RESUMEN
Iridoid synthases belong to the family of short-chain dehydrogenase/reductase involved in the biosynthesis of iridoids. Despite having high sequence and structural homology with progesterone 5ß-reductase, these enzymes exhibit differential substrate specificities. Previously, two loops, L1 and L2 at substrate-binding pocket, were suggested to be involved in generating substrate specificity. However, the structural basis of specificity determinants was elusive. Here, combining sequence and structural analysis, site-directed mutagenesis, and molecular dynamics simulations, we have shown that iridoid synthase contains two channels for substrate entry whose geometries are altered by L1-L2 dynamics, primarily orchestrated by interactions of residues Glu161 and Gly162 of L1 and Asn358 of L2. A complex interplay of these interactions confer the substrate specificity to the enzyme.
Asunto(s)
Iridoides/farmacocinética , Simulación de Dinámica Molecular , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Sitios de Unión/genética , Cristalografía por Rayos X , Iridoides/química , Iridoides/metabolismo , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Oxidorreductasas/genética , Progesterona Reductasa/química , Progesterona Reductasa/genética , Progesterona Reductasa/metabolismo , Estructura Secundaria de Proteína/fisiología , Especificidad por Sustrato/genéticaRESUMEN
Search vectors composed of Gly, Ala, Arg, and Pro (GARP) residues retrieve 98% of each of the ribosomal proteins in prokaryotic species with no false hits. Different combinations of G, A, R and P and insertions differentiate each ribosomal protein from all others. Amino acids in two sequence positions separate Gram+ from Gram- bacteria. Specific residues separate proteins of cyanobacteria and chloroplasts from all other species. Structural information played an essential role in developing a GARP based technique to achieve perfect sequence alignment. It is possible to understand why GARP residues are 100% conserved in specific positions in families of proteins present in all species.
Asunto(s)
Proteínas Bacterianas/genética , Evolución Molecular , Proteínas Ribosómicas/genética , Secuencia de Aminoácidos , Aminoácidos/genética , Cloroplastos/metabolismo , Secuencia Conservada , Cianobacterias/genética , Cianobacterias/metabolismo , Datos de Secuencia Molecular , Alineación de SecuenciaRESUMEN
The characteristic oxidation or reduction reaction mechanisms of short-chain oxidoreductase (SCOR) enzymes involve a highly conserved Asp-Ser-Tyr-Lys catalytic tetrad. The SCOR enzyme Q9HYA2 from the pathogenic bacterium Pseudomonas aeruginosa was recognized to possess an atypical catalytic tetrad composed of Lys118-Ser146-Thr159-Arg163. Orthologs of Q9HYA2 containing the unusual catalytic tetrad along with conserved substrate and cofactor recognition residues were identified in 27 additional species, the majority of which are bacterial pathogens. However, this atypical catalytic tetrad was not represented within the Protein Data Bank. The crystal structures of unligated and NADPH-complexed Q9HYA2 were determined at 2.3 A resolution. Structural alignment to a polyketide ketoreductase (KR), a typical SCOR, demonstrated that Q9HYA2's Lys118, Ser146, and Arg163 superimposed upon the KR's catalytic Asp114, Ser144, and Lys161, respectively. However, only the backbone of Q9HYA2's Thr159 overlapped KR's catalytic Tyr157. The Thr159 hydroxyl in apo Q9HYA2 is poorly positioned for participating in catalysis. In the Q9HYA2-NADPH complex, the Thr159 side chain was modeled in two alternate rotamers, one of which is positioned to interact with other members of the tetrad and the bound cofactor. A chloride ion is bound at the position normally occupied by the catalytic tyrosine hydroxyl. The putative active site of Q9HYA2 contains a chemical moiety at each catalytically important position of a typical SCOR enzyme. This is the first observation of a SCOR protein with this alternate catalytic center that includes threonine replacing the catalytic tyrosine and an ion replacing the hydroxyl moiety of the catalytic tyrosine.
