Active Site Zinc Research Articles

X-ray Structure-Guided Discovery of a Potent Benzimidazole Glutaminyl Cyclase Inhibitor That Shows Activity in a Parkinson's Disease Mouse Model.

The secretory glutaminyl cyclase (sQC) and Golgi-resident glutaminyl cyclase (gQC) are responsible for N-terminal protein pyroglutamation and associated with various human diseases. Although several sQC/gQC inhibitors have been reported, only one inhibitor, PQ912, is currently undergoing clinic trials for the treatment of Alzheimer's disease. We report an X-ray crystal structure of sQC complexed with PQ912, revealing that the benzimidazole makes "anchor" interactions with the active site zinc ion and catalytic triad. Structure-guided design and optimization led to a series of new benzimidazole derivatives exhibiting nanomolar inhibition for both sQC and gQC. In a MPTP-induced Parkinson's disease (PD) mouse model, BI-43 manifested efficacy in mitigating locomotor deficits through reversing dopaminergic neuronal loss, reducing microglia, and decreasing levels of the sQC/gQC substrates, α-synuclein, and CCL2. This study not only offers structural basis and new leads for drug discovery targeting sQC/gQC but also provides evidence supporting sQC/gQC as potential targets for PD treatment.

Crosslinking of base-modified RNAs by synthetic DYW-KP base editors implicates an enzymatic lysine as the nitrogen donor for U-to-C RNA editing

Sequence-specific cytidine to uridine (C-to-U) and adenosine to inosine editing tools can alter RNA and DNA sequences and utilize a hydrolytic deamination mechanism requiring an active site zinc ion and a glutamate residue. In plant organelles, DYW-PG domain containing enzymes catalyze C-to-U edits through the canonical deamination mechanism. Proteins developed from consensus sequences of the related DYW-KP domain family catalyze what initially appeared to be uridine to cytidine (U-to-C) edits leading to this investigation into the U-to-C editing mechanism. The synthetic DYW-KP enzyme KP6 was found sufficient for C-to-U editing activity stimulated by the addition of carboxylic acids invitro. Despite addition of putative amine/amide donors, U-to-C editing by KP6 could not be observed in vitro. C-to-U editing was found not to be concomitant with U-to-C editing, discounting a pyrimidine transaminase mechanism. RNAs containing base modifications were highly enriched in interphase fractions consistent with covalent crosslinks to KP6, KP2, and KP3 proteins. Mass spectrometry of purified KP2 and KP6 proteins revealed secondary peaks with mass shifts of 319Da. A U-to-C crosslinking mechanism was projected to explain the link between crosslinking, RNA base changes, and the ∼319Da mass. In this model, an enzymatic lysine attacks C4 of uridine to form a Schiff base RNA-protein conjugate. Sequenced RT-PCR products from the fern Ceratopteris richardii indicate U-to-C base edits do not preserve proteinaceous crosslinks in planta. Hydrolysis of a protonated Schiff base conjugate releasing cytidine is hypothesized to explain the completed pathway in plants.

Design and Biological Evaluation of Cephalosporin Based Metallo-β-lactamase (MBL) Inhibitors#

Background: Prevalence of microbial resistance due to Metallo-β-lactamase (MBL) enzyme pose a serious threat to human life. MBLs depend on active site zinc for their hydrolytic activity; hence, the investigation of zinc chelators emerged as an attractive strategy for the development of potent MBL inhibitors. Method: To prove that such chelators selectively target MBLs, in the present investigation, novel cephalosporins based MBL inhibitors (Cef-MBLi) were designed as a conjugate of cephalosporins with a potent zinc binder 8-thioquinoline (8-TQ). Result: Cef-MBLi showed site specific release of conjugate only in the presence of a Veronaintegron encoded metallo-β-lactamase 2 (VIM-2) bacterial enzyme through hydrolytic cleavage mechanism. A total of 6 (4a-e and 6f) New Chemical Entities (NCE’s) were prepared, characterized and subjected for in vitro study. Conclusion: Among tested NCE’s, 4c showed potent MBL inhibitory activity against the VIM-2 enzyme.

Structural, Biochemical, and Computational Characterization of Sulfamides as Bimetallic Peptidase Inhibitors.

