Molecular Dynamics Simulation and Molecular Docking Studies of Angiotensin Converting Enzyme with Inhibitor Lisinopril and Amyloid Beta Peptide

Angiotensin converting enzyme (ACE) cleaves amyloid beta peptide. So far this cleavage mechanism has not been studied in detail at atomic level. Keeping this view in mind, we performed molecular dynamics simulation of crystal structure complex of testis truncated version of ACE (tACE) and its inhibitor lisinopril along with Zn²⁺ to understand the dynamic behavior of active site residues of tACE. Root mean square deviation results revealed the stability of tACE throughout simulation. The residues Ala 354, Glu 376, Asp 377, Glu 384, His 513, Tyr 520 and Tyr 523 of tACE stabilized lisinopril by hydrogen bonding interactions. Using this information in subsequent part of study, molecular docking of tACE crystal structure with Aβ-peptide has been made to investigate the interactions of Aβ-peptide with enzyme tACE. The residues Asp 7 and Ser 8 of Aβ-peptide were found in close contact with Glu 384 of tACE along with Zn²⁺. This study has demonstrated that the residue Glu 384 of tACE might play key role in the degradation of Aβ-peptide by cleaving peptide bond between Asp 7 and Ser 8 residues. Molecular basis generated by this attempt could provide valuable information towards designing of new therapies to control Aβ concentration in Alzheimer’s patient.     

Testicular Angiotensin-converting enzyme with different glycan modification: characterization on glycosylphosphatidylinositol-anchored protein releasing and dipeptidase activities

We have previously found that the angiotensin-converting enzyme (ACE) carries GPI-anchored protein releasing activity (GPIase) as well as dipeptidase activity. Testicular ACE (tACE), the male germinal specific isozyme, plays a crucial role in male fertilization. The amino-terminal region of this isozyme is different from that of somatic isozyme (sACE) and contains potential O-linked glycosylation sites. By multiple mutagenesis after an in silico prediction, amino acid residues acquiring O-glycans were assigned. Both GPIase and dipeptidase activities were compared between O-glycan null mutant and wild-type molecules, but no differences were found. Furthermore, the wild-type tACE was produced in two different cells (COS7 and CHO) and its activities compared. The GPIase activity, but not dipeptidase, was apparently higher for CHO-derived molecule than COS7. Sensitivity to neuraminidase and O-glycosidase digestions and the profile of glycosylation were quite different between these two molecules. Moreover, serial digestions with neuraminidase and O-glycosidase have no influence on GPIase activity of both molecules, suggesting that the sialylation and the presence of O-glycan has no influence on tACE enzyme activities, while the set of glycans modulate GPIase activity.     

Mapping of conformational mAb epitopes to the C domain of human angiotensin I-converting enzyme

Angiotensin I-converting enzyme (ACE, CD143) has two homologous domains, each having a functional active site. Fine epitope mapping of 8 mAbs to the C-terminal domain of human ACE was carried out using plate precipitation assays, mAbs' cross-reactivity with ACE from different species, site-directed mutagenesis, and antigen- and cell-based ELISAs. Almost all epitopes contained potential glycosylation sites. Therefore, these mAbs could be used to distinguish different glycoforms of ACE expressed in different tissues or cell lines. mAbs 1B8 and 3F10 were especially sensitive to the composition of the N-glycan attached to Asn 731; mAbs 2H9 and 3F11 detected the glycosylation status of the glycan attached to Asn 685 and perhaps Asn1162; and mAb 1E10 and 4E3 recognized the glycan on Asn 666. The epitope of mAb 1E10 is located at the N-terminal end of the C domain, close to the unique 36 amino acid residues of testicular ACE (tACE). Moreover, it binds preferentially to tACE on the surface of human spermatozoa and thus may find application as an immunocontraceptive drug. mAb 4E3 was the best mAb for quantification of ACE-expressing somatic cells by flow cytometry. In contrast to the other mAbs, binding of mAb 2B11 was not markedly influenced by ACE glycosylation or by the cell culture conditions or cell types, making this mAb a suitable reference antibody. Epitope mapping of these C-domain mAbs, particularly those that compete with N-domain mAbs, enabled us to propose a model of the two-domain somatic ACE that might explain the interdomain cooperativity. Our findings demonstrated that mAbs directed to conformational epitopes on the C-terminal domain of human ACE are very useful for the detection of testicular and somatic ACE, quantification using flow cytometry and ELISA assays, and for the study of different aspects of ACE biology.     