Asunto(s)
Proteínas Bacterianas/química , Oxidorreductasas/química , Pseudomonas aeruginosa/enzimología , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Secuencia Conservada , Cristalografía por Rayos X , Bases de Datos de Proteínas , Escherichia coli/genética , Modelos Moleculares , Datos de Secuencia Molecular , NADP/química , NADP/metabolismo , Oxidorreductasas/genética , Oxidorreductasas/metabolismo , Unión Proteica , Pseudomonas aeruginosa/genética , Alineación de SecuenciaRESUMEN
We have identified a highly conserved fingerprint of 40 residues in the TGYK subfamily of the short-chain oxidoreductase enzymes. The TGYK subfamily is defined by the presence of an N-terminal TGxxxGxG motif and a catalytic YxxxK motif. This subfamily contains more than 12,000 members, with individual members displaying unique substrate specificities. The 40 fingerprint residues are critical to catalysis, cofactor binding, protein folding, and oligomerization but are substrate independent. Their conservation provides critical insight into evolution of the folding and function of TGYK enzymes. Substrate specificity is determined by distinct combinations of residues in three flexible loops that make up the substrate-binding pocket. Here, we report the structure determinations of the TGYK enzyme A3DFK9 from Clostridium thermocellum in its apo form and with bound NAD(+) cofactor. The function of this protein is unknown, but our analysis of the substrate-binding loops putatively identifies A3DFK9 as a carbohydrate or polyalcohol metabolizing enzyme. C. thermocellum has potential commercial applications because of its ability to convert biomaterial into ethanol. A3DFK9 contains 31 of the 40 TGYK subfamily fingerprint residues. The most significant variations are the substitution of a cysteine (Cys84) for a highly conserved glycine within a characteristic VNNAG motif, and the substitution of a glycine (Gly106) for a highly conserved asparagine residue at a helical kink. Both of these variations occur at positions typically participating in the formation of a catalytically important proton transfer network. An alternate means of stabilizing this proton wire was observed in the A3DFK9 crystal structures.
Asunto(s)
Clostridium thermocellum/enzimología , Oxidorreductasas/química , Oxidorreductasas/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Cristalografía por Rayos X , Modelos Moleculares , Mapeo Peptídico/métodos , Unión Proteica , Agua/químicaRESUMEN
beta-ketoacyl (acyl carrier protein) reductase (beta-k-ACPR) enzymes are essential to fatty acid synthesis in bacteria. The analyses revealed the most primitive member of the beta-k-ACPRs family was a NADP reductase where NADP was recognised by a Thr residue in the beta2alpha3 turn. Aromatic residue stacking at the dimer interface and a previously undetected conserved sequence at the C-terminus, stabilise the oligomeric assembly of these proteins. Our analysis indicates that the primordial members of the beta-k-ACPR family probably arose in the alpha-proteobacteria and are characterised by the presence of multiple open reading frames and an extreme codon and amino acid bias.
Asunto(s)
Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/genética , Evolución Molecular , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Sitios de Unión , Variación Genética , Modelos Moleculares , Sistemas de Lectura Abierta , Conformación Proteica , Pliegue de ProteínaRESUMEN
The human type 1 (placenta, breast tumors) and type 2 (gonads, adrenals) isoforms of 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) are key enzymes in steroidogenic pathways leading to the production of all active steroid hormones. Kinetic analyses of purified 3beta-HSD1 show that the Michaelis-Menten constants (Km) for substrates and cofactor are decreased dramatically (three- to eight-fold) by the addition of beta-mercaptoethanol (BME), which suggest that a disulfide bond may be critical to ligand utilization. Western immunoblots and SDS-PAGE of purified 3beta-HSD1 in the presence or absence of BME showed a lack of intersubunit disulfide bonds in the dimeric enzyme. The Rossmann-fold domain of 3beta-HSD1 contains two Cys residues, Cys72 and Cys111, which are capable of forming an intrasubunit disulfide bond based on their proximity in our structural model. Our structural model also predicts that Cys83 may affect the orientation of substrate and cofactor. To test these predictions, the C72S, C72F, C111S, C111A, C83S and C83A mutants of 3beta-HSD1 were produced, expressed, and purified. BME failed to diminish the Km values of substrate and cofactor for C72S, C72F, C111S and C111A but produced a 2.5 decrease in Km values for C83A ligands similar to wild-type 3beta-HSD. Thus, our results support the presence of an intrasubunit disulfide bond between Cys72 and Cys111 that participates in the tertiary structure of the Rossmann-fold domain. Although C83S had no enzyme activity, the C83A mutant enzyme exhibited two- to five-fold higher Km values for substrate and cofactor but had similar K(cat) values compared to wild-type 3beta-HSD. These data characterize the roles of Cys residues in 3beta-HSD and validate the predictions of our structural model.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , Coenzimas/metabolismo , Cisteína/metabolismo , Modelos Moleculares , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Sitios de Unión , Cisteína/genética , Humanos , Mutagénesis Sitio-Dirigida , Conformación Proteica , Especificidad por SustratoRESUMEN
