Heme-thiolate Proteins Research Articles

Microarray-based analysis of gene expression in very large gene families: the cytochrome P450 gene superfamily of Arabidopsis thaliana

Cytochrome P450 (P450s) are heme-thiolate protein products of a very large gene superfamily, present in all kingdoms and involved in a variety of metabolic reactions. P450s are classified according to the degree of amino acid sequence identity, with P450s of the same family defined as having >40% identity, and P450s of the same subfamily having >55% identity. Currently, 273 P450 genes distributed over 45 families have been identified in Arabidopsis, and its genome is estimated to contain as many as 286. Genome-wide DNA microarrays make it possible to broadly correlate P450 gene activity with alterations in physiological or developmental states. A potential problem with microarray research is that sequence similarity between and within these families of closely related genes may lead to cross-hybridization. We designed experiments to systematically evaluate the specificity of P450 microarrays, and showed that conditions could be optimized to provide a very high degree of hybridization specificity. Under these conditions, and employing a 20% intensity value of maximum hybridization intensity as a cut-off, labeled P450 genes exhibited essentially no cross-hybridization between families and within subfamilies. We also compared the gene transcription levels of microarray probes derived from EST clones and from genomic DNA sequences for which ESTs were not available, using cDNA produced from RNA from various Arabidopsis tissue as the target. Many of the P450 genes displayed tissue-specific expression, leading to hypotheses as to the function of individual genes and their regulation. We also observed that several of the genomic sequences reported high levels of expression, highlighting the limitations of expression analysis based on ESTs alone.

Reactivity and Redox Potential of Heme-Thiolate Proteins – Results from Enzymes and Enzyme Models

The redox potential is a characteristic parameter of various intermediates of the catalytic cycle of heme-thiolate proteins (cytochromes P450, NO-synthase, chloroperoxidase) which significantly influences catalytic turnover. E0 values of these proteins are surprisingly positive compared to synthetic active site analogues that have an arylthiolate or an alkylthiolate coordinating to the iron. This report examines factors underlying this phenomenon and describes the design of enzyme mimics having redox potentials close to those of heme-thiolate proteins.

Synthetic active site analogues of heme–thiolate proteins: Characterization and identification of intermediates of the catalytic cycles of cytochrome P450 cam and chloroperoxidase

In view of recent results from different sources, the reaction mechanisms of two heme–thiolate proteins, cytochrome P450 cam and chloroperoxidase (CPO), are discussed. In this context a mechanism of CPO is proposed which includes H 2O 2 cleavage, subsequent formation of compound I and the identification of two elusive intermediates. The HOCl adduct of the iron(III)porpyhrin is the catalytically competent Cl + donor chlorinating activated C–H bonds of substrates bound to the enzyme. Pulse-EPR characterization of an enzyme model of the resting state of P450 cam suggests a role of the electric field of the protein for stabilizing the low-spin state of the cofactor of the enzyme. It is further suggested that the same effect of the protein may trigger the reactivity of compound I such that both concerted and two-step reactions are feasible within the concept of a Two-State-Reactivity. This review emphasizes the value of synthetic enzyme models complementing investigations of the native proteins.

Autocatalytic nitration of P450 CAM by peroxynitrite

Peroxynitrite (PN) gains high selectivity as a physiological oxidizing and nitrating agent through catalysis by metal ions. This was established for the heme-thiolate (P450) enzyme prostacyclin synthase which was tyrosine nitrated and inhibited at low PN levels [FEBS Lett. 382 (1996) 101]. Other P450 proteins reacted in a similar manner and a ferryl species (Compound II) has been identified as an intermediate during reactions with PN [Nitric Oxide 3 (1999) 142]. Here we investigated cytochrome P450 CAM and found that it catalyzes the decomposition of PN as well as an increased nitration of phenol. The latter at the expense of phenol hydroxylation is characteristic for the proton-assisted PN action. PN also caused self-nitration of P450 CAM at several tyrosine residues. Two of them, Y 96 and Y 305 were largely protected in the presence of the ligand metyrapone. Unlike other heme-thiolate proteins P450 CAM did not form distinct spectral intermediates characteristic for Compound II. We conclude that P450 CAM serves as a model for the nitration of prostacyclin synthase with respect to its autocatalytic tyrosine nitration and its prevention by blocking the active site.