Structural details on the binding of antihypertensive drugs captopril and enalaprilat to human testicular angiotensin I-converting enzyme

Angiotensin converting enzyme (ACE) plays a critical role in the circulating or endocrine renin-angiotensin system (RAS) as well as the local regulation that exists in tissues such as the myocardium and skeletal muscle. Here we report the high-resolution crystal structures of testis ACE (tACE) in complex with the first successfully designed ACE inhibitor captopril and enalaprilat, the Phe-Ala-Pro analogue. We have compared these structures with the recently reported structure of a tACE-lisinopril complex [Natesh et al. (2003) Nature 421, 551-554]. The analyses reveal that all three inhibitors make direct interactions with the catalytic Zn(2+) ion at the active site of the enzyme: the thiol group of captopril and the carboxylate group of enalaprilat and lisinopril. Subtle differences are also observed at other regions of the binding pocket. These are compared with N-domain models and discussed with reference to published biochemical data. The chloride coordination geometries of the three structures are discussed and compared with other ACE analogues. It is anticipated that the molecular details provided by these structures will be used to improve the binding and/or the design of new, more potent domain-specific inhibitors of ACE that could serve as new generation antihypertensive drugs.     

Structural characterization of angiotensin I-converting enzyme in complex with a selenium analogue of captopril

Human somatic angiotensin I-converting enzyme (ACE), a zinc-dependent dipeptidyl carboxypeptidase, is central to the regulation of the renin-angiotensin aldosterone system. It is a well-known target for combating hypertension and related cardiovascular diseases. In a recent study by Bhuyan and Mugesh [Org. Biomol. Chem. (2011) 9, 1356-1365], it was shown that the selenium analogues of captopril (a well-known clinical inhibitor of ACE) not only inhibit ACE, but also protect against peroxynitrite-mediated nitration of peptides and proteins. Here, we report the crystal structures of human testis ACE (tACE) and a homologue of ACE, known as AnCE, from Drosophila melanogaster in complex with the most promising selenium analogue of captopril (SeCap) determined at 2.4 and 2.35 ? resolution, respectively. The inhibitor binds at the active site of tACE and AnCE in an analogous fashion to that observed for captopril and provide the first examples of a protein-selenolate interaction. These new structures of tACE-SeCap and AnCE-SeCap inhibitor complexes presented here provide important information for further exploration of zinc coordinating selenium-based ACE inhibitor pharmacophores with significant antioxidant activity.     

Structure and physiological importance of angiotensin converting enzyme domains

Somatic angiotensin converting enzyme (ACE) consists of two homologous catalytic domains (N- and C-domain), exhibiting different biochemical properties. The catalytically active ACE isoforms consisted of just one domain have been also detected in mammals. Substantial progress in ACE domain research was achieved during the last years, when their crystal structures were determined. The crystal structures of domains in complex with diverse potent ACE inhibitors provided new insights into structure-based differences of the domain active sites. Physiological functions of ACE are not limited by regulation of the cardiovascular system. Recent evidence suggests that the ACE domains may be also involved into control of different physiological functions. The C-terminal catalytic domain plays an important role in the regulation of blood pressure: it catalyzes angiotensin I cleavage in vivo. The N-domain contributes to the processing of other bioactive peptides for which it exhibits high affinity. The role of the N-domain is not ultimately associated with functioning of the rennin-angiotensin system and it contributes processing of other bioactive peptides for which it exhibits high affinity (goralatide, luliberin, enkephalin heptapeptide, beta-amyloid peptide). Domain-selective inhibitors selectively blocking either the N- or C-domain of ACE have been developed.     

The identity of the active site of oxalate decarboxylase and the importance of the stability of active-site lid conformations

Oxalate decarboxylase (EC 4.1.1.2) catalyses the conversion of oxalate into carbon dioxide and formate. It requires manganese and, uniquely, dioxygen for catalysis. It forms a homohexamer and each subunit contains two similar, but distinct, manganese sites termed sites 1 and 2. There is kinetic evidence that only site 1 is catalytically active and that site 2 is purely structural. However, the kinetics of enzymes with mutations in site 2 are often ambiguous and all mutant kinetics have been interpreted without structural information. Nine new site-directed mutants have been generated and four mutant crystal structures have now been solved. Most mutants targeted (i) the flexibility (T165P), (ii) favoured conformation (S161A, S164A, D297A or H299A) or (iii) presence (Delta162-163 or Delta162-164) of a lid associated with site 1. The kinetics of these mutants were consistent with only site 1 being catalytically active. This was particularly striking with D297A and H299A because they disrupted hydrogen bonds between the lid and a neighbouring subunit only when in the open conformation and were distant from site 2. These observations also provided the first evidence that the flexibility and stability of lid conformations are important in catalysis. The deletion of the lid to mimic the plant oxalate oxidase led to a loss of decarboxylase activity, but only a slight elevation in the oxalate oxidase side reaction, implying other changes are required to afford a reaction specificity switch. The four mutant crystal structures (R92A, E162A, Delta162-163 and S161A) strongly support the hypothesis that site 2 is purely structural.     