The gene product of fabG from Aquifex aeolicus has been heterologously expressed in Escherichia coli. Purification of the protein took place using anion-exchange and size-exclusion chromatography and the protein was then crystallized. Diffraction data were collected to a maximum resolution of 1.8 A and the initial phases were determined by molecular replacement. The A. aeolicus FabG protein is a putative beta-ketoacyl-acyl carrier protein reductase. Structure-function studies of this protein are being performed as part of a larger project investigating naturally occurring deviations from highly conserved residues within the short-chain oxidoreductase (SCOR) family.
Asunto(s)
Oxidorreductasas de Alcohol/química , Bacterias/enzimología , Oxidorreductasas de Alcohol/genética , Bacterias/genética , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , Cristalización , Cristalografía por Rayos X , Electroforesis en Gel de Poliacrilamida , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismoRESUMEN
Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.
Asunto(s)
Complejos Multienzimáticos/metabolismo , Progesterona Reductasa/metabolismo , Proteómica , Esteroide Isomerasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Catálisis , Cartilla de ADN , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , Progesterona Reductasa/química , Progesterona Reductasa/genética , Homología de Secuencia de Aminoácido , Esteroide Isomerasas/química , Esteroide Isomerasas/genética , Especificidad por SustratoRESUMEN
The Escherichia coli enterobactin synthetic cluster is composed of six proteins, EntA-EntF, that form the enterobactin molecule from three serine molecules and three molecules of 2,3-dihydroxybenzoic acid (DHB). EntC, EntB and EntA catalyze the three-step synthesis of DHB from chorismate. EntA is a member of the short-chain oxidoreductase (SCOR) family of proteins and catalyzes the final step in DHB synthesis, the NAD+-dependent oxidation of 2,3-dihydro-2,3-dihydroxybenzoic acid to DHB. The structure of EntA has been determined by multi-wavelength anomalous dispersion methods. Here, the 2.0 A crystal structure of EntA in the unliganded form is presented. Analysis of the structure in light of recent structural and bioinformatic analysis of other members of the SCOR family provides insight into the residues involved in cofactor and substrate binding.
Asunto(s)
Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Hidroxibenzoatos/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/química , Sitios de Unión , Dimerización , Enterobactina/química , Enterobactina/metabolismo , Proteínas de Escherichia coli/metabolismo , Modelos Moleculares , Estructura Molecular , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/fisiología , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
The short-chain oxidoreductase (SCOR) family of enzymes includes over 6000 members, extending from bacteria and archaea to humans. Nucleic acid sequence analysis reveals that significant numbers of these genes are remarkably free of stopcodons in reading frames other than the coding frame, including those on the antisense strand. The genes from this subset also use almost entirely the GC-rich half of the 64 codons. Analysis of a million hypothetical genes having random nucleotide composition shows that the percentage of SCOR genes having multiple open reading frames exceeds random by a factor of as much as 1 x 10(6). Nevertheless, screening the content of the SWISS-PROT TrEMBL database reveals that 15% of all genes contain multiple open reading frames. The SCOR genes having multiple open reading frames and a GC-rich coding bias exhibit a similar GC bias in the nucleotide triple composition of their DNA. This bias is not correlated with the GC content of the species in which the SCOR genes are found. One possible explanation for the conservation of multiple open reading frames and extreme bias in nucleic acid composition in the family of Rossman folds is that the primordial member of this family was encoded early using only very stable GC-rich DNA and that evolution proceeded with extremely limited introduction of any codons having two or more adenine or thymine nucleotides. These and other data suggest that the SCOR family of enzymes may even have diverged from a common ancestor before most of the AT-rich half of the genetic code was fully defined.