Rapid Reactions of Peroxynitrite with Heme–Thiolate Proteins as the Basis for Protection of Prostacyclin Synthase from Inactivation by Nitration

Prostacyclin (PGI2) synthase is a heme–thiolate (P450) protein which reacts with low levels of peroxynitrite (PN) under tyrosine nitration and inactivation. Studying heme proteins as models, we have found the heme–thiolate protein NADH–NO reductase (P450NOR) to be highly efficient in decomposing PN under concomitant nitration of phenol. The present study investigates two other P450 proteins, P450BM-3 and chloroperoxidase, in order to test for the specific role of the thiolate ligand in the reaction with PN. A comparison with horseradish peroxidase and microperoxidase gives evidence of kinetic differences that classify heme–thiolate proteins, but not other heme proteins, as effective inhibitors of PGI2 synthase nitration and inactivation. P450BM-3 with PN catalyzes phenol nitration and nitration of its own tyrosine below 10 μM PN, whereas chloroperoxidase and P450NOR at such concentrations also nitrate phenol but not enzyme-bound tyrosine residues. We conclude that heme–thiolate proteins in general exhibit high reactivity with PN and turnover, probably due to the special electronic structure of the presumed thiolate–ferryl intermediate.

Cytochromes P450: a success story.

Cytochrome P450 proteins, named for the absorption band at 450 nm of their carbon-monoxide-bound form, are one of the largest superfamilies of enzyme proteins. The P450 genes (also called CYP) are found in the genomes of virtually all organisms, but their number has exploded in plants. Their amino-acid sequences are extremely diverse, with levels of identity as low as 16% in some cases, but their structural fold has remained the same throughout evolution. P450s are heme-thiolate proteins; their most conserved structural features are related to heme binding and common catalytic properties, the major feature being a completely conserved cysteine serving as fifth (axial) ligand to the heme iron. Canonical P450s use electrons from NAD(P)H to catalyze activation of molecular oxygen, leading to regiospecific and stereospecific oxidative attack of a plethora of substrates. The reactions carried out by P450s, though often hydroxylation, can be extremely diverse and sometimes surprising. They contribute to vital processes such as carbon source assimilation, biosynthesis of hormones and of structural components of living organisms, and also carcinogenesis and degradation of xenobiotics. In plants, chemical defense seems to be a major reason for P450 diversification. In prokaryotes, P450s are soluble proteins. In eukaryotes, they are usually bound to the endoplasmic reticulum or inner mitochondrial membranes. The electron carrier proteins used for conveying reducing equivalents from NAD(P)H differ with subcellular localization. P450 enzymes catalyze many reactions that are important in drug metabolism or that have practical applications in industry; their economic impact is therefore considerable.

Concise review of the cytochrome P450s and their roles in toxicology.

The cytochrome P450s are hemoproteins that play critical roles in the bioactivation and detoxication of a wide variety of xenobiotic substances. The enzymes are encoded by a superfamily of genes. The term “cytochrome P450” originates from the observation that the reduced state of the proteins form complexes with carbon monoxide that exhibit absorbance maxima at 450 nm (Omura and Sato, 1964). The P450 enzymes are now referred to as heme-thiolate proteins. P450s have been characterized in many species of organisms, including bacteria, fungi, plants, fish, and mammalian systems (Nebert and McKinnon, 1994). In mammalian cells, P450s are localized predominantly in the smooth endoplasmic reticulum of the cell and are entirely distinct from the cytochrome proteins that comprise the mitochondrial electron-transport function. Currently, .700 P450s have been characterized, inclusive of the many different species of organisms that have been studied (Nelson et al., 1996). Based primarily on sequence similarities, a standardized nomenclature has been adopted that categorizes the individual P450s into respective families and subfamilies. P450 proteins exhibiting .40% similarity in protein sequence are classified within the same family; proteins exhibiting .55% sequence similarity are grouped into the same subfamily (Nebert and McKinnon, 1994; Nelson et al., 1996). Although the catalog is updated regularly, the current P450 nomenclature and classification scheme was most recently reviewed in 1996 (Nelson et al., 1996). The purpose of this current review is to very briefly highlight some of the more important mammalian P450s with respect to their expression, inducibility and inhibition character, and substrate preferences, especially as they relate to human toxicology. Due to this limited scope, it is impossible to thoroughly reference all the important and relevant literature pertaining to this topic. The reader is referred to other recent reviews for more detailed information (Dogra et al., 1998; Meyer and Zanger, 1997; Pelkonen and Raunio, 1997; Rendic and Di-Carlo, 1997). Information from these and other references has been used as source material to compile the contents of Table 1, summarizing some of the salient features of several relevant P450s.