Novel mechanism of inhibition of human angiotensin-I-converting enzyme (ACE) by a highly specific phosphinic tripeptide

Human ACE (angiotensin-I-converting enzyme) has long been regarded as an excellent target for the treatment of hypertension and related cardiovascular diseases. Highly potent inhibitors have been developed and are extensively used in the clinic. To develop inhibitors with higher therapeutic efficacy and reduced side effects, recent efforts have been directed towards the discovery of compounds able to simultaneously block more than one zinc metallopeptidase (apart from ACE) involved in blood pressure regulation in humans, such as neprilysin and ECE-1 (endothelin-converting enzyme-1). In the present paper, we show the first structures of testis ACE [C-ACE, which is identical with the C-domain of somatic ACE and the dominant domain responsible for blood pressure regulation, at 1.97? (1 ?=0.1 nm)] and the N-domain of somatic ACE (N-ACE, at 2.15?) in complex with a highly potent and selective dual ACE/ECE-1 inhibitor. The structural determinants revealed unique features of the binding of two molecules of the dual inhibitor in the active site of C-ACE. In both structures, the first molecule is positioned in the obligatory binding site and has a bulky bicyclic P(1)' residue with the unusual R configuration which, surprisingly, is accommodated by the large S(2)' pocket. In the C-ACE complex, the isoxazole phenyl group of the second molecule makes strong pi-pi stacking interactions with the amino benzoyl group of the first molecule locking them in a 'hand-shake' conformation. These features, for the first time, highlight the unusual architecture and flexibility of the active site of C-ACE, which could be further utilized for structure-based design of new C-ACE or vasopeptidase inhibitors.     

Homologous substitution of ACE C-domain regions with N-domain sequences: effect on processing, shedding, and catalytic properties

Angiotensin-converting enzyme (ACE) exists as two isoforms: somatic ACE (sACE), comprised of two homologous N and C domains, and testis ACE (tACE), comprised of the C domain only. The N and C domains are both active, but show differences in substrate and inhibitor specificity. While both isoforms are shed from the cell surface via a sheddase-mediated cleavage, tACE is shed much more efficiently than sACE. To delineate the regions of tACE that are important in catalytic activity, intracellular processing, and regulated ectodomain shedding, regions of the tACE sequence were replaced with the corresponding N-domain sequence. The resultant chimeras C1-163Ndom-ACE, C417-579Ndom-ACE, and C583-623Ndom-ACE were processed to the cell surface of transfected Chinese hamster ovary (CHO) cells, and were cleaved at the identical site as that of tACE. They also showed acquisition of N-domain-like catalytic properties. Homology modelling of the chimeric proteins revealed structural changes in regions required for tACE-specific catalytic activity. In contrast, C164-416Ndom-ACE and C191-214Ndom-ACE demonstrated defective intracellular processing and were neither enzymatically active nor shed. Therefore, critical elements within region D164-V416 and more specifically I191-T214 are required for the processing, cell-surface targeting, and enzyme activity of tACE, and cannot be substituted for by the homologous N-domain sequence.     

Adaptability and flexibility of HIV-1 protease.

Even though more than 200 three-dimensional structures of HIV-1 protease complexed to a variety of inhibitors are available in the Protein Data Bank; very few structures of unliganded protein have been determined. We have recently solved structures of unliganded HIV-1 protease tethered dimer mutants to resolutions of 1.9 A and 2.1 A, and have found that the flaps assume closed-flap conformation even in the absence of any bound ligand. We report comparison of the unliganded closed-flap structure with structures of HIV-1 protease inhibitor complexes with a view to accurately identifying structural changes that the ligand can induce on binding to HIV-1 protease in the crystal. These studies reveal that the least flexible region present in the active site of HIV-1 protease need not also be the least adaptable to external stress, thus highlighting the conceptual difference between flexibility and adaptability of proteins in general.     

