Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.1.1.3 (HSD)
3,464 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Homogeneous 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD, EC 1.1.1.50) of rat liver cytosol is potently inhibited at its active site by nonsteroidal anti-inflammatory drugs (NSAIDs). Using 3 alpha-bromoacetoxy-5 alpha-androstan-17-one (BrAnd, a substrate analog) and 11 alpha-bromoacetoxyprogesterone (Br11P, a glucocorticoid analog) as affinity-labeling agents, kinetic evidence was obtained that these agents alkylate this site. Inactivation of 3 alpha-HSD with either [14C]BrAnd or [14C] Br11P led to the incorporation of 1 mol of affinity-labeling agent per enzyme monomer. Complete acid hydrolysis of 3 alpha-HSD radiolabeled with either agent followed by amino acid analysis led to the identification of [14C]carboxymethylcysteine indicating that [14C]BrAnd and [14C]Br11P covalently tag discrete reactive cysteine(s) at the enzyme active site. Trypsin digestion of [14C]BrAnd-inactivated 3 alpha-HSD followed by peptide mapping led to the purification of a single radiolabeled peptide (3A1) which gave the following sequence: H2N-Ser-Ile-Gly-Val-Ser-Asn-Phe-Asn-X-Arg-CO2H. Identical experiments on [14C] Br11P-inactivated 3 alpha-HSD led to the purification of three radiolabeled peptides (11P1-11P3). The major radiolabeled peptide (11P1) had an identical sequence to 3A1 which was tagged with [14C]BrAnd. The minor radiolabeled peptides had the following sequences: H2N-Ser-Lys-Asp-Ile-Ile-Leu-Val-Ser-Tyr-X-Thr-Leu-Gly-Ser-Ser-Arg-CO2H (11P2) and H2N-Ser-Pro-Val-Leu-Leu-Asp-Asp-Pro-Val-Leu-X-Ala-Ile-Ala-Lys-CO2H (11P3). In each peptide group X was identified as carboxymethylcysteine. Alignment of the peptide sequences with the primary structure of 3 alpha-HSD, deduced from its cDNA clone, assigned peptide 11P1 to residues 162-171, peptide 11P2 to residues 208-223, and peptide 11P3 to residues 232-246 of the amino acid sequence. The reactive cysteines correspond to Cys170, Cys217, and Cys242. We propose that Cys170 labeled by BrAnd may lie within the catalytic pocket of the enzyme. By contrast the 11 alpha-bromoacetoxy group in Br11P labeled several reactive cysteines which may be involved in the binding of glucocorticoids and NSAIDs.
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PMID:Affinity labeling of 3 alpha-hydroxysteroid dehydrogenase with 3 alpha-bromoacetoxyandrosterone and 11 alpha-bromoacetoxyprogesterone. Isolation and sequence of active site peptides containing reactive cysteines; sequence confirmation using nucleotide sequence from a cDNA clone. 202 97

The presence of a single aspartokinase was demonstrated in Rhodospirillum tenue. The enzyme has been purified about 60-fold. No physical association exists in this species between aspartokinase and homoserine dehydrogenase. The general properties of the enzyme are described. Inhibition by l-lysine, by l-threonine, and concerted inhibition by these two end products are regulatory characters which have also been found in many other species. In R. tenue, aspartokinase is also subject to a hitherto not encountered type of concerted feedback inhibition, by l-threonine plus l-methionine. The inhibition caused by lysine can be reversed either by glycine, l-isoleucine, l-methionine, or l-phenylalanine. The concerted inhibition by lysine plus threonine is reversed by glycine, l-isoleucine, or l-phenylalanine, but not by l-methionine, which exerts in conjunction with threonine the independent concerted inhibition referred to above. Addition of single or several metabolites to cultures of R. tenue caused inhibition of growth and reversal of growth inhibition, compatible with the effects observed in vitro on aspartokinase activity. The regulation of this enzyme in relation to that of other bacterial aspartokinases is discussed.
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PMID:Concerted feedback inhibition of the aspartokinase of Rhodospirillum tenue by threonine and methionine: a novel pattern. 507 65