Peroxynitrite Reaction with Heme Proteins

We have reported that low levels of peroxynitrite (PN) can cause inactivation of the heme-thiolate protein prostacyclin (PGI2)-synthase by nitration of a tyrosine residue. To prove that iron catalysis is involved we studied the interaction of PN with microperoxidase and P450nor, a heme-thiolate protein of known structure. Spectral and kinetic analyses allow to conclude on a ferryl nitrogen dioxide complex as an intermediate which decomposes in the presence of an excess of PN under formation of dioxygen, nitrite, and nitrate. This occurs in a catalytic cycle which was more efficient with P450northan with microperoxidase. If phenol was added to the reaction mixtures of PN and the ferric complexes the ratio of hydroxylated to nitrated phenols decreased compared to the metal-free system. Phenol competed with the formation of dioxygen indicating that the ferryl intermediate was involved in both pathways. One therefore can postulate that the ferryl complex reacts with phenol to give the phenoxyradical which is nitrated in the presence of nitrogen dioxide but does not give hydroxylated products as with metal-free PN. Alternately, the ferryl nitrogen dioxide complex can oxidize a second PN molecule to the radical, ·OONO, which can decompose to dioxygen and NO. The latter forms N2O3with the remaining ·NO2radical. A third pathway consists in the isomerization to nitrate which also is catalyzed by the heme proteins since the ratio of nitrite/nitrate does not change significantly during the catalytic reaction with excess of PN. Our data explain the mechanism of nitration of PGI2-synthase, suggest a role of P450noras a PN scavenger, and favor heme-thiolate complexes for trapping PN.

Nitric oxide-generated P420 nitric oxide synthase: characterization and roles for tetrahydrobiopterin and substrate in protecting against or reversing the P420 conversion.

The neuronal NO synthase (nNOS) heme binds self-generated NO, and this negatively regulates NO synthesis. Here we utilized the nNOS oxygenase domain and full-length nNOS along with various spectroscopic methods to (1) study formation of the six-coordinate ferrous NO complex and its conversion to a five-coordinate NO complex and (2) investigate the spectral and catalytic properties of the five-coordinate NO complex following its air oxidation to a ferric enzyme. NO bound quickly to ferrous nNOS oxygenase to form a six-coordinate NO complex (kon and koff values of 1.25 x 10(-)3 mM-1 s-1 and 128 s-1 at 10 degreesC, respectively) that was stable in the presence of L-arginine or tetrahydrobiopterin (BH4) but was converted to a five-coordinate NO complex in a biphasic process (k = 0.1 and 0.01 s-1 at 10 degreesC) in the absence of these molecules. Air oxidation of the ferrous six-coordinate NO complex generated an enzyme with full activity and ferrous-CO Soret absorbance at 444 nm. In contrast, oxidation of the five-coordinate NO complex generated an inactive dimer with ferrous-CO Soret absorbance at 420 nm, indicating nNOS was converted to a ferric P420 form. Incubation of ferric P420 nNOS with BH4 alone or BH4 and L-arginine resulted in time-dependent reactivation of catalysis and associated recovery of P450 character. Thus, nNOS is a heme-thiolate protein that can undergo a reversible P450-P420 conversion. BH4 has important roles in preventing P420 formation during NO synthesis, and in rescuing P420 nNOS.

THE ROLE OF CYP2B6 IN HUMAN XENOBIOTIC METABOLISM*

The cytochromes P450 (CYP) comprise a large multigene family of heme-thiolate proteins which are of considerable importance in the metabolism of xenobiotics and endobiotics. Over 500 CYP genes have...

Role of α-Helix Conformation Cooperating with NH···S Hydrogen Bond in the Active Site of Cytochrome P-450 and Chloroperoxidase: Synthesis and Properties of [MIII(OEP)(Cys-Helical Peptide)] (M = Fe and Ga)

Heme-thiolate protein model complexes, [FeIII(OEP)(Ac-LcXAF-LLLLL-ALFL-OMe)] {OEP = octhaethylporphinato, X = Leu (1) and Pro (2)}, having α-helical structure were synthesized and characterized as P-450 and CPO models. The stable ligation of cysteinyl thiolate at the axial position of model complexes was established using MCD, UV−visible, 1H NMR, and ESI-MS. These results indicate that 1 and 2 possess high-spin 5-coordinated Fe(III) ions. The helical contents were examined by CD measurements in THF which revealed the contents of 1 and [GaIII(OEP)(Ac-LcLAF-LLLLL-ALFL-OMe)] (3) to be both 49%, though the thiolate anion (Et4N){Ac-LC(S-)LAF-LLLLL-ALFL-OMe} (5) has the value of 57%. Thus, the α-helical NH···OC hydrogen bond network is intercepted by the NH···S hydrogen bond between NH(Leu3) and Sγ(Cys2). However, the helical percentages of 2 (59%) and [GaIII(OEP)(Ac-LcPAF-LLLLL-ALFL-OMe)] (4) (57%) are similar to that of (Et4N){Ac-LC(S-)LAF-LLLLL-ALFL-OMe} (6) (52%). The NMR analysis of the solution structure ...

Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring

Cytochromes P450 (P450s or CYPs) constitute a superfamily of heme-thiolate proteins that play important roles in oxidative metabolism of endogenous and exogenous compounds. This review provides some limited history but addresses mainly the research progress on the cytochrome P450s in rainbow trout ( Oncorhynchus mykiss), their purification, structures at the primary level, role in metabolism, responses to chemicals and environmental pollutants, application to biomonitoring and the effect of various factors on their expression or activities. Information obtained to date suggests that the rainbow trout P450 systems are as complex as those seen in mammals. Fourteen P450s have been purified from liver or trunk kidney to relatively high specific content. cDNAs belonging to seven different P450 families have been documented from trout liver, kidney and ovary. Two CYP1A genes, nine cDNAs containing open reading frames, and a cDNA fragment were entered into GenBank. Among them, CYP2K1, CYP2K3, CYP2K4, CYP2M1, CYP3A27 and CYP4T1 are the most recently described forms. CYP2K1, CYP2M1 and CYP4T1 represent newly identified P450 subfamilies first described in the rainbow trout. In many cases, the cloned rainbow trout P450s have subsequently been expressed in heterologous expressions systems such as COS-7 cells, yeast and baculovirus infected insect cells. Some of the overexpressed P450 isoforms have been partially characterized. Potential future research directions are discussed.

Drug metabolism by Escherichia coli expressing human cytochromes P450.

The broad substrate specificity of the cytochrome P450 (P450) enzyme superfamily of heme-thiolate proteins lends itself to diverse environmental and pharmaceutical applications. Until recently, the primary drawback in using living bacteria to catalyze mammalian P450-mediated reactions has been the paucity of electron transport from NADPH to P450 via endogenous flavoproteins. We report the functional expression in Escherichia coli of bicistronic constructs consisting of a human microsomal P450 enzyme encoded by the first cistron and the auxiliary protein NADPH-P450 reductase by the second. Expression levels of P450s ranged from 35 nmol per liter culture to 350 nmol per liter culture, with expression of NADPH-P450 reductase typically ranging from 50% to 100% of that of P450. Transformed bacteria metabolized a number of typical P450 substrates at levels comparable to isolated bacterial membranes fortified with an NADPH-generating system. These rates compare favorably with those obtained using human liver microsomes as well as those of reconstituted in vitro systems composed of purified proteins, lipids, and cofactors.

Identification of intermediates in the catalytic cycle of chloroperoxidase

Background: Chloroperoxidase (CPO) is the most versatile of the hemethiolate proteins, catalyzing the chlorination of activated CH bonds and reactions reminiscent of peroxidase, catalase, and cytochrome P450. Despite 30 years of continuous efforts, no intermediates of the enzyme's catalytic cycle have been identified except for compound I. Thus, in the absence of conclusive evidence it is generally believed that the halogenation of substrates proceeds by means of ‘free HOCl’ in solution. Results: The pH profile of chloroperoxidase from Caldariomyces fumago revealed a new active-site complex that can be detected only at pH 4.4. According to ultra-violet (UV) spectroscopy, and by comparison with suitable enzyme models, this intermediate is the HOCl adduct of the iron(III) protoporphyrin(IX). Inactivation of chloroperoxidase by diethyl pyrocarbonate, which interrupts the proton shuttle by modification of the distal histidine, led to the formation of the −OCl adduct of the iron complex, which was identified by comparison with a corresponding active site analogue. Conclusions: The availability of enzyme models of heme-thiolate proteins allowed the identification by UV spectroscopy of both the −OCl adduct and the HOCI adduct of the iron(III) protoporphyrin(IX) of chloroperoxidase. The existence of these previously elusive intermediates suggests that the chlorination catalyzed by CPO, and its corresponding active site analogue, proceeds by Cl + transfer from the HOCl adduct to the substrate bound in the distal pocket of the enzyme.