The role of glycosylation and domain interactions in the thermal stability of human angiotensin-converting enzyme

Abstract The N and C domains of somatic angiotensin-converting enzyme (sACE) differ in terms of their substrate specificity, inhibitor profiling, chloride dependency and thermal stability. The C domain is thermally less stable than sACE or the N domain. Since both domains are heavily glycosylated, the effect of glycosylation on their thermal stability was investigated by assessing their catalytic and physicochemical properties. Testis ACE (tACE) expressed in mammalian cells, mammalian cells in the presence of a glucosidase inhibitor and insect cells yielded proteins with altered catalytic and physicochemical properties, indicating that the more complex glycans confer greater thermal stabilization. Furthermore, a decrease in tACE and N-domain N-glycans using site-directed mutagenesis decreased their thermal stability, suggesting that certain N-glycans have an important effect on the protein's thermodynamic properties. Evaluation of the thermal stability of sACE domain swopover and domain duplication mutants, together with sACE expressed in insect cells, showed that the C domain contained in sACE is less dependent on glycosylation for thermal stabilization than a single C domain, indicating that stabilizing interactions between the two domains contribute to the thermal stability of sACE and are decreased in a C-domain-duplicating mutant.     

The structure of testis angiotensin-converting enzyme in complex with the C domain-specific inhibitor RXPA380

Angiotensin I-converting enzyme (ACE) is central to the regulation of the renin-angiotensin system and is a key therapeutic target for combating hypertension and related cardiovascular diseases. Currently available drugs bind both active sites of its two homologous domains, although it is now understood that these domains function differently in vivo. The recently solved crystal structures of both domains (N and C) open the door to new domain-specific inhibitor design, taking advantage of the differences between these two large active sites. Here we present the first crystal structure at a resolution of 2.25 A of testis ACE (identical to the C domain of somatic ACE) with the highly C-domain-specific phosphinic inhibitor, RXPA380. Testis ACE retains the same conformation as seen in previously determined inhibitor complexes, but the RXPA380 central backbone conformation is more similar to that seen for the inhibitor captopril than enalaprilat. The RXPA380 molecule occupies more subsites of the testis ACE active site than the previously determined inhibitors and possesses bulky moieties that extend into the S2' and S2 subsites. Thus the high affinity of RXPA380 for the testis ACE/somatic ACE C domain is explained by the interaction of these bulky moieties with residues unique to these domains, specifically Phe 391, Val 379, and Val 380, that are not found in the N domain. The characterization of the extended active site and the binding of a potent C-domain-selective inhibitor provide the first structural data for the design of truly domain-specific pharmacophores.     

A model of the ACE2 structure and function as a SARS-CoV receptor

The angiotensin-converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system and was very recently identified as a functional receptor for the SARS virus. The ACE2 sequence is similar (sequence identities 43% and 35%, and similarities 61% and 55%, respectively) to those of the testis-specific form of ACE (tACE) and the Drosophila homolog of ACE (AnCE). The high level of sequence similarity allowed us to build a robust homology model of the ACE2 structure with a root-mean-square deviation from the aligned crystal structures of tACE and AnCE less than 0.5A. A prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site. Negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (RBD) of the S-glycoprotein, which we recently identified [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Several distinct patches of hydrophobic residues at the ACE2 surface were noted at close proximity to the charged ridges that could contribute to binding. These results suggest a possible binding region for the SARS-CoV S-glycoprotein on ACE2 and could help in the design of experiments to further elucidate the structure and function of ACE2.     

High-Resolution Crystal Structures of Drosophila melanogaster Angiotensin-Converting Enzyme in Complex with Novel Inhibitors and Antihypertensive Drugs

Angiotensin I-converting enzyme (ACE), one of the central components of the renin-angiotensin system, is a key therapeutic target for the treatment of hypertension and cardiovascular disorders. Human somatic ACE (sACE) has two homologous domains (N and C). The N- and C-domain catalytic sites have different activities toward various substrates. Moreover, some of the undesirable side effects of the currently available and widely used ACE inhibitors may arise from their targeting both domains leading to defects in other pathways. In addition, structural studies have shown that although both these domains have much in common at the inhibitor binding site, there are significant differences and these are greater at the peptide binding sites than regions distal to the active site. As a model system, we have used an ACE homologue from Drosophila melanogaster (AnCE, a single domain protein with ACE activity) to study ACE inhibitor binding. In an extensive study, we present high-resolution structures for native AnCE and in complex with six known antihypertensive drugs, a novel C-domain sACE specific inhibitor, lisW-S, and two sACE domain-specific phosphinic peptidyl inhibitors, RXPA380 and RXP407 (i.e., nine structures). These structures show detailed binding features of the inhibitors and highlight subtle changes in the orientation of side chains at different binding pockets in the active site in comparison with the active site of N- and C-domains of sACE. This study provides information about the structure-activity relationships that could be utilized for designing new inhibitors with improved domain selectivity for sACE.     