Bovine liver NADP(+)-dependent dihydrodiol dehydrogenase (DD3) is extremely sensitive to SH reagents such as N-ethylmaleimide (NEM) and 5,5'-dithiobis(2-nitrobenzoic acid). NEM produced time- and concentration-dependent inactivation of DD3 in a pseudo-first-order reaction manner. This inactivation was prevented by NADP+, 3-acetylpyridine-adenine dinucleotide phosphate, 2',5'-ADP and 2'-AMP but not by substrates, NAD+, nicotinamide mononucleotide or 5'-ADP.DD3 was absorbed by an affinity column of thiopropyl-Sepharose 6B, but enzyme incubated with both NEM and NADP+ was not. Moreover, one [14C]NEM molecule was incorporated into a cysteine of DD3 in the presence, and two cysteines of DD3 in the absence, of NADP+. These results suggested that two cysteine residues were modified per enzyme molecule by NEM, one was protected by NADP+ and the other had no significant function for the enzyme activity. Two radiolabelled peptides (P1 and P2) produced by the digestion with lysyl endopeptidase of [14C]NEM-modified DD3 could be separated by reverse-phase HPLC. P1, which was radiolabelled by [14C]NEM only in the absence of NADP+, showed the following sequence; H2N-Tyr-Lys-Pro-Val-Xaa-Asn-Gln-Val-Glu- NEM.Cys-His-Pro-Tyr-Phe-Asn-Gln-Ser-Lys-COOH (Xaa indicates a possible cysteine residue). This sequence was very similar to that of rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD/DD) (residues 184 to 201) and was also highly conserved in the aldo-keto reductase superfamily. The sequence of P2, which had radioactivity in both the absence and presence of NADP+, also contained an NEM-modified cysteine and was similar in sequence to the regions located in loop A of rat 3 alpha-HSD/DD. The present study suggests that P1, which may have a cysteine residue corresponding to Cys-193 of rat 3 alpha-HSD/DD, functions in the alteration of DD3 activity depending on the modulation of NADP(+)-binding ability through a thiol/disulphide exchange reaction similar to that of rat 3 alpha-HSD/DD shown in our previous results; while P2, which may have a cysteine residue corresponding to Cys-145 of rat 3 alpha-HSD/DD, may be located near the surface of the enzyme molecule.
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PMID:The role of cysteine in the alteration of bovine liver dihydrodiol dehydrogenase 3 activity. 764 30

We have cloned the homoserine dehydrogenase genes (hom) from the gram-negative obligate methylotrophs Methylobacillus glycogenes ATCC 21276 and ATCC 21371 by complementation of an Escherichia coli homoserine dehydrogenase-deficient mutant. The 4.15-kb DNA fragment cloned from M. glycogenes ATCC 21371 also complemented an E. coli threonine synthase-deficient mutant, suggesting the DNA fragment contained the thrC gene in addition to the hom gene. The homoserine dehydrogenases expressed in the E. coli recombinants were hardly inhibited by L-threonine, L-phenylalanine, or L-methionine. However, they became sensitive to the amino acids after storage at 4 degrees C for 4 days as in M. glycogenes. The structures of the homoserine dehydrogenases overexpressed in E. coli were thought to be different from those in M. glycogenes, probably in subunit numbers of the enzyme, and were thought to have converted to the correct structures during the storage. The nucleotide sequences of the hom and thrC genes were determined. The hom genes of M. glycogenes ATCC 21276 and ATCC 21371 encode peptides with M(r)s of 48,225 and 44,815, respectively. The thrC genes were located 50 bp downstream of the hom genes. The thrC gene of ATCC 21371 encodes a peptide with an M(r) of 52,111, and the gene product of ATCC 21276 was truncated. Northern (RNA) blot analysis suggests that the hom and thrC genes are organized in an operon. Significant homology between the predicted amino acid sequences of the hom and thrC genes and those from other microorganisms was found.
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PMID:Cloning and nucleotide sequences of the homoserine dehydrogenase genes (hom) and the threonine synthase genes (thrC) of the gram-negative obligate methylotroph Methylobacillus glycogenes. 811 70