The sulfonamide function is used extensively as a general building block in various inhibitory scaffolds and, more specifically, as a zinc-binding group (ZBG) of metalloenzyme inhibitors. Here, we provide biochemical, structural, and computational characterization of a metallopeptidase in complex with inhibitors, where the mono- and bisubstituted sulfamide functions are designed to directly engage zinc ions of a bimetallic enzyme site. Structural data showed that while monosubstituted sulfamides coordinate active-site zinc ions via the free negatively charged amino group in a canonical manner, their bisubstituted counterparts adopt an atypical binding pattern divergent from expected positioning of corresponding tetrahedral reaction intermediates. Accompanying quantum mechanics calculations revealed that electroneutrality of the sulfamide function is a major factor contributing to the markedly lower potency of bisubstituted compounds by considerably lowering their interaction energy with the enzyme. Overall, while bisubstituted uncharged sulfamide functions can bolster favorable pharmacological properties of a given inhibitor, their use as ZBGs in metalloenzyme inhibitors might be less advantageous due to their suboptimal metal-ligand properties.

Molecular Docking, ADMET Study, Synthesis, Characterization and Preliminary Antiproliferative Activity of Potential Histone Deacetylase Inhibitors with Isoxazole as New Zinc Binding Group

Histone acetylation is a highly interesting epigenetic target for drug therapy. Histone deacetylase enzymes (HDACs) are overexpressed in several diseases, including cancers. Most of the clinically used HDAC inhibitors involve hydroxamate group as a zinc-binding group (ZBG). Hydroxamates have a poor pharmacokinetic properties and high toxicity profile. Therefore, developing non-hydroximate HDAC inhibitors is a promising strategy for potency and selectivity enhancement. In this work, we designed new HDAC inhibitors with isoxazole moiety as ZBG using Ligand Designer from Glide (Schrodinger LLC). The cap group and the linker were optimized through trying various aliphatic and aromatic residues. The potential inhibition over HDAC8 for the optimally designed products was virtually evaluated using licensed Schrodinger modelling software. The results showed that the isoxazole has a potential bidentate interaction with HDAC8 active site zinc ion with acceptable fitness. ADMET/tox study performed to predict the pharmacokinetic properties for the final compounds. The final compounds showed a decent estimated drug-like properties. The intermediates and final compounds were successfully synthesized and purified using column chromatography. The chemical structure for intermediates and final compounds were characterized by IR and NMR spectroscopy.

Phenolic compounds as histone deacetylase inhibitors: binding propensity and interaction insights from molecular docking and dynamics simulations.

Histone deacetylases are well-established target enzymes involved in the pathology of different diseases including cancer and neurodegenerative disorders. The approved HDAC inhibitor drugs are associated with cellular toxicities. Different phenolic compounds have been shown to possess inhibitory activities against HDACs and are, therefore, considered safer alternatives to synthetic compounds. Here, we elucidated the binding mode and calculated the binding propensity of some of the top phenolic compounds against different isoforms representing different classes of Zn2+ ion-containing HDACs using the molecular docking approach. Our data reaffirmed the activity of the studied phenolic compounds against HDACs. Binding interaction analysis suggested that these compounds can block the activity of HDACs with or without binding to the active site zinc metal ion. Furthermore, molecular dynamics (MD) simulations were carried out on the selected crystal and docking complexes of each selected HDAC isoform. Analysis of root-mean-square displacement (RMSD) showed that the phenolic compounds demonstrated a stable binding mode over 50ns in a way that is comparable to the cocrystal ligands. Together, these findings can aid future efforts in the search for natural inhibitors of HDACs.

Targeting Aggrecanases for Osteoarthritis Therapy: From Zinc Chelation to Exosite Inhibition.

Osteoarthritis (OA) is the most common degenerative joint disease. In 1999, two members of the A Disintegrin and Metalloproteinase with Thrombospondin Motifs (ADAMTS) family of metalloproteinases, ADAMTS4 and ADAMTS5, or aggrecanases, were identified as the enzymes responsible for aggrecan degradation in cartilage. The first aggrecanase inhibitors targeted the active site by chelation of the catalytic zinc ion. Due to the generally disappointing performance of zinc-chelating inhibitors in preclinical and clinical studies, inhibition strategies tried to move away from the active-site zinc in order to improve selectivity. Exosite inhibitors bind to proteoglycan-binding residues present on the aggrecanase ancillary domains (called exosites). While exosite inhibitors are generally more selective than zinc-chelating inhibitors, they are still far from fulfilling their potential, partly due to a lack of structural and functional data on aggrecanase exosites. Filling this gap will inform the design of novel potent, selective aggrecanase inhibitors.