Asunto(s)
Codón/genética , Evolución Molecular , Código Genético , Sistemas de Lectura Abierta , Oxidorreductasas/química , Oxidorreductasas/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , ADN sin Sentido/química , ADN sin Sentido/genética , Oxidorreductasas/genética , Programas InformáticosRESUMEN
Significant sequence homology has been detected between prokaryotic beta-ketoacyl-[acyl carrier protein] reductases (BKR) and eukaryotic 17beta-hydroxysteroid dehydrogenases type 8 (17beta-HSD_8). Three-dimensional models of ternary complexes of human 17beta-HSD_8 with NAD cofactor and two chemically distinct substrates, the BKR substrate {CH3-(CH2)(12)-CO-CH(2)-CO-S-[ACP]} and the HSD substrate {estradiol} have been constructed (the atomic coordinates are available on request; e-mail: pletnev@hwi.buffalo.edu). The more extensive and specific interactions of 17beta-HSD_8 with the BKR substrate compared to interactions with estradiol raise a serious question about the enzyme's primary function in vivo and suggest that it is likely to be involved in the regulation of fatty acid metabolism rather than in the steroid-dependent activity that has been demonstrated in vitro.
Asunto(s)
Oxidorreductasas/metabolismo , Proteínas/metabolismo , Proteómica/métodos , 3-Oxoacil-(Proteína Transportadora de Acil) Reductasa , Oxidorreductasas de Alcohol/química , Oxidorreductasas de Alcohol/metabolismo , Secuencia de Aminoácidos , Animales , Brassica napus , Biología Computacional , Estradiol/química , Estradiol/metabolismo , Humanos , Isoenzimas/metabolismo , Ratones , Modelos Moleculares , Conformación Molecular , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , Oxidorreductasas/química , Proteínas/química , Alineación de Secuencia , Especificidad por SustratoRESUMEN
The short-chain oxidoreductase (SCOR) family of enzymes includes over 6,000 members identified in sequenced genomes. Of these enzymes, approximately 300 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are steroid dehydrogenases involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown and to determine their three-dimensional structure and mechanism of action. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30-40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including cofactor preference, catalytic residues, and substrate specificity. Human type 1 3beta-hydroxysteroid dehydrogenase isomerase (3beta-HSDI) has 30% sequence identity with a human UDP galactose 4-epimerase (UDPGE), a SCOR family enzyme for which an X-ray structure has been reported. Both UDPGE and 3-HSDI appear to trace their origins back to bacterial 3alpha,20beta-HSD. Combining three-dimensional structural information and sequence data on the 3alpha,20beta-HSD, UDPGE, and 3beta-HSDI subfamilies with mutational analysis, we were able to identify the residues critical to the dehydrogenase function of 3-HSDI. We also identified the residues most probably responsible for the isomerase activity of 3beta-HSDI. We test our predictions by specific mutations based on sequence analysis and our structure-based model.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/genética , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Análisis Mutacional de ADN/métodos , Glycyrrhiza , Humanos , Hidroxiesteroide Deshidrogenasas/genética , Hidroxiesteroide Deshidrogenasas/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Proteómica , Análisis de Secuencia de Proteína , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The dominant role of long-range electrostatic interatomic interactions in nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NAD/NADP) cofactor recognition has been shown for enzymes of the short-chain oxidoreductase (SCOR) family. An estimation of cofactor preference based only on the contribution of the electrostatic energy term to the total energy of enzyme-cofactor interaction has been tested for approximately 40 known three-dimensional (3D) crystal complexes and approximately 330 SCOR enzymes, with cofactor preference predicted by the presence of Asp or Arg recognition residues at specific 3D positions in the beta2alpha3 loop (Duax et al., Proteins 2003;53:931-943). The results obtained were found to be consistent with approximately 90% reliable cofactor assignments for those subsets. The procedure was then applied to approximately 170 SCOR enzymes with completely uncertain NAD/NADP dependence, due to the lack of Asp and Arg marker residues. The proposed 3D electrostatic approach for cofactor assignment ("3D_DeltaE(el)") has been implemented in an automatic screening procedure, and together with the use of marker residues proposed earlier (Duax et al., Proteins 2003;53:931-943), increases the level of reliable predictions for the putative SCORs from approximately 70% to approximately 90%. It is expected to be applicable for any NAD/NADP-dependent enzyme subset having at least 25-30% sequence identity, with at least one enzyme of known 3D crystal structure.
Asunto(s)
Oxidorreductasas/química , Oxidorreductasas/metabolismo , Proteómica/métodos , Animales , Proteínas Bacterianas/química , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Humanos , Enlace de Hidrógeno , Proteínas de Insectos/química , Proteínas de Insectos/metabolismo , Ratones , Modelos Moleculares , NAD/metabolismo , NADP/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Proteínas Protozoarias/química , Proteínas Protozoarias/metabolismo , Ratas , Especificidad por SustratoRESUMEN
Separate genes encode the human type 1 (placenta, breast tumors, other peripheral tissues) and type 2 (gonad, adrenal) isoforms of 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD1, 3 beta-HSD2). Mutagenesis of 3 beta-HSD1 produced the Y154F, H156Y and K158Q mutant enzymes in the probable Y(154)-P-H(156)-S-K(158) catalytic motif. The H(156)Y mutant of the 3 beta-HSD1 created a chimera of the 3 beta-HSD2 motif (Y(154)-P-Y(156)-S-K(158)) in 3 beta-HSD1. The D241N, D257L, D258L and D265N mutants are in the potential isomerase site of the 3 beta-HSD1 enzyme. Homology modeling with UDP-galactose-4-epimerase predicted that Asp(36) in the Rossmann-fold domain is responsible for the NAD(H) specificity of human 3 beta-HSD1, and our D36A/K37R mutant tested that assignment. The H(156)Y mutant of the 3 beta-HSD1 enzyme shifted the substrate (DHEA) kinetics to the 14-fold higher K(m) value measured for the 3 beta-HSD2 activity. From Dixon analysis, epostane inhibited the 3 beta-HSD1 activity with 17-fold greater affinity compared to 3 beta-HSD2 and H(156)Y. The mutants of Tyr(154) and Lys(158) exhibited no dehydrogenase activity and appear to be catalytic 3 beta-HSD residues. The D257L and D258L mutations eliminated isomerase activity, suggesting that Asp(257) or Asp(258) may be catalytic residues for isomerase activity. The D36A/K37R mutant shifted the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H). In addition to characterizing catalytic residues, these studies have identified the structural basis (His(156)) for an exploitable difference in the substrate and inhibition kinetics of 3 beta-HSD1 and 3 beta-HSD2. Hence, it may be possible to selectively inhibit human 3 beta-HSD1 to slow the growth of hormone-sensitive breast tumor cells and control placental steroidogenesis near term to prevent premature labor.
Asunto(s)
3-Hidroxiesteroide Deshidrogenasas/química , 3-Hidroxiesteroide Deshidrogenasas/metabolismo , Mutación , NADP/metabolismo , NAD/metabolismo , 3-Hidroxiesteroide Deshidrogenasas/genética , Secuencia de Aminoácidos , Humanos , Concentración de Iones de Hidrógeno , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Homología de Secuencia de Aminoácido , Relación Estructura-ActividadRESUMEN
The short-chain oxidoreductase (SCOR) family of enzymes includes over 2000 members identified in sequenced genomes. Of these enzymes, approximately 200 have been characterized functionally, and the three-dimensional crystal structures of approximately 40 have been reported. Since some SCOR enzymes are involved in hypertension, diabetes, breast cancer, and polycystic kidney disease, it is important to characterize the other members of the family for which the biological functions are currently unknown. Although the SCOR family appears to have only a single fully conserved residue, it was possible, using bioinformatics methods, to determine characteristic fingerprints composed of 30-40 residues that are conserved at the 70% or greater level in SCOR subgroups. These fingerprints permit reliable prediction of several important structure-function features including NAD/NADP cofactor preference. For example, the correlation of aspartate or arginine residues with NAD or NADP binding, respectively, predicts the cofactor preference of more than 70% of the SCOR proteins with unknown function. The analysis of conserved residues surrounding the cofactor has revealed the presence of previously undetected CH em leader O hydrogen bonds in the majority of the SCOR crystal structures, predicts the presence of similar hydrogen bonds in 90% of the SCOR proteins of unknown function, and suggests that these hydrogen bonds may play a critical role in the catalytic functions of these enzymes.