The nucleotide sequence of the Serratia marcescens threonine operon (thrA1A2BC) was determined. Three long open reading frames were identified; these open reading frames code for aspartokinase I (AKI)-homoserine dehydrogenase I (HDI), homoserine kinase, and threonine synthase, in that order. The predicted amino acid sequences of these enzymes were similar to the amino acid sequences of the corresponding enzymes in Escherichia coli. The AKI-HDI protein is apparently a tetramer composed of monomer polypeptides that are 819 amino acids long. A deletion analysis revealed that the central and C-terminal region was responsible for threonine-resistant HDI activity, a monomeric fragment extending from the N terminus to residue 306 was responsible for threonine-resistant AKI activity, and an N-terminal portion containing 468 residues was responsible for threonine-sensitive AKI activity. The thrA(1)1A(2)1 and thrA(1)5A(2)5 mutations of threonine-excreting strains HNr21 and TLr156, which result in the loss of threonine-mediated feedback inhibition of both AKI activity and HDI activity, cause single amino acid substitutions (Gly to Asp at position 330 and Ser to Phe at position 352, respectively) in the central region of the AKI-HDI protein. The thrA1+A(2)2 mutation of strain HNr59, which results in a threonine-sensitive AKI and a threonine-resistant HDI, also causes a single amino acid substitution (Ala to Thr at position 479).
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PMID:Nucleotide sequence of the Serratia marcescens threonine operon and analysis of the threonine operon mutations which alter feedback inhibition of both aspartokinase I and homoserine dehydrogenase I. 842 51

Aspartokinase I and homoserine dehydrogenase I (AKI-HDI) from Serratia marcescens Sr41 are encoded by the thrA gene as a single polypeptide chain. Previously, a single amino acid substitution of Ser-352 with Phe was shown to produce an AKI-HDI enzyme that is not subject to threonine-mediated feedback inhibition. To determine the role of Ser-352 in the allosteric response, the thrA gene was modified by using site-directed mutagenesis so that Ser-352 of the wild-type AKI-HDI was replaced by Ala, Arg, Asn, Gln, Glu, His, Leu, Met, Pro, Thr, Trp, Tyr, or Val. The Thr-352 and Pro-352 replacements rendered AKIs sensitive to threonine. The Tyr-352 and Asn-352 substitutions led to activation, rather than inhibition, of AKI by threonine. The other replacements conferred threonine insensitivity on AKI. The threonine sensitivity of HDI was also changed by the amino acid substitutions at Ser-352. The HDI carried by the Tyr-352 mutant AKI-HDI was activated by threonine. Single amino acid replacements at Ser-352 by Ala, Asn, Gln, His, Phe, Pro, Thr, or Tyr were introduced into truncated AKI-HDIs containing the AKI and the central regions. The AKI activity of the truncated AKI-HDI containing the first 468 amino acid residues was sensitive to threonine, and introduction of the amino acid replacements did not alter the threonine sensitivity of the AKI. Another truncated AKI-HDI containing the first 462 amino acid residues possessed threonine-resistant AKI, whereas the substitutions of Ser-352 with Ala and Pro rendered AKI sensitive to threonine. The replacement of GIn-351 with Phe activated AK1 of the truncated AKI-HDI in the presence of L-threonine. These findings suggest that Ser-352 of the central region of AKI-HDI is possibly a key residue involved with the allosteric regulation of both AKI and HDI activities.
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PMID:Role of serine 352 in the allosteric response of Serratia marcescens aspartokinase I-homoserine dehydrogenase I analyzed by using site-directed mutagenesis. 843 19