Ibuprofen: a weak inhibitor of carbonic anhydrase II.

Carbonic anhydrases (CAs) are drug targets for a variety of diseases. While many clinically relevant CA inhibitors are sulfonamide-based, novel CA inhibitors are being developed that incorporate alternative zinc-binding groups, such as carboxylic acid moieties, to develop CA isoform-specific inhibitors. Here, the X-ray crystal structure of human CA II (hCA II) in complex with the carboxylic acid ibuprofen [2-(4-isobutylphenyl)propanoic acid, a common over-the-counter nonsteroidal anti-inflammatory drug] is reported to 1.54 Å resolution. The binding of ibuprofen is overlaid with the structures of other carboxylic acids in complex with hCA II to compare their inhibition mechanisms by direct or indirect (via a water) binding to the active-site zinc. Additionally, enzyme-inhibition assays using ibuprofen, nicotinic acid and ferulic acid were performed with hCA II to determine their IC50 values and were compared with those of other carboxylic acid binders. This study discusses the potential development of CA inhibitors utilizing the carboxylic acid moiety.

Actions of Novel Angiotensin Receptor Blocking Drugs, Bisartans, Relevant for COVID-19 Therapy: Biased Agonism at Angiotensin Receptors and the Beneficial Effects of Neprilysin in the Renin Angiotensin System.

Angiotensin receptor blockers (ARBs) used in the treatment of hypertension and potentially in SARS-CoV-2 infection exhibit inverse agonist effects at angiotensin AR1 receptors, suggesting the receptor may have evolved to accommodate naturally occurring angiotensin ‘antipeptides’. Screening of the human genome has identified a peptide (EGVYVHPV) encoded by mRNA, complementary to that encoding ANG II itself, which is an inverse agonist. Thus, opposite strands of DNA encode peptides with opposite effects at AR1 receptors. Agonism and inverse agonism at AR1 receptors can be explained by a receptor ‘switching’ between an activated state invoking receptor dimerization/G protein coupling and an inverse agonist state mediated by an alternative/second messenger that is slow to reverse. Both receptor states appear to be driven by the formation of the ANG II charge-relay system involving TyrOH-His/imidazole-Carboxylate (analogous to serine proteases). In this system, tyrosinate species formed are essential for activating AT1 and AT2 receptors. ANGII is also known to bind to the zinc-coordinated metalloprotease angiotensin converting enzyme 2 (ACE2) used by the COVID-19 virus to enter cells. Here we report in silico results demonstrating the binding of a new class of anionic biphenyl-tetrazole sartans (‘Bisartans’) to the active site zinc atom of the endopeptidase Neprilysin (NEP) involved in regulating hypertension, by modulating humoral levels of beneficial vasoactive peptides in the RAS such as vasodilator angiotensin (1–7). In vivo and modeling evidence further suggest Bisartans can inhibit ANG II-induced pulmonary edema and may be useful in combatting SARS-CoV-2 infection by inhibiting ACE2-mediated viral entry to cells.

Fragment-based screening and hit-based substructure search: Rapid discovery of 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic, nanomolar metallo β-lactamase inhibitor.

Metallo-β-lactamases (MBLs) are zinc-containing carbapenemases that inactivate a broad range of β-lactam antibiotics. There is a lack of β-lactamase inhibitors for restoring existing β-lactam antibiotics arsenals against common bacterial infections. Fragment-based screening of a non-specific metal chelator library demonstrates 8-hydroxyquinoline as a broad-spectrum nanomolar inhibitor against VIM-2 and NDM-1. A hit-based substructure search provided an early structure-activity relationship of 8-hydroxyquinolines and identified 8-hydroxyquinoline-7-carboxylic acid as a low-cytotoxic β-lactamase inhibitor that can restore β-lactam activity against VIM-2-expressing E.coli. Molecular modeling further shed structural insight into its potential mode of binding within the dinuclear zinc active site. 8-Hydroxyquinoline-7-carboxylic acid is highly stable in human plasma and human liver microsomal study, making it an ideal lead candidate for further development.

