Regulation of expression of a cDNA from barley roots encoding a high affinity sulphate transporter

A cDNA encoding a high-affinity sulphate transporter has been isolated from barley by complementation of a yeast mutant. The cDNA, designated HVST1, encodes a polypeptide of 660 amino acids (M_r = 72 550), which is predicted to have 12 membrane-spanning domains and has extensive sequence homology with other identified eukaryotic sulphate transporters. The K_m for sulphate was 6.9 µM when the HVST1 cDNA was expressed in a yeast mutant deficient in the gene encoding for the yeast SUL1 sulphate transporter. The strong pH-dependency of sulphate uptake when HVST1 was expressed heterologously in yeast suggests that the HVST1 polypeptide is a proton/sulphate co-transporter. The gene encoding HVST1 is expressed specifically in root tissues and the abundance of the mRNA is strongly influenced by sulphur nutrition. During sulphur-starvation of barley, the abundance of mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, both increase. Upon re-supply of sulphate, the abundance of the mRNA corresponding to HVST1, and the capacity of the roots to take up sulphate, decrease rapidly, concomitant with rises in tissue sulphate, cysteine and glutathione contents. Addition of the cysteine precursor, O-acetylserine, to plants grown with adequate sulphur supply, leads to increases in sulphate transporter mRNA, sulphate uptake rates and tissue contents of glutathione and cysteine. It is suggested, that whilst sulphate, cysteine and glutathione may be candidates for negative metabolic regulators of sulphate transporter gene expression, this regulation may be overridden by O-acetylserine acting as a positive regulator.     

Activities and kinetic mechanisms of native and soluble NADPH-cytochrome P450 reductase

A highly thermostable xylanase (Xyl I) produced by Thermomonospora sp. was purified to homogeneity and was classified as a family 10 xylanase based on its molecular weight (38,000 Da) and isoelectric point (4.1). K2d analysis showed that the secondary structure of Xyl I was made up of 38% alpha-helix and 10% beta-sheet. The optimal temperature for the activity of Xyl I was 80 degrees C. Xyl I was highly thermostable with half-lives of 86, 30, and 15 min at 80, 90, and 100 degrees C respectively. Xyl I was stable in an expansive pH range of 5 to 10 with more than 75% residual activity. Our present investigation using o-phthalaldehyde (OPTA) as the chemical initiator for fluorescent chemoaffinity labeling and trinitrobenzenesulphonic acid (TNBS) as chemical modifier have revealed the presence of a single lysine residue in the active site of Xyl I. The high pK value for the basic limb of the pH profile reflects the ionization of a lysine residue. The higher K(m) values and similar k(cat) values of the TNBS modified enzyme in comparison to native enzyme and the substrate protection against OPTA and TNBS, suggested the presence of the lysine residue in the substrate-binding site.     

Arbovirus of marine mammals: a new alphavirus isolated from the elephant seal louse, Lepidophthirus macrorhini

A novel alphavirus was isolated from the louse Lepidophthirus macrorhini, collected from southern elephant seals, Mirounga leonina, on Macquarie Island, Australia. The virus displayed classic alphavirus ultrastructure and appeared to be serologically different from known Australasian alphaviruses. Nearly all Macquarie Island elephant seals tested had neutralizing antibodies against the virus, but no virus-associated pathology has been identified. Antarctic Division personnel who have worked extensively with elephant seals showed no serological evidence of exposure to the virus. Sequence analysis illustrated that the southern elephant seal (SES) virus segregates with the Semliki Forest group of Australasian alphaviruses. Phylogenetic analysis of known alphaviruses suggests that alphaviruses might be grouped according to their enzootic vertebrate host class. The SES virus represents the first arbovirus of marine mammals and illustrates that alphaviruses can inhabit Antarctica and that alphaviruses can be transmitted by lice.     

Design,synthesis and antibacterial properties of pyrimido[4,5-b]indol-8-amine inhibitors of DNA gyrase

According to the World Health Organization (WHO), approximately 1.7 million deaths per year are caused by tuberculosis infections. Furthermore, it has been predicted that, by 2050, antibacterial resistance will be the cause of approximately 10 million deaths annually if the issue is not tackled. As a result, novel approaches to treating broad-spectrum bacterial infections are of vital importance. During the course of our wider efforts to discover unique methods of targeting multidrug-resistant (MDR) pathogens, we identified a novel series of amide-linked pyrimido[4,5-b]indol-8-amine inhibitors of bacterial type II topoisomerases. Compounds from the series were highly potent against gram-positive bacteria and mycobacteria, with excellent potency being retained against a panel of relevant Mycobacterium tuberculosis drug-resistant clinical isolates.     