Asunto(s)
Oxidorreductasas/química , Oxidorreductasas/genética , Proteómica/métodos , Secuencia de Aminoácidos , Sitios de Unión/genética , Dominio Catalítico , Enlace de Hidrógeno , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , NAD/química , NAD/metabolismo , NADP/química , NADP/metabolismo , Niacinamida/química , Niacinamida/metabolismo , Oxidorreductasas/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Agua/químicaRESUMEN
Human type 1 3 beta-hydroxysteroid dehydrogenase/isomerase (3 beta-HSD/isomerase) catalyzes the two sequential enzyme reactions on a single protein that converts dehydroepiandrosterone or pregnenolone to androstenedione or progesterone, respectively, in placenta, mammary gland, breast tumors, prostate, prostate tumors, and other peripheral tissues. Our earlier studies show that the two enzyme reactions are linked by the coenzyme product, NADH, of the 3 beta-HSD activity. NADH activates the isomerase activity by inducing a time-dependent conformational change in the enzyme protein. The current study tested the hypothesis that the 3 beta-HSD and isomerase activities shared a common coenzyme domain, and it characterized key amino acids that participated in coenzyme binding and the isomerase reaction. Homology modeling with UDP-galactose-4-epimerase predicts that Asp36 is responsible for the NAD(H) specificity of human 3 beta-HSD/isomerase and identifies the Rossmann-fold coenzyme domain at the amino terminus. The D36A/K37R mutant in the potential coenzyme domain and the D241N, D257L, D258L, and D265N mutants in the potential isomerase domain (previously identified by affinity labeling) were created, expressed, and purified. The D36A/K37R mutant shifts the cofactor preference of both 3 beta-HSD and isomerase from NAD(H) to NADP(H), which shows that the two activities utilize a common coenzyme domain. The D257L and D258L mutations eliminate isomerase activity, whereas the D241N and D265N mutants have nearly full isomerase activity. Kinetic analyses and pH dependence studies showed that either Asp257 or Asp258 plays a catalytic role in the isomerization reaction. These observations further characterize the structure/function relationships of human 3 beta-HSD/isomerase and bring us closer to the goal of selectively inhibiting the type 1 enzyme in placenta (to control the timing of labor) or in hormone-sensitive breast tumors (to slow their growth).
Asunto(s)
Coenzimas/metabolismo , Complejos Multienzimáticos/química , Complejos Multienzimáticos/metabolismo , NAD/metabolismo , Progesterona Reductasa/química , Progesterona Reductasa/metabolismo , Esteroide Isomerasas/química , Esteroide Isomerasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Arginina , Ácido Aspártico , Femenino , Humanos , Cinética , Masculino , Modelos Moleculares , Datos de Secuencia Molecular , Complejos Multienzimáticos/genética , Mutagénesis Sitio-Dirigida , NAD/química , Placenta/enzimología , Embarazo , Progesterona Reductasa/genética , Conformación Proteica , Proteínas Recombinantes/metabolismo , Esteroide Isomerasas/genética , Especificidad por Sustrato , Células Tumorales CultivadasRESUMEN
Structural homology of class II aminoacyl-tRNA synthetases to the HSP70 family and the existence of a gene whose sense and antisense strands code for a dehydrogenase and an HSP70 chaperonin justify reconsideration of a possible sense-antisense ancestry for the two synthetase classes.