New Active‐Site Analogues of Chloraperoxidase—Syntheses and Catalytic Reactions

The iron(III) porphyrin 1 catalyzes the chlorination of activated CH bonds. It is therefore a model compound for the as yet unidentified intermediate of the catalytic cycle of the heme–thiolate protein chloroperoxidase (CPO). Neither “free HOCl” nor Cl• are involved in the CPO-catalyzed chlorination, but rather an iron-bound HOCl is the source of Cl+.

Catalytic characteristics of CYP3A4: requirement for a phenolic function in ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor.

CYP3A4 is the major human cytochrome P-450 in a superfamily of heme-thiolate proteins that catalyze the oxidation of numerous lipophilic compounds. In this investigation, we report that CYP3A4 requires a phenolic function for ortho hydroxylation of estradiol and mono-O-demethylated methoxychlor and that CYP3A4 aromatic hydroxylation in general may be dependent on the presence of a free phenolic group. Indeed, when methoxyls were present instead of phenolic hydroxyls, CYP3A4 essentially failed to catalyze ortho hydroxylation. By contrast, of eight additional cDNA-expressed P-450s (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2D6, and 2E1) examined, only CYP1A2 and CYP2B6 could catalyze ortho hydroxylation of [o-3H]methoxychlor (7.2 and 14.6 pmol/90 min/pmol P-450, respectively), indicating that these isoforms do not require a phenolic hydroxyl for aromatic hydroxylation and that methoxyls do not sterically hinder catalysis by these CYPs. However, with [o-3H]mono-O-demethylated methoxychlor, containing a phenolic group, five isoforms (CYP1A2, 2B6, 2D6, 2E1, and 3A4) supported ortho hydroxylation. Of these, CYP3A4 exhibited by far the highest rate of hydroxylation at 87.8 pmol/90 min/pmol P-450. Further studies with [2-(3)H]estradiol 3-methyl ether and with [2-(3)H]estradiol revealed a similar and dramatic augmentation of CYP3A4-mediated C2 hydroxylase activity of approximately 75-fold by the presence of the phenolic group in the 3-position. The mechanism of augmentation by the phenolic hydroxyl does not appear to involve the acidic proton of estradiol, since CYP3A4-catalyzed estradiol 2-hydroxylation and testosterone 6-beta-hydroxylation were diminished to an equal extent when incubations were performed at increasing buffer pH values from 7 to 9. Both estradiol and its 3-methoxy derivative bound with similar affinity to cDNA-expressed, microsomal CYP3A4: spectral dissociation constants were 270 and 370 microM, respectively, and both compounds exhibited type I spectra. Thus, the disparities in aromatic hydroxylation rates between compounds containing phenolic hydroxyls and those with methoxyls cannot be explained by differences in their binding affinities. To explain the mode via which the phenolic hydroxyl facilitates ortho hydroxylation, a mechanism in which the phenolic moiety attacks the iron-oxo double bond of CYP3A4, resulting in oxygen transfer to the ortho position, is proposed. It is anticipated that these findings will assist in forecasting the CYP-mediated metabolic fate of phenolic compounds.

The human drug metabolizing cytochromes P450.

The superfamily of heme-thiolate proteins known as the cytochromes P450 is responsible for the oxidative metabolism of the majority of drugs. Thus, the phenotypes of individuals with respect to their levels of catalytically active cytochromes P450 determines to a large part the substantial interindividual variation observed in the metabolic clearance of drugs. Over the past 10 years 15 different human cytochromes P450 involved in drug metabolism have been isolated and characterized to varying degrees. This brief review discusses the characterization of these cytochromes P450 and how this knowledge has been used by the pharmaceutical industry to aid in the development of new drugs.

Cytochrome P450-catalyzed hydroxylation of taxa-4(5),11(12)-diene to taxa-4(20),11(12)-dien-5a-o1: the first oxygenation step in taxol biosynthesis

Background: The structural complexity of taxol dictates continued reliance on biological production methods, which may be improved by a detailed understanding of taxol biosynthesis, especially the rate-limiting steps. The biosynthesis of taxol involves the cyclization of the common isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by extensive, largely oxidative, modification of this diterpene olefin. We set out to define the first oxygenation step in taxol biosynthesis. Results: Microsomal enzymes from Taxus stem and cultured cells were used to define the first hydroxylation of taxadiene. We confirmed the structure of the reaction product (taxa-4(20),11(12)-dien-5α-ol) by synthesizing this compound. The responsible biological catalyst was characterized as a cytochrome P450 (heme thiolate protein). In vivo studies confirmed that taxadienol is a biosynthetic intermediate and indicated that the hydroxylation step that produces this product is slow relative to subsequent metabolic transformations. Conclusions: The structure of the first oxygenated intermediate on the taxol pathway establishes that the hydroxylation reaction proceeds with an unusual double bond migration that limits the mechanistic possibilities for subsequent elaboration of the oxetane moiety of taxol. The reaction is catalyzed by a cytochrome P450, suggesting that the seven remaining oxygenation steps in taxol biosynthesis may involve similar catalysts. Because the first oxygenation step is slow relative to subsequent metabolic transformations, it may be possible to speed taxol biosynthesis by isolating and manipulating the gene for the taxadiene-5-hydroxylase that catalyzes this reaction.

Localization of cytochromes p450 in human tissues: implications for chemical toxicity

Cytochromes P450 comprise a remarkably diverse superfamily of heme-thiolate proteins critical in the metabolism of numerous endogenous ligands and xenobiotics. Among the myriad of P450 substrates are many compounds of toxicological and pharmacological significance. The precise complement of cytochrome P450 isoforms in any given tissue may therefore be an important determinant of susceptibility to chemical-mediated toxicity. We have used a histological approach to study the distribution of individual P450s in human and rabbit gastro-intestinal tissues. We have focused primarily on P450 enzymes of importance in the metabolism of carcinogens, namely CYP1A1, CYP1A2, CYP2E1, CYP3A4/3A5 and CYP4B1. Here we give an overview of the distribution of these enzymes in human and rabbit tissues and discuss the possible toxicological implications of the results. In addition we will discuss the value of archival human tissue specimens for histological analysis of P450 distribution.

The Primary Sequence of Cytochrome P450tyr, the MultifunctionalN-Hydroxylase Catalyzing the Conversion ofL-Tyrosine top-Hydroxyphenylacetaldehyde Oxime in the Biosynthesis of the Cyanogenic Glucoside Dhurrin inSorghum bicolor(L.) Moench

The heme thiolate protein cytochrome P450tyr is a multifunctionalN-hydroxylase convertingL-tyrosine top-hydroxyphenylacetaldehyde oxime in the biosynthesis of the cyanogenic glucoside dhurrin inSorghum bicolor(Sibbesenet al.(1995)J. Biol. Chem.270, 3506–3511). Using a polyclonal antibody toward cytochrome P450tyr and oligonucleotide probes designed on the basis of amino acid sequences of tryptic fragments, a full-length cDNA clone encoding cytochrome P450tyr has been isolated and sequenced. The open reading frame encodes a protein with a molecular mass of 61,887 Da. A comparison with the amino acid sequencing data demonstrates that the protein is not subjected to posttranslational modification at the N- and C-terminal ends except for the removal of the N-terminal methionine residue. Highest positional identity (30.8%) is found to the 3′,5′-flavonoid hydroxylase of petunia (CYP75A1) and to a cytochrome P450 sequence from avocado of unknown function (CYP71A1). Consequently, cytochrome P450tyr is assigned as the first member of a new cytochrome P450 family denoted CYP79. The N-terminal region of cytochrome P450tyr contains the four domains characteristic for cytochrome P450 enzymes of the endoplasmic reticulum (ER) in animals. The amino acid sequence before the proline-rich domain is longer in cytochrome P450tyr and in four cytochrome P450s presently avail able from other monocotyledoneous plants compared to the sequences from dicotyledoneous plants but is concluded to contain a single transmembrane helix with the N-terminal located in the lumen of the ER and the bulk of the protein protruding into the cytoplasm. The heme-binding cysteine residue of cytochrome P450tyr is recognizable at position 493 but this region deviates from the consensus sequence by having an unusual alanine residue at position 495. The central region of helix I contains three residues, Ala-352, Asn-355, and Pro-356, deviating from the consensus sequence. CYP56 is the only other known cytochrome P450 using tyrosine as substrate and contains the same Asn–Pro substitution in the consensus sequence of helix I indicating the importance of these residues in defining substrate specificity. The conserved threonine residue which normally helps to form the oxygen binding pocket is absent. The cytochrome P450tyr sequence represents the first amino acid sequence of a functionally characterized cytochrome P450 enzyme from a monocotyledoneous plant and the first sequence of a membrane-boundN-hydroxylase with high substrate specificity. MultifunctionalN-hydroxylases of the cytochrome P450 type have not been previously demonstrated to catalyze biosynthetic pathways in living organisms.