The First Virally Encoded Cytochrome P450

The genome sequence of the giant virus Acanthamoeba polyphaga mimivirus revealed the presence of two putative cytochrome P450 (CYP) genes. The product of one of the two predicted CYP genes ({"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162) showed low-level homology to sterol 14-demethylase (CYP51) and contained a C-terminal polypeptide domain of unknown function. {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 expression (without an N-terminal membrane binding domain) in Escherichia coli yields a CYP protein which gives a reduced CO difference maximum at 448 nm and was formally demonstrated as the first viral cytochrome P450. Analysis of binding of lipid and sterol substrates indicated no perturbation in CYP heme environment, and an absence of activity was seen when 14-methyl sterols were used as a substrate. The function of the CYP protein and its C-terminal domain remain unknown.Cytochromes P450 (CYP) are a superfamily of heme-thiolate enzymes that are distributed widely throughout Eukarya, Archaea, and Bacteria (http://drnelson.utmem.edu/CytochromeP450.html). Viruses are the most abundant biological entities in nature and are also responsible for many diseases in plants and animals. To date, 2,180 viral genome sequencing projects have been completed and annotated and no CYP open reading frames have been observed (http://www.ncbi.nlm.nih.gov/genomes/GenomesGroup.cgi?taxid=10239&opt=Virus).Acanthamoeba polyphaga mimivirus is the largest known virus, which grows in amoeba (5). In 2004, the 1.2-Mbp genome of mimivirus (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AY653733","term_id":"55416625","term_text":"AY653733"}}AY653733) was sequenced (9). Its genome is larger than that of several bacteria and archaea and is predicted to encode 911 proteins, among which only 298 have predicted functions. Many atypical proteins are predicted to be encoded by the mimivirus genome, including key protein translation enzymes, a full complement of DNA repair pathway components, and the unique presence of three different topoisomerases (9). Interestingly, among genes never yet reported to occur in a virus, mimivirus contained two putative gene sequences predicted to encode cytochrome P450 enzymes (GenBank accession no. {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 and {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162, also known as MIMI_L532 and MIMI_L808, respectively). First, {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 is a putative protein of 468 amino acids in length. In a BLASTP search, this putative CYP protein showed homology to a range of bacterial P450 proteins, including a P450 protein from Chloroflexus aurantiacus (23% identity) and CYP171 from Streptomyces peucetius (23% identity). Additionally, {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 also showed homology at the same level to nematode P450 proteins, including Caenorhabditis briggsae CYP37B1 (25% identity) and a P450 protein from the sea squirt Ciona intestinalis similar to the CYP4 family (24% identity). Efforts in our laboratory to express the {"type":"entrez-protein","attrs":{"text":"YP_142886","term_id":"55819402","term_text":"YP_142886"}}YP_142886 gene and verify that it indeed encodes a cytochrome P450 have been unsuccessful, but additional attempts are in progress. The mimivirus protein {"type":"entrez-protein","attrs":{"text":"YP_143886","term_id":"55980589","term_text":"YP_143886"}}YP_143886 was designated CYP5254A1 by David Nelson (http://drnelson.utmem.edu/CytochromeP450.html).The second putative mimivirus CYP protein ({"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162) showed in a BLASTP search the strongest homology to CYP51 proteins (7) from a variety of organisms, including protozoal CYP51 proteins from, e.g., Leishmania major (23% identity); plant CYP51 proteins from, e.g., Arabidopsis thaliana (22% identity); and fungal CYP51 proteins from, e.g., Aspergillus fumigatus (21% identity). This homology is low, strongly suggesting the absence of a functional link. Further analysis of the {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 709-amino-acid sequence revealed this putative CYP protein to be approximately 200 residues longer than its closest CYP homologues, and this protein was proposed to comprise a fused protein domain of unknown function, with the best homologies to lipopolysaccharide core biosynthesis glycosyl transferase from Proteus mirabilis HI4320 (26% identity), integral membrane sensor signal transduction histidine kinase from Dinoroseobacter shibae DFL 12 (24% identity), a short region of dysferlin from Strongylocentrotus purpuratus (35% identity), and pierisin-1 (NAD-DNA ADP-ribosyltransferase) from Pieris rapae (24% identity). Interestingly, several putative posttranslational modifications, including one N glycosylation site, a protein kinase C phosphorylation site, four casein kinase II phosphorylation sites, and three myristoylation sites, were predicted to exist in this C-terminal extension peptide, representing the first time these specific modifications were present in a P450 molecule.Historically, a protein can be identified as a cytochrome P450 through the production of the carbon monoxide (CO)-bound form of the reduced (sodium dithionite-treated) pigment, which has an intense absorption band at 450 nm (8). Following isopropyl β-d-thiogalactopyranoside-induced expression of the full-length {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 gene, utilizing the T7 promoter of the Escherichia coli expression vector pET17b (Novagen), only a protein producing a Soret maximum at 420 nm, recognized as the misfolded, incorrect form of CYP, was detected in reduced-difference CO spectrophotometry. Alterations in temperature, coexpression with molecular chaperones GroES and GroEL of E. coli (which allow production of active and correctly folded human P450s [4]), and the use of different E. coli strains for recombinant protein expression did not produce correctly folded {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (data not shown). Analysis of the primary sequence revealed the presence of a putative membrane-spanning segment located from residue 2 to 19 which may interfere with the expression of correctly folded P450 (1). A modified {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 gene sequence encoding an insertion of alanine at amino acid position 2 was generated by PCR. This N-terminally truncated enzyme (Fig. ​(Fig.1A),1A), expressed as a correctly folded CYP protein, generated a characteristic reduced-CO-difference spectrum with a maximum at 448 nm (Fig. ​(Fig.1B).1B). Cell fractionation revealed the truncated protein to be associated with the membrane fraction following ultracentrifugation at 100,000 × g, and no CYP protein was detected in the E. coli cytoplasm, thus necessitating the use of detergents to purify the enzyme. The truncated but membrane-bound enzyme was expressed at CYP levels of >1,000 nmol P450/liter of culture, with supplementation of the growth medium with the heme precursor δ-aminolevulinic acid increasing heme-incorporated CYP expression levels approximately twofold, to 2,000 to 3,000 nmol P450/liter of culture. Truncated {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 was the major band observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (Fig. ​(Fig.1C),1C), with the molecular mass estimated to be 78 kDa, in agreement with the predicted molecular mass of the truncated protein. The absolute absorbance spectrum of the purified (oxidized, Fe³⁺) {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 protein showed a Soret band at 419 nm and α and β bands at 572 and 536 nm, while reduction with sodium dithionite (Fe²⁺) resulted in a typical Soret peak shift to 417 nm. Most CYP enzymes are purified in a low-spin state with a water molecule hexacoordinated to the CYP heme iron, as indicated by a peak at 390 nm in the absolute spectrum. Substrate or inhibitor addition shifts the heme to the high-spin state, as indicated by a peak at 419 nm in the absolute spectrum, which can be the case when imidazole, used to purify the protein, binds to the heme iron. Continued dialysis for removal of the imidazole from the protein resulted in a shift from a high- to a low-spin state. Furthermore, quantification of the iron content (2) of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (0.97 ± 0.06 atoms of iron per heme-containing molecule of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162) indicated that there is one atom of iron per heme-containing molecule of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162, confirming one atom of iron associated with the heme of this P450 protein. Given the weak homology of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 to sterol-metabolizing CYP proteins, the binding of the sterols lanosterol and obtusifoliol as well as the final A. polyphaga sterol end product ergosterol to purified enzyme was investigated as previously described (3). No evidence of sterol binding or metabolism was obtained (data not shown). Such data can be confirmed by the fact that the key motif aGQHTSs (which is involved in catalysis, includes an invariant H in all CYP51 proteins to date [6], and corresponds to a negatively charged residue [D/E] in other P450 families) is missing in {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (Fig. ​(Fig.2A).2A). Additionally, {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 did not cluster with any CYP51 proteins but mapped to a distinct and separate branch on the tree (Fig. ​(Fig.2B).2B). A homology model was generated for mimivirus {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 protein on the basis of the resolved P450 crystal structures of Mycobacterium tuberculosis CYP51 (Protein Data Bank accession no. 1E9X) and flavocytochrome CYP102A1 from Bacillus megaterium (Protein Data Bank accession no. 1JPZ). {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 is predicted to adopt a typical P450 fold reflecting the similarity in helix assignment. It was possible to confirm the likely heme-coordinating residue (C425), and the EXXR motif, present in nearly all P450 proteins and involved in heme binding and P450 architecture, is also conserved in {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 (Fig. ​(Fig.3).3). Consequently, the mimivirus protein {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 was designated CYP5253A1 (http://drnelson.utmem.edu/CytochromeP450.html).Open in a separate windowFIG. 1.Purification and spectral characterization of mimivirus {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162. (A) N-terminal sequence of the full-length native enzyme, with the hydrophobic stretch that most likely forms a transmembrane α helix underlined. Below is shown the N-terminal sequence of the truncated CYP protein used to obtain the correctly folded P450 protein. (B) Absolute oxidized and reduced CO difference (inset) spectra at 1 μM P450 concentration. (C) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel (10%). Lane 1, rainbow marker; lane 2, purified mimivirus CYP51-like protein.Open in a separate windowFIG. 2.Alignment of mimivirus {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162 with 143 CYP51 family members (only two representatives of each biological kingdom are shown). (A) The fragments shown are the BC loop (SRS1 region, helices F and G [SRS2 and SRS3] and helix I [SRS4]). (B) Representative phylogenetic tree of CYP51 sequences showing the position of {"type":"entrez-protein","attrs":{"text":"YP_143162","term_id":"55819674","term_text":"YP_143162"}}YP_143162.Open in a separate windowFIG. 3.Mimivirus P450 model with marked secondary structural elements. The resultant homology models were validated by cross-reference to the secondary structure predictions. Homology models were generated with 10 iterations of the MODELLER program, and the model structure was clipped to the first 480 amino acids, for which the sequence identity with the resolved crystal structures of MTCYP51 and flavocytochrome P450-2 of Bacillus megaterium for the CYP domain was 16%. The energy-minimized model has very good ProsaII and Profiles 3D scores.The presence of genes encoding CYP in the mimivirus genome is intriguing from the standpoint of P450 evolutionary discussion. Phylogenetically, mimivirus and other giant viruses are very old and are thought to have existed prior to cellular organisms (9). Furthermore, it was previously proposed that a form of cytochrome P450 has been present in life forms for billions of years and before the advent of free atmospheric oxygen (10). Subsequently P450 had protective value in detoxifying reactive oxygen species and was retained in aerobic organisms as a monooxygenase in biosynthetic processes and in the degradation of complex molecules. Consequently, it can be speculated that P450 may have been present in a viral genome prior to the establishment of the three domains of life (eukaryotes and prokaryotes, which consist of bacteria and archaea). It is possible that mimivirus acquired CYP genes from a more ancient progenitor. Conversely, Moreira and Brochier-Armanet (7) hypothesize that the diverse mimivirus genes, many with eukaryotic homology, including CYP, were obtained by horizontal gene transfer. The basis of this theory suggests that the mimivirus host, A. polyphaga, is also host to parasitic, bacterial endosymbionts, including many Mycobacteria spp. which have high numbers of CYP genes within their genomes. Consequently, mimivirus may have picked up genes from such endosymbionts or from the amoeba host itself. At present, no Acanthamoeba genome has been sequenced to allow comparisons, but Acanthamoeba genomes certainly contain CYP51 for synthesis of sterols. Finally, although mimivirus was first isolated from A. polyphaga, additional hosts acting as a source of new genetic material for this virus cannot be ruled out. For example, mimivirus has been implicated as a causative agent of influenza in mice and humans, suggesting mammalian cellular hosts for mimivirus. It can also be argued that mimivirus CYP has evolved into a gene encoding a protein with a totally different function, unrelated to its being a P450. It is probable that we will never know the nature of the ancient P450 ancestor or how it evolved into the superfamily that we see today (>8,500 genes). However our confirmation of a viral gene encoding a cytochrome P450 protein invigorates this continuing debate.     

Streptomyces coelicolor A3(2) CYP102 Protein,a Novel Fatty Acid Hydroxylase Encoded as a Heme Domain without an N-Terminal Redox Partner

The gene from Streptomyces coelicolor A3(2) encoding CYP102B1, a recently discovered CYP102 subfamily which exists solely as a single P450 heme domain, has been cloned, expressed in Escherichia coli, purified, characterized, and compared to its fusion protein family members. Purified reconstitution metabolism experiments with spinach ferredoxin, ferredoxin reductase, and NADPH revealed differences in the regio- and stereoselective metabolism of arachidonic acid compared to that of CYP102A1, exclusively producing 11,12-epoxyeicosa-5,8,14-trienoic acid in addition to the shared metabolites 18-hydroxy arachidonic acid and 14,15-epoxyeicosa-5,8,11-trienoic acid. Consequently, in order to elucidate the physiological function of CYP102B1, transposon mutagenesis was used to generate an S. coelicolor A3(2) strain lacking CYP102B1 activity and the phenotype was assessed.Streptomycetes produce a vast array of antibiotics applied in human and veterinary medicine and agriculture, as well as antiparasitic agents, herbicides, and pharmacologically active metabolites (e.g., immunosuppressants). Streptomycetes also catalyze numerous transformations of xenobiotics of industrial and environmental importance (1). The most significant of these biocatalytic reactions include aromatic and aliphatic hydroxylations, O and N dealkylations, N oxidation, and C-C coupling and fission catalyzed by heme-containing cytochrome P450 (CYP) enzyme systems (25). Streptomyces coelicolor A3(2) is the most-studied member of the genus in molecular genetic and biochemical investigations (1). Eighteen cytochrome P450, six ferredoxin, and four ferredoxin reductase genes were shown to be distributed across the linear chromosome, with nine P450 genes arranged in polycistronic organization with other genes (1, 14).Arguably, the most biochemically and structurally characterized CYP to date is CYP102A1 (P450 BM3) from Bacillus megaterium. CYP102A1 was first isolated as a fatty acid monooxygenase in the laboratory of Armand Fulco more than 20 years ago (16). It was established as a catalytically self-sufficient monooxygenase consisting of a CYP heme domain fused at the carboxy terminus to a flavin mononucleotide (FMN)/flavin adenine dinucleotide (FAD)-containing flavin reductase domain in a single polypeptide chain. It has been proposed that such architecture affords the CYP domain optimal catalytic activity in the turnover of substrates. Indeed, of all the CYPs characterized functionally, CYP102A1 exhibits the highest P450 turnover frequency, with rates of >15,000 min⁻¹ obtained in exogenous arachidonate hydroxylation (17, 18). Furthermore, CYP102A1-like P450 reductase fusion proteins have also been found in certain fungi, including Fusarium oxysporum (13) and Phanerochaete chrysosporium (3).CYP102B1 was the first member of a new CYP102 subfamily discovered outside Bacillus spp. (1) and is intriguing since it exists and functions as a single CYP protein domain. Here we report the cloning, expression, and characterization of CYP102B1 and demonstrate that the enzyme has activity in metabolizing arachidonic acid but with very different product profiles and with enzymatic rates orders of magnitude lower than those of CYP102A1. To address the question of the contribution of CYP102B1 to S. coelicolor A3(2) physiology, a CYP102B1 transposon mutant was generated and isolated and the phenotype of the subsequent mutant strains was analyzed.     

Modulation of cyanoalanine synthase and O-acetylserine (thiol) lyases A and B activity by beta-substituted alanyl and anion inhibitors

The reaction mechanisms of three enzymes belonging to a single gene family are compared: a cyanoalanine synthase and two isoforms of O-acetylserine (thiol) lyase (O-ASTL) isolated from spinach (Spinacea oleracea L. cv. Medina). O-ASTL represents a major regulatory point in the S-assimilatory pathway, and the related cyanoalanine synthase, which is specific to the mitochondrial compartment, has evolved an independent function of cyanide detoxification. All three enzymes catalysed both the cysteine synthesis and cyanoalanine synthesis reactions although with different efficiencies, and which may be explained by a single amino acid substitution in the substrate-binding pocket of the enzyme. Substituted alanine and nucleophillic inhibitors caused predominantly non-competitive inhibition, indicating binding to both E- and F-forms of the enzyme in a bi-bi ping-pong kinetic model. Michaelis-Menten kinetics were observed when the alanyl substrate was varied in the presence and absence of inhibitors. The use of alanyl inhibitors has shown that the alanyl half-cycle of both the cysteine synthesis and cyanoalanine synthesis reactions of cyanoalanine synthase and O-acetylserine (thiol) lyases are similar. This is in contrast to the results observed with nucleophillic inhibitors, which have shown that the mechanisms of anion binding and processing differ between cyanoalanine synthase and O-ASTLs.     

Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase

Prothioconazole is a new triazolinthione fungicide used in agriculture. We have used Candida albicans CYP51 (CaCYP51) to investigate the in vitro activity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment of C. albicans cells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts from C. albicans grown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.