Structural and biophysical characterization of the Burkholderia pseudomallei IspF inhibitor L-tryptophan hydroxamate

The enzyme 2-methylerythritol 2,4-cyclodiphosphate synthase, IspF, is essential for the biosynthesis of isoprenoids in most bacteria, some eukaryotic parasites, and the plastids of plant cells. The development of inhibitors that target IspF may lead to novel classes of anti-infective agents or herbicides. Enantiomers of tryptophan hydroxamate were synthesized and evaluated for binding to Burkholderia pseudomallei (Bp) IspF. The L-isomer possessed the highest potency, binding BpIspF with a KD of 36 µM and inhibited BpIspF activity 55% at 120 µM. The high-resolution crystal structure of the L-tryptophan hydroxamate (3)/BpIspF complex revealed a non-traditional mode of hydroxamate binding where the ligand interacts with the active site zinc ion through the primary amine. In addition, two hydrogen bonds are formed with active site groups, and the indole group is buried within the hydrophobic pocket composed of side chains from the 60 s/70 s loop. Along with the co-crystal structure, STD NMR studies suggest the methylene group and indole ring are potential positions for optimization to enhance binding potency.

Interrogating the Role of the Two Distinct Fructose-Bisphosphate Aldolases of Bacillus methanolicus by Site-Directed Mutagenesis of Key Amino Acids and Gene Repression by CRISPR Interference.

The Gram-positive Bacillus methanolicus shows plasmid-dependent methylotrophy. This facultative ribulose monophosphate (RuMP) cycle methylotroph possesses two fructose bisphosphate aldolases (FBA) with distinct kinetic properties. The chromosomally encoded FBAC is the major glycolytic aldolase. The gene for the major gluconeogenic aldolase FBAP is found on the natural plasmid pBM19 and is induced during methylotrophic growth. The crystal structures of both enzymes were solved at 2.2 Å and 2.0 Å, respectively, and they suggested amino acid residue 51 to be crucial for binding fructose-1,6-bisphosphate (FBP) as substrate and amino acid residue 140 for active site zinc atom coordination. As FBAC and FBAP differed at these positions, site-directed mutagenesis (SDM) was performed to exchange one or both amino acid residues of the respective proteins. The aldol cleavage reaction was negatively affected by the amino acid exchanges that led to a complete loss of glycolytic activity of FBAP. However, both FBAC and FBAP maintained gluconeogenic aldol condensation activity, and the amino acid exchanges improved the catalytic efficiency of the major glycolytic aldolase FBAC in gluconeogenic direction at least 3-fold. These results confirmed the importance of the structural differences between FBAC and FBAP concerning their distinct enzymatic properties. In order to investigate the physiological roles of both aldolases, the expression of their genes was repressed individually by CRISPR interference (CRISPRi). The fbaC RNA levels were reduced by CRISPRi, but concomitantly the fbaP RNA levels were increased. Vice versa, a similar compensatory increase of the fbaC RNA levels was observed when fbaP was repressed by CRISPRi. In addition, targeting fbaP decreased tktP RNA levels since both genes are cotranscribed in a bicistronic operon. However, reduced tktP RNA levels were not compensated for by increased RNA levels of the chromosomal transketolase gene tktC.

A de novo binuclear zinc enzyme with DNA cleavage activity.

Metallohydrolases are broadly used throughout biology, often to catalyze the degradation of macromolecules such as DNA and proteins. Many of these enzymes function with zinc in their active site, and an important subset of these enzymes utilize a binuclear zinc active site. Mimics of these enzymes have been developed, some of which catalyze the digestion of DNA. However, the majority of the mimics that utilize zinc are small molecules, and most are mononuclear. Herein, we report DNA cleavage activity by the de novo designed Due Ferri single-chain (DFsc) protein containing a binuclear zinc active site. This binuclear zinc-protein complex is able to digest plasmid DNA at rates up to 50ng/h, and these cleavage rates are affected by changes to amino acid residues near the zinc-binding site. These results indicate that the DFsc scaffold is a good model system to carry out careful structure-function relationship studies to understand key structural features that influence reactivity in natural binuclear zinc hydrolases, as it is the first report of a binuclear model system in a protein scaffold.