The role of N-linked oligosaccharides in glycoprotein function.

N-linked glycoproteins include such biologically important molecules as cell-surface receptors, cell-adhesion molecules, immunoglobulins and other serum proteins, and tumor antigens. Investigating the role of carbohydrate in glycoprotein function has included the use of glycosylation inhibitors or site-directed mutagenesis of specific glycosylation sites to prevent the addition of carbohydrate, or glycosylation processing inhibitors or animal cell glycosylation mutants to alter carbohydrate structure. In some proteins, glycosylation plays an important role in recognition, while in others, it may stabilize and/or control the conformation of the protein. The cloning of genes in bacteria or lower eukaryotes--with the goal of producing biologically active proteins for biotechnological purposes--necessitates a better understanding of the role of specific carbohydrate structures.     

ACE for all – a molecular perspective

Angiotensin-I converting enzyme (ACE, EC 3.4.15.1) is a zinc dependent dipeptidyl carboxypeptidase with an essential role in mammalian blood pressure regulation as part of the renin-angiotensin aldosterone system (RAAS). As such, it has long been targeted in the treatment of hypertension through the use of ACE inhibitors. Although ACE has been studied since the 1950s, only recently have the full range of functions of this enzyme begun to truly be appreciated. ACE homologues have been found in a host of other organisms, and are now known to be conserved in insects. Insect ACE homologues typically share over 30 % amino acid sequence identity with human ACE. Given that insects lack a mammalian type circulatory system, they must have crucial roles in other physiological processes. The first ACE crystal structures were reported during the last decade and have enabled these enzymes to be studied from an entirely different perspective. Here we review many of these key developments and the implications that they have had on our understanding of the diverse functions of these enzymes. Specifically, we consider how structural information is being used in the design of a new generation of ACE inhibitors with increased specificity, and how the structures of ACE homologues are related to their functions. The Anopheles gambiae genome is predicted to code for ten ACE homologues, more than any genome studied so far. We have modelled the active sites of some of these as yet uncharacterised enzymes to try and infer more about their potential roles at the molecular level.     

The N Domain of Human Angiotensin-I-converting Enzyme: THE ROLE OF N-GLYCOSYLATION AND THE CRYSTAL STRUCTURE IN COMPLEX WITH AN N DOMAIN-SPECIFIC PHOSPHINIC INHIBITOR, RXP407*

Angiotensin-I-converting enzyme (ACE) plays a critical role in the regulation of blood pressure through its central role in the renin-angiotensin and kallikrein-kinin systems. ACE contains two domains, the N and C domains, both of which are heavily glycosylated. Structural studies of ACE have been fraught with severe difficulties because of surface glycosylation of the protein. In order to investigate the role of glycosylation in the N domain and to create suitable forms for crystallization, we have investigated the importance of the 10 potential N-linked glycan sites using enzymatic deglycosylation, limited proteolysis, and mass spectrometry. A number of glycosylation mutants were generated via site-directed mutagenesis, expressed in CHO cells, and analyzed for enzymatic activity and thermal stability. At least eight of 10 of the potential glycan sites are glycosylated; three C-terminal sites were sufficient for expression of active N domain, whereas two N-terminal sites are important for its thermal stability. The minimally glycosylated Ndom389 construct was highly suitable for crystallization studies. The structure in the presence of an N domain-selective phosphinic inhibitor RXP407 was determined to 2.0 Å resolution. The Ndom389 structure revealed a hinge region that may contribute to the breathing motion proposed for substrate binding.     