Rat liver 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase (3 alpha-HSD) inactivates circulating steroid hormones and is involved in polycyclic aromatic hydrocarbon (PAH) carcinogenesis. It is the only HSD of known structure in the aldo-keto reductase (AKR) superfamily and may provide a paradigm for other mammalian HSDs in this family. The structure of the 3 alpha-HSD.NADP+ binary complex has been determined at 2.7 A resolution and refined to a crystallographic R-factor of 23.4% with good geometry. The model is similar to other binary complexes in the AKR superfamily in that NADP+ binds at the C-terminal end of an alpha/beta barrel. However, it is unique in that NADP+ is bound in two alternate conformations, probably because of the lack of a salt-linked "safety belt" over the pyrophosphate bridge. The structure supports a previously proposed catalytic mechanism for carbonyl reduction in which Tyr 55 is the general acid, and its effective pKa is lowered by the adjacent Lys 84. We present evidence that the structurally distinct short-chain dehydrogenase/reductase (SDR) superfamily may have convergently evolved a similar catalytic mechanism. Insight into substrate binding is offered by a crystal packing contact in which a neighboring molecule inserts a tryptophan residue (Trp 227) into an apolar cleft in 3 alpha-HSD. This cleft is proximal to the bound NADP+ cofactor and contains a surface of apolar residues (Leu 54, Trp 86, Leu 122, Phe 128, Phe 129, Leu 137, Phe 139), making it a likely candidate for the substrate-binding site. Thus, in forming this crystal contact, Trp 227 may mimic a portion of a bound steroid. In addition, we propose that a water molecule in the active site indicates the position of the hydroxyl oxygen in a 3 alpha-hydroxysteroid substrate. Knowledge of the position of this water molecule, combined with the stereochemistry of hydride transfer, suggests that the alpha face of a bound steroid will be oriented toward the side of the apolar cleft containing Trp 86.
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PMID:Structure of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase complexed with NADP+. 871 59

Naturally occurring chlorophylls (Chl) have shown anti-mutagenic activity but little is known about their chemoprotective properties in vivo. This study examined the effect of Chl on formation in vivo of DNA adducts by the potent environmental carcinogen dibenzo[a,l]pyrene (DBP), using rainbow trout as the animal model. Fingerling trout were fed diets containing 200 p.p.m. DBP alone or with one of the following preparations incorporated at 3000 p.p.m. total chlorins: purified pheophytin a (Phe a) (94%); semi-purified Chl a (77%, 23% Phe a), commercial Chl a (88%, 12% other Chl a-related compounds); crude spinach extract (53% Chl a, 19% Chl b, 14% Phe a, 9% carotenoids); commercial Cu-chlorophyllin (55% chlorins, 45% neutral salts), as a known inhibitory control. After 2 weeks dietary treatment, the animals were killed and organs were collected. Stable DBP-DNA adducts from liver were quantified after 33P-post-labeling and separation by reversed-phase HPLC. Total DBP-DNA adducts in the DBP-only group were 2.46 +/- 0.32 adducts/10(6) nucleotides. All chlorophyll treatment groups showed significantly lower adduct levels (P < 0.001, Tukey's HSD test), as follows: crude spinach extract, 0.64 +/- 0.14; semi-pure Chl a, 0.5 +/- 0.11; commercial Chl a, 1.26 +/- 0.17; Phe a, 0.95 +/- 0.01; chlorophyllin, 0.78 +/- 0.09. The various treatments suppressed DBP-DNA adducts essentially uniformly across the HPLC profile, which is consistent with complex formation and reduced carcinogen uptake as the predominant protective mechanism. The chlorophyll-mediated reduction in DBP-DNA adducts in vivo is the first demonstration of anti-genotoxic activity of these common dietary phytochemicals in any vertebrate animal model.
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PMID:Chemoprotection by natural chlorophylls in vivo: inhibition of dibenzo[a,l]pyrene-DNA adducts in rainbow trout liver. 968 96

Candidate attenuators were identified that regulate operons responsible for biosynthesis of branched amino acids, histidine, threonine, tryptophan, and phenylalanine in gamma- and alpha-proteobacteria, and in some cases in low-GC Gram-positive bacteria, Thermotogales and Bacteroidetes/Chlorobi. This allowed us not only to describe the evolutionary dynamics of regulation by attenuation of transcription, but also to annotate a number of hypothetical genes. In particular, orthologs of ygeA of Escherichia coli were assigned the branched chain amino acid racemase function. Three new families of histidine transporters were predicted, orthologs of yuiF and yvsH of Bacillus subtilis, and lysQ of Lactococcus lactis. In Pasteurellales, the single bifunctional aspartate kinase/homoserine dehydrogenase gene thrA was predicted to be regulated not only by threonine and isoleucine, as in E. coli, but also by methionine. In alpha-proteobacteria, the single acetolactate synthase operon ilvIH was predicted to be regulated by branched amino acