide. With recent successful experiences in synthesizing 1-(phenylsulfonyl)indole-based compounds, in this study, we further combined two features of the above chemical compounds and generated indolyl benzamides. Compounds were screened in different cancer cell lines, and enzyme activity was examined to demonstrate their potential for anti-HDAC activity. Various biological functional assays evidenced that two of these compounds could suppress cancer growth and migration capacity, through regulating epithelial–mesenchymal transition (EMT), cell cycle, and apoptosis mechanisms. Data from 3D cancer cells and the in vivo zebrafish model suggested the potential of these compounds in cancer therapy in the future.

Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in Streptomyces.

Streptomyces antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. Streptomyces coelicolor AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the afs box overlapping the -35 element and the σHrdB region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β' zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.

The catalytic domain of free or ligand bound histone deacetylase 4 occurs in solution predominantly in closed conformation.

Human histone deacetylase 4 (HDAC4) is a key epigenetic regulator involved in a number of important cellular processes. This makes HDAC4 a promising target for the treatment of several cancers and neurodegenerative diseases, in particular Huntington's disease. HDAC4 is highly regulated by phosphorylation and oxidation, which determine its nuclear or cytosolic localization, and exerts its function through multiple interactions with other proteins, forming multiprotein complexes of varying composition. The catalytic domain of HDAC4 is known to interact with the SMRT/NCOR corepressor complex when the structural zinc-binding domain (sZBD) is intact and forms a closed conformation. Crystal structures of the HDAC4 catalytic domain have been reported showing an open conformation of HDAC4 when bound to certain ligands. Here, we investigated the relevance of this HDAC4 conformation under physiological conditions in solution. We show that proper zinc chelation in the sZBD is essential for enzyme function. Loss of the structural zinc ion not only leads to a massive decrease in enzyme activity, but it also has serious consequences for the overall structural integrity and stability of the protein. However, the Zn2+ free HDAC4 structure in solution is incompatible with the open conformation. In solution, the open conformation of HDAC4 was also not observed in the presence of a variety of structurally divergent ligands. This suggests that the open conformation of HDAC4 cannot be induced in solution, and therefore cannot be exploited for the development of HDAC4-specific inhibitors.

Characterization of Two Gonadal Genes, zar1 and wt1b, in Hermaphroditic Fish Asian Seabass (Lates calcarifer)

Zygote arrest-1 (Zar1) and Wilms’ tumor 1 (Wt1) play an important role in oogenesis, with the latter also involved in testicular development and gender differentiation. Here, Lczar1 and Lcwt1b were identified in Asian seabass (Lates calcarifer), a hermaphrodite fish, as the valuable model for studying sex differentiation. The cloned cDNA fragments of Lczar1 were 1192 bp, encoding 336 amino acids, and contained a zinc-binding domain, while those of Lcwt1b cDNA were 1521 bp, encoding a peptide of 423 amino acids with a Zn finger domain belonging to Wt1b family. RT-qPCR analysis showed that Lczar1 mRNA was exclusively expressed in the ovary, while Lcwt1b mRNA was majorly expressed in the gonads in a higher amount in the testis than in the ovary. In situ hybridization results showed that Lczar1 mRNA was mainly concentrated in oogonia and oocytes at early stages in the ovary, but were undetectable in the testis. Lcwt1b mRNA was localized not only in gonadal somatic cells (the testis and ovary), but also in female and male germ cells in the early developmental stages, such as those of previtellogenic oocytes, spermatogonia, spermatocytes and spermatids. These results indicated that Lczar1 and Lcwt1b possibly play roles in gonadal development. Therefore, the findings of this study will provide a basis for clarifying the mechanism of Lczar1 and Lcwt1b in regulating germ cell development and the sex reversal of Asian seabass and even other hermaphroditic species.

Multiscale simulations reveal the driving forces of p53C phase separation accelerated by oncogenic mutations.

Liquid-Liquid phase separation (LLPS) of p53 to form liquid condensates has been implicated in cellular functions and dysfunctions. The p53 condensates may serve as amyloid fibril precursors to initiate p53 aggregation, which is associated with oncogenic gain-of-function and various human cancers. M237I and R249S mutations located in p53 core domain (p53C) have been detected respectively in glioblastomas and hepatocellular carcinoma. Interestingly, these p53C mutants can also undergo LLPS and liquid-to-solid phase transition, which are faster than wild type p53C. However, the underlying molecular basis governing the accelerated LLPS and liquid-to-solid transition of p53C remain poorly understood. Herein, we explore the M237I/R249S mutation-induced structural alterations and phase separation behavior of p53C by employing multiscale molecular dynamics simulations. All-atom simulations revealed conformational disruptions in the zinc-binding domain of the M237I mutant and in both loop3 and zinc-binding domain of the R249S mutant. The two mutations enhance hydrophobic exposure of those regions and attenuate intramolecular interactions, which may hasten the LLPS and aggregation of p53C. Martini 3 coarse-grained simulations demonstrated spontaneous phase separation of p53C and accelerated effects of M237I/R249S mutations on the phase separation of p53C. Importantly, we find that the regions with enhanced intermolecular interactions observed in coarse-grained simulations coincide with the disrupted regions with weakened intramolecular interactions observed in all-atom simulations, indicating that M237I/R249S mutation-induced local structural disruptions expedite the LLPS of p53C. This study unveils the molecular mechanisms underlying the two cancer-associated mutation-accelerated LLPS and aggregation of p53C, providing avenues for anticancer therapy by targeting the phase separation process.

PHD finger proteins function in plant development and abiotic stress responses: an overview.

The plant homeodomain (PHD) finger with a conserved Cys4-His-Cys3 motif is a common zinc-binding domain, which is widely present in all eukaryotic genomes. The PHD finger is the "reader" domain of methylation marks in histone H3 and plays a role in the regulation of gene expression patterns. Numerous proteins containing the PHD finger have been found in plants. In this review, we summarize the functional studies on PHD finger proteins in plant growth and development and responses to abiotic stresses in recent years. Some PHD finger proteins, such as VIN3, VILs, and Ehd3, are involved in the regulation of flowering time, while some PHD finger proteins participate in the pollen development, for example, MS, TIP3, and MMD1. Furthermore, other PHD finger proteins regulate the plant tolerance to abiotic stresses, including Alfin1, ALs, and AtSIZ1. Research suggests that PHD finger proteins, as an essential transcription regulator family, play critical roles in various plant biological processes, which is helpful in understanding the molecular mechanisms of novel PHD finger proteins to perform specific function.

Investigation of the impact of R273H and R273C mutations on the DNA binding domain of P53 protein through molecular dynamic simulation

The P53 protein, a cancer-associated transcriptional factor and tumor suppressor, houses a Zn2+ ion in its DNA-binding domain (DBD), essential for sequence-specific DNA binding. However, common mutations at position 273, specifically from Arginine to Histidine and Cysteine, lead to a loss of function as a tumor suppressor, also called DNA contact mutations. The mutant (MT) P53 structure cannot stabilize DNA due to inadequate interaction. To investigate the conformational changes, we performed a comparative molecular dynamic simulation (MDS) to study the effect of the P53-Wildtype (P53-WT) and the DNA contact mutations (R273H and R273C) on the DBD. Our research indicated that the DNA binding bases lose Hydrogen bonds (H bonds) when mutated to P53-R273H and P53-R273C during the simulation. We employed tools, such as PDIviz to highlight the contacts with DNA bases and backbone, major and minor grooves, and various pharmacophore forms of atoms. The contact maps for R273H and R273C were generated using the COZOID tool, which displayed changes in the frequency of the amino acids and DNA bases interaction in the DNA binding domain. These residues have diminished interactions, and the zinc-binding domain shows significant movements by Zn2+ ion binding to the phosphate group of the DNA, moving away from its binding sites. In conclusion, our research suggests that R273H and R273C each have unique stability and self-assembly properties. This understanding might assist researchers in better comprehending the function of the p53 protein and its importance in cancer. Communicated by Ramaswamy H. Sarma

A novel loss of function mutation in the PHD domain of the RAG2 gene, affecting zinc-binding affinity.

Severe combined immune deficiencies (SCIDs) are genetically heterogeneous disorders that lead to the absence or malfunction of adaptive immune cells, including T- and B-cells. Pathogenic variants in the RAG2 gene are associated with this disease. A couple with consanguineous marriage from the Iranian-Azeri-Turkish ethnic group was referred to the genetic lab. Two children of this family died due to SCID disease with symptoms of skin granulomas, lack of developed T- and B-cells, and intact NK cells. To infer their genotypes, DNA samples obtained from the parents were subjected to whole-exome sequencing (WES). WES data analysis revealed that both parents were carriers of a pathogenic variant, NC_000011.10 (NM_000536.4):c.1268G > C, in the RAG2 gene. This variant was absent in our cohort of 400 healthy individuals from the same ethnic group. To gain insight into the consequence of the variant on the protein function, further analysis was performed by applying bioinformatics tools. This study revealed that the replacement of cysteine with serine at the zinc-binding domain diminished the domain's affinity to zinc ion, resulting in the loss of the mutant protein's ability to bind to the recombination signal sequence (RSS). The formation of the RAG2-RSS complex is vital for T- and B-cell development. The identification of a novel pathogenic variant, c.1268G > C, revealed that this variant in the zinc-binding domain diminished the affinity of the zinc ion to the mutant protein and consequently led to the loss of its ability to bind to the RSS.

An iron–sulfur cluster in the zinc-binding domain of the SARS-CoV-2 helicase modulates its RNA-binding and -unwinding activities

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of COVID-19, uses an RNA-dependent RNA polymerase along with several accessory factors to replicate its genome and transcribe its genes. Nonstructural protein (nsp) 13 is a helicase required for viral replication. Here, we found that nsp13 ligates iron, in addition to zinc, when purified anoxically. Using inductively coupled plasma mass spectrometry, UV-visible absorption, EPR, and Mössbauer spectroscopies, we characterized nsp13 as an iron-sulfur (Fe-S) protein that ligates an Fe4S4 cluster in the treble-clef metal-binding site of its zinc-binding domain. The Fe-S cluster in nsp13 modulates both its binding to the template RNA and its unwinding activity. Exposure of the protein to the stable nitroxide TEMPOL oxidizes and degrades the cluster and drastically diminishes unwinding activity. Thus, optimal function of nsp13 depends on a labile Fe-S cluster that is potentially targetable for COVID-19 treatment.

Conformational exchange at a C2H2 zinc-binding site facilitates redox sensing by the PML protein

The promyelocytic leukemia protein, PML, plays a vital role in the cellular response to oxidative stress; however, the molecular mechanism of its action remains poorly understood. Here, we identify redox-sensitive sites of PML. A molecule of PML is cysteine-rich and contains three zinc-binding domains including RING, B-box1, and B-box2. Using invitro assays, we have compared the sensitivity of the isolated RING and B-box1 domains and shown that B-box1 is more sensitive to oxidation. NMR studies of PML dynamics showed that one of the Zn-coordination sites within the B-box1 undergoes significant conformational exchange, revealing a hotspot for exposure of reactive cysteines. In agreement with the invitro data, enhancement of the B-box1 Zn-coordination dynamics led to more efficient recruitment of PML into PML nuclear bodies in cells. Overall, our results suggest that the increased sensitivity of B-box1 to oxidative stress makes this domain an important redox-sensing component of PML.

Identification and functional analysis of CG3526 in spermatogenesis of Drosophila melanogaster.

Spermatogenesis is a critical part of reproduction in insects; however, its molecular mechanism is still largely unknown. In this study, we identified a testis-specific gene CG3526 in Drosophila melanogaster. Bioinformatics analysis showed that CG3526 contains a zinc binding domain and 2 C2 H2 type zinc fingers, and it is clustered to the vertebrate really interesting new gene (RING) family E3 ubiquitin-protein ligases. When CG3526 was knocked down by RNA interference (RNAi), the testis became much smaller in size, and the apical tip exhibited a sharp and thin end instead of the blunt and round shape in the control testis. More importantly, compared to the control flies, only a few mature sperm were present in the seminal vesicle of C587-Gal4>CG3526 RNAi flies. Immunofluorescence staining of the testis from CG3526 RNAi flies showed that the homeostasis of testis stem cell niche was disrupted, cell distribution in the apical tip was scattered, and the process of spermatogenesis was not completed. Furthermore, we found that the phenotype of CG3526 RNAi flies' testis was similar to that of testis of Stat92E RNAi flies, the expression level of CG3526 was significantly downregulated in the Stat92EF06346 mutant flies, and the promoter activity of CG3526 was upregulated by STAT92E. Taken together, our results indicated that CG3526 is a downstream effector gene in the JAK-STAT signaling pathway that plays a key role in the spermatogenesis of Drosophila.

Isolation and Characterization of Phages That Bypass the Requirement for RNA-Mediated Antitermination.

The rpoCY75N mutation in the zinc-binding domain of the β' subunit of Escherichia coli RNA polymerase blocks the RNA-based mechanism of transcription antitermination utilized by bacteriophage HK022. Mutant phages that overcome the block imposed by the rpoCY75N mutation are described. These phages, designated "orc" (overcomes rpoC), carry mutations that create new promoters. Promoter activity was assessed by cloning the respective regions from the wild-type and orc phages into a promoterless lacZ reporter vector. Reporter assays showed that the sequence originating from orc phages had significant promoter activity when compared with the equivalent sequence cloned from the parental phage. The newly created promoters facilitate the expression of phage genes that are essential for growth on the rpoCY75N strain by bypassing transcription terminators. The small plaque phenotype of orc phages, when grown on the mutant host, suggests that suppression of the rpoCY75N mutation is incomplete.

Cryo-EM structure of the human Sirtuin 6-nucleosome complex.

Sirtuin 6 (SIRT6) is a multifaceted protein deacetylase/deacylase and a major target for small-molecule modulators of longevity and cancer. In the context of chromatin, SIRT6 removes acetyl groups from histone H3 in nucleosomes, but the molecular basis for its nucleosomal substrate preference is unknown. Our cryo-electron microscopy structure of human SIRT6 in complex with the nucleosome shows that the catalytic domain of SIRT6 pries DNA from the nucleosomal entry-exit site and exposes the histone H3 N-terminal helix, while the SIRT6 zinc-binding domain binds to the histone acidic patch using an arginine anchor. In addition, SIRT6 forms an inhibitory interaction with the C-terminal tail of histone H2A. The structure provides insights into how SIRT6 can deacetylate both H3 K9 and H3 K56.

B-box1 Domain of MID1 Interacts with the Ube2D1 E2 Enzyme Differently Than RING E3 Ligases.

The MID1 TRIM protein is important for ventral midline development in vertebrates, and mutations of its B-box1 domain result in several birth defects. The B-box1 domain of the human MID1 protein binds two zinc atoms and adopt a similar ββα-RING structure. This domain is required for the efficient ubiquitination of protein phosphatase 2A, alpha4, and fused kinase. Considering the structural similarity, the MID1 B-box1 domain exhibits mono-autoubiquitination activity, in contrast to poly-autoubiquitination observed for RING E3 ligases. To understand its mechanism of action, the interaction of the B-box1 domain with Ube2D1 (UbcH5a, E2), a preferred E2 ligase, is investigated. Using isothermal titration calorimetry, the MID1 RING and B-box1 domains were observed to have similar binding affinities with the Ube2D1 protein. However, NMR 15N-1H Heteronuclear Single Quantum Coherence titration, 15N relaxation data, and High Ambiguity Driven protein-protein DOCKing (HADDOCK) calculations show the B-box1 domain binding on a surface distinct from where RING domains bind. The novel binding interaction shows the B-box1 domain partially overlapping the noncovalent Ube2D1 and a ubiquitin binding site that is necessary for poly-autoubiquitination activity. The B-box1 domain also displaces the ubiquitin from the Ube2D1 protein. These studies reveal a novel binding interaction between the zinc-binding ββα-fold B-box1 domain and the Ube2D enzyme family and that this difference in binding, compared to RING E3 ligases, provides a rationale for its auto-monoubiquitination E3 ligase activity.

Human IFT-A complex structures provide molecular insights into ciliary transport

Intraflagellar transport (IFT) complexes, IFT-A and IFT-B, form bidirectional trains that move along the axonemal microtubules and are essential for assembling and maintaining cilia. Mutations in IFT subunits lead to numerous ciliopathies involving multiple tissues. However, how IFT complexes assemble and mediate cargo transport lacks mechanistic understanding due to missing high-resolution structural information of the holo-complexes. Here we report cryo-EM structures of human IFT-A complexes in the presence and absence of TULP3 at overall resolutions of 3.0–3.9 Å. IFT-A adopts a “lariat” shape with interconnected core and peripheral subunits linked by structurally vital zinc-binding domains. TULP3, the cargo adapter, interacts with IFT-A through its N-terminal region, and interface mutations disrupt cargo transport. We also determine the molecular impacts of disease mutations on complex formation and ciliary transport. Our work reveals IFT-A architecture, sheds light on ciliary transport and IFT train formation, and enables the rationalization of disease mutations in ciliopathies.

Investigation of Multi-Subunit Mycobacterium tuberculosis DNA-Directed RNA Polymerase and Its Rifampicin Resistant Mutants.

Emerging Mycobacterium tuberculosis (Mtb) resistant strains have continued to limit the efficacies of existing antitubercular therapies. More specifically, mutations in the RNA replicative machinery of Mtb, RNA polymerase (RNAP), have been widely linked to rifampicin (RIF) resistance, which has led to therapeutic failures in many clinical cases. Moreover, elusive details on the underlying mechanisms of RIF-resistance caused by Mtb-RNAP mutations have hampered the development of new and efficient drugs that are able to overcome this challenge. Therefore, in this study we attempt to resolve the molecular and structural events associated with RIF-resistance in nine clinically reported missense Mtb RNAP mutations. Our study, for the first time, investigated the multi-subunit Mtb RNAP complex and findings revealed that the mutations commonly disrupted structural-dynamical attributes that may be essential for the protein's catalytic functions, particularly at the βfork loop 2, β'zinc-binding domain, the β' trigger loop and β'jaw, which in line with previous experimental reports, are essential for RNAP processivity. Complementarily, the mutations considerably perturbed the RIF-BP, which led to alterations in the active orientation of RIF needed to obstruct RNA extension. Consequentially, essential interactions with RIF were lost due to the mutation-induced repositioning with corresponding reductions in the binding affinity of the drug observed in majority of the mutants. We believe these findings will significantly aid future efforts in the discovery of new treatment options with the potential to overcome antitubercular resistance.

Hydroxyurea as a promising ADAM17 inhibitor

A disintegrin and metalloproteinase-17 (ADAM17) is a multi-domain, type 1 trans-membrane protein with a zinc-binding catalytic domain and a rich history of research scrutinizing its cellular activation and presentation, shedding activity, and prominent physiological and pathological roles. Presently, despite the advancement in the inhibitory approaches focusing on ADAM17, clinically approved therapies targeting its inhibition are not available. A principal approach to inhibit ADAM17 involves ligating the catalytic zinc atom using a zinc binding group (ZBG), with the hydroxamate functionality being the principal ZBG directing the synthesis of a vast number of zinc-binders as ADAM17 inhibitors. In addition, multiple biological and pathophysiological pathways are involved in the activation and upregulation of ADAM17 with a capacity for pharmacological interventions to modulate these pathways and hence indirectly inhibit the activity of ADAM17. Thus, we hypothesize that hydroxyurea (HU), as the simplest hydroxamate, has the intrinsic ability to chelate the zinc atom of the active site region of ADAM17, which shares similarity with matrix metalloproteinases (MMPs) and carbonic anhydrases (CAs) as zinc-confining enzymes. Furthermore, our hypothetical argument sheds light on the structural characteristics, the nitric oxide (NO) donation properties, and the iron-chelating attributes of HU in addition to its immunomodulatory and anti-angiogenic activities; hence, a multi-faceted strategic targeting of ADAM17 is proposed that can be employed to put forward an approved hydroxamate-bearing compound as an inhibitor of ADAM17. Finally, a novel mechanism of action of HU is hypothesized that mandates further research within the field of therapeutics.

Candida parapsilosis carbonyl reductase as a tool for preliminary screening of inhibitors for alcohol dehydrogenase induced skin sensitization

Human alcohol dehydrogenases, specifically ADH1 oxidize primary alcohols to aldehydes in the skin. These aldehydes act as haptens, leading to sensitization. Inhibition of these ADH enzymes may help in combating skin allergies. In the present study, the recombinantly purified stereospecific enzyme from Candida parapsilosis ATCC 7330, Candida parapsilosis Carbonyl Reductase (CpCR) has 23.31 % sequence similarity with human alcohol dehydrogenase ADH1B1. These two enzymes have perfectly overlapping cofactor and zinc binding domains. CpCR was found to oxidize primary alcohols to their corresponding aldehydes. Oxidation of primary alcohols by CpCR was further optimized with cinnamyl alcohol as the model substrate that initially showed a conversion of 56 % ± 0.25, and upon optimization increased to 98.2 % ± 0.23. An increase of 20–50 % in the conversion rate has been observed for various primary alcohols under the optimized reaction conditions. A simple and efficient model was designed for the screening of compounds that inhibit CpCR with the possibility of mitigating the action of skin allergens by inhibiting ADH1. p-Nitrophenylglyoxal (PNG) was found to be a good inhibitor for CpCR which showed inhibitory activity at very low concentration (IC50,100 mM ± 1.27) as compared to the standard inhibitor 4-methyl pyrazole (4MP) (IC50, 400 mM ± 2.05).