Cephalosporin Prodrug Inhibitors Overcome Metallo-β-Lactamase Driven Antibiotic Resistance.

The increasing prevalence of metallo‐β‐lactamase (MBL)‐expressing bacteria presents a worrying trend in antibiotic resistance. MBLs rely on active site zinc ions for their hydrolytic activity and the pursuit of MBL‐inhibitors has therefore involved the investigation of zinc chelators. To ensure that such chelators specifically target MBLs, a series of cephalosporin prodrugs of two potent zinc‐binders: dipicolinic acid (DPA) and 8‐thioquinoline (8‐TQ) was prepared. Although both DPA and 8‐TQ bind free zinc very tightly (K d values in the low nm range), the corresponding cephalosporin conjugates do not. The cephalosporin conjugates are efficiently hydrolyzed by MBLs to release DPA or 8‐TQ, as confirmed by using both NMR and LC‐MS studies. Notably, the cephalosporin prodrugs of DPA and 8‐TQ show potent inhibitory activity against NDM, VIM, and IMP classes of MBLs and display potent synergy with meropenem against MBL‐expressing clinical isolates of K. pneumoniae and E. coli.

Preparation of SNS Cobalt(II) Pincer Model Complexes of Liver Alcohol Dehydrogenase.

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each possessing two sulfur and one nitrogen donor atom functionalities (SNS) and based on bis-imidazole or bis-triazole compounds) are metallated with CoCl2·6H2O to afford tridentate SNS pincer cobalt(II) complexes. Preparation of the cobalt(II) model complexes for liver alcohol dehydrogenase is facile. Based on a quick color change upon adding the CoCl2·6H2O to acetonitrile solution that contains the ligand precursor, the complex forms rapidly. Formation of the metal complex is complete after allowing the solution to reflux overnight. These cobalt(II) complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH). The complexes are characterized using single crystal X-ray diffraction, electrospray mass spectrometry, ultra-violet visible spectroscopy, and elemental analysis. To accurately determine the structure of the complex, its single crystal structure must be determined. Single crystals of the complexes that are suitable for X-ray diffraction are then grown via slow vapor diffusion of diethyl ether into an acetonitrile solution that contains the cobalt(II) complex. For high quality crystals, recrystallization typically takes place over a 1 week period, or longer. The method can be applied to the preparation of other model coordination complexes and can be used in undergraduate teaching laboratories. Finally, it is believed that others may find this recrystallization method to obtain single crystals beneficial to their research.

Phenylthiourea Binding to Human Tyrosinase-Related Protein 1.

Tyrosinase-related protein 1 (TYRP1) is one of the three human melanogenic enzymes involved in the biosynthesis of melanin, a pigment responsible for the color of the skin, hair, and eyes. It shares high sequence identity with tyrosinase, but has two zinc ions in its active site rather than two copper ions as in tyrosinase. Typical tyrosinase inhibitors do not directly coordinate to the zinc ions of TYRP1. Here, we show, from an X-ray crystal structure determination, that phenylthiourea, a highly potent tyrosinase inhibitor, does neither coordinate the active site zinc ions, but binds differently from other structurally characterized TYRP1-inhibitor complexes. Its aromatic ring is directed outwards from the active site, apparently as a result from the absence of polar oxygen substituents that can take the position of water molecules bound in the active site. The compound binds via hydrophobic interactions, thereby blocking substrate access to the active site.

Discovery of mercaptopropanamide-substituted aryl tetrazoles as new broad-spectrum metallo-β-lactamase inhibitors.

β-Lactam antibiotic resistance mediated by metallo-β-lactamases (MBL) has threatened global public health. There are currently no available inhibitors of MBLs for clinical use. We previously reported the ruthenium-catalyzed meta-selective C–H nitration synthesis method, leading to some meta-mercaptopropanamide substituted aryl tetrazoles as new potent MBL inhibitors. Here, we described the structure–activity relationship of meta- and ortho-mercaptopropanamide substituted aryl tetrazoles with clinically relevant MBLs. The resulting most potent compound 13a showed IC50 values of 0.044 μM, 0.396 μM and 0.71 μM against VIM-2, NDM-1 and IMP-1 MBL, respectively. Crystallographic analysis revealed that 13a chelated to active site zinc ions via the thiol group and interacted with the catalytically important residues Asn233 and Tyr67, providing further structural information for the development of thiol based MBL inhibitors.