A crucial role in fertility for the oyster angiotensin-converting enzyme orthologue CgACE

Angiotensin-converting enzyme (ACE) is a highly conserved metallopeptidase. In mammals, the somatic isoform governs blood pressure whereas the germinal isoform (tACE) is required for fertility. In Ecdysozoans, ACE-like enzymes are implicated in reproduction. Despite ACE orthologues being present from bacteria to humans, their function(s) remain(s) unknown in distant organisms such as Lophotrochozoans. In silico analysis of an oyster (Crassostrea gigas) EST library suggested the presence of an ACE orthologue in molluscs. Primer walking and 5'-RACE revealed that the 1.9 kb cDNA encodes CgACE, a 632 amino acid protein displaying a conserved single active site and a putative C-terminal transmembrane anchor, thus resembling human tACE, as supported by molecular modelling. FRET activity assays and Maldi-TOF spectrometry indicated that CgACE is a functional dipeptidyl-carboxypeptidase which is active on Angiotensin I and sensitive to ACE inhibitors and chloride ion concentration. Immunocytochemistry revealed that, as its human counterpart, recombinant CgACE is synthesised as a transmembrane enzyme. RT-qPCR, in-situ hybridization and immunohistochemistry shed light on a tissue, and development stage, specific expression pattern for CgACE, which is increased in the gonad during spermatogenesis. The use of ACE inhibitors in vivo indicates that the dipeptidase activity of CgACE is crucial for the oyster fertilization. Our study demonstrates that a transmembrane active ACE is present in the oyster Crassostrea gigas, and for the first time ascribes a functional role for ACE in Lophotrochozoans. Its biological function in reproduction is conserved from molluscs to humans, a finding of particular evolutionary interest especially since oysters represent the most important aquaculture resource worldwide.     

Structural diversity of angiotensin-converting enzyme

The crystal structure of a Drosophila angiotensin-converting enzyme (ANCE) has recently been solved, revealing features important for the binding of ACE inhibitors and allowing molecular comparisons with the structure of human testicular angiotensin-converting enzyme (tACE). ACER is a second Drosophila ACE that displays both common and distinctive properties. Here we report further functional differences between ANCE and ACER and have constructed a homology model of ACER to help explain these. The model predicts a lack of the Cl(-)-binding sites, and therefore the strong activation of ACER activity towards enkephalinamide peptides by NaCl suggests alternative sites for Cl(-) binding. There is a marked difference in the electrostatic charge of the substrate channel between ANCE and ACER, which may explain why the electropositive peptide, MKRSRGPSPRR, is cleaved efficiently by ANCE with a low K(m), but does not bind to ACER. Bradykinin (BK) peptides are excellent ANCE substrates. Models of BK docked in the substrate channel suggest that the peptide adopts an N-terminal beta-turn, permitting a tight fit of the peptide in the substrate channel. This, together with ionic interactions between the guanidino group of Arg9 of BK and the side chains of Asp360 and Glu150 in the S(2)' pocket, are possible reasons for the high-affinity binding of BK. The replacement of Asp360 with a histidine in ACER would explain the higher K(m) recorded for the hydrolysis of BK peptides by this enzyme. Other differences in the S(2)' site of ANCE and ACER also explain the selectivity of RXPA380, a selective inhibitor of human C-domain ACE, which also preferentially inhibits ACER. These structural and enzymatic studies provide insight into the molecular basis for the distinctive enzymatic features of ANCE and ACER.     

Functional conservation of the active sites of human and Drosophila angiotensin I-converting enzyme

Human somatic angiotensin I-converting enzyme (sACE) has two active sites present in two homologous protein domains, resulting from a tandem gene duplication. It has been proposed that the N- and C-terminal active sites can have specific in vivo roles. In Drosophila melanogaster, Ance and Acercode for two ACE-like single-domain proteins, also predicted to have distinct physiological roles. We have investigated the relationship of Ance and Acer to the N- and C-domains of human sACE by genomic sequence analysis and by using domain-selective inhibitors, including RXP 407, a selective inhibitor of the human N-domain. These phosphinic peptides were potent inhibitors of Acer, but not of Ance. We conclude that the active sites of the N-domain and of Acer share structural features that permit the binding of the unusual RXP407 inhibitor and the hydrolysis of a broader range of peptide structures. In comparison, Ance, like the human C-domain of ACE, displays greater inhibitor selectivity. From the analysis of the published sequence of the Adh region of Drosophila chromosome 2, which carries Ance, Acer, and four additional ACE-like genes, we also suggest that this functional conservation is reflected in an ancestral gene structure identifiable in both protostome and deuterostome lineages and that the duplication seen in vertebrate genomes predates the divergence of these lineages. The conservation of ACE enzymes with distinct active sites in the evolution of both vertebrate and invertebrate species provides further evidence that these two kinds of active sites have different physiological functions.