Crystal structure of mature myroilysin and implication for its activation mechanism

Myroilysin is a novel bacterial member of M12A metalloproteases family with an uncommon “cysteine switch” activation mechanism and a unique “cap” structure. However, activation of pro-myroilysin is elusive. Here, mature myroilysin was obtained for structure determination by treating pro-myroilysin with trypsin. The structure of mature myroilysin showed that the active-site zinc ion of the mature protein is coordinated by three histidine residues, a water molecule, and a tyrosine residue (Tyr208) in the conserved Met-turn motif (SIMHY). The “cap” structure moves away from the active-site to leave the active cleft open; the newly formed N-terminus is deeply buried in myroilysin, and Glu151 forms a salt bridge directly with the first amino acid residue (Gly38), whereas they are far from each other in the pro-myroilysin. The mutation of Tyr208 indicates that Tyr208 plays an important role in activity of myroilysin. The proteolytic activity and thermostability of mutant E151A decreased dramatically, implying that Glu151 is not only important for catalysis, but also crucial for structural stability in myroilysin. Structural comparison also reveals differences existed between myroilysin and astacin. Our biochemical and structural data provide new insights into the activation of myroilysin and functional involvement of crucial residues Tyr208 and Glu151.

Discovery of a New Isoxazole-3-hydroxamate-Based Histone Deacetylase 6 Inhibitor SS-208 with Antitumor Activity in Syngeneic Melanoma Mouse Models.

Isoxazole is a five-membered heterocycle that is widely used in drug discovery endeavors. Here, we report the design, synthesis, and structural and biological characterization of SS-208, a novel HDAC6-selective inhibitor containing the isoxazole-3-hydroxamate moiety as a zinc-binding group as well as a hydrophobic linker. A crystal structure of the Danio rerio HDAC6/SS-208 complex reveals a bidentate coordination of the active-site zinc ion that differs from the preferred monodentate coordination observed for HDAC6 complexes with phenylhydroxamate-based inhibitors. While SS-208 has minimal effects on the viability of murine SM1 melanoma cells in vitro, it significantly reduced in vivo tumor growth in a murine SM1 syngeneic melanoma mouse model. These findings suggest that the antitumor activity of SS-208 is mainly mediated by immune-related antitumor activity as evidenced by the increased infiltration of CD8+ and NK+ T cells and the enhanced ratio of M1 and M2 macrophages in the tumor microenvironment.

Structure of Ddi2, a highly inducible detoxifying metalloenzyme from Saccharomyces cerevisiae

Cyanamide (H2N-CN) is used to break bud dormancy in woody plants and to deter alcohol use in humans. The biological effects of cyanamide in both these cases require the enzyme catalase. We previously demonstrated that Saccharomyces cerevisiae exposed to cyanamide resulted in strong induction of DDI2 gene expression. Ddi2 enzymatically hydrates cyanamide to urea and belongs to the family of HD-domain metalloenzymes (named after conserved active-site metal-binding His and Asp residues). Here, we report the X-ray structure of yeast Ddi2 to 2.6 Å resolution, revealing that Ddi2 is a dimeric zinc metalloenzyme. We also confirm that Ddi2 shares structural similarity with other known HD-domain proteins. HD residues His-55, His-88, and Asp-89 coordinate the active-site zinc, and the fourth zinc ligand is a water/hydroxide molecule. Other HD domain enzymes have a second aspartate metal ligand, but in Ddi2 this residue (Thr-157) does not interact with the zinc ion. Several Ddi2 active-site point mutations exhibited reduced catalytic activity. We kinetically and structurally characterized H137N and T157V mutants of Ddi2. A cyanamide soak of the Ddi2-T157V enzyme revealed cyanamide bound directly to the Zn2+ ion, having displaced the zinc-bound water molecule. The mode of cyanamide binding to Ddi2 resembles cyanamide binding to the active-site zinc of carbonic anhydrase, a known cyanamide hydratase. Finally, we observed that the sensitivity of ddi2Δ ddi3Δ to cyanamide was not rescued by plasmids harboring ddi2-H137N or ddi2-TI57V variants, demonstrating that yeast cells require a functioning cyanamide hydratase to overcome cyanamide-induced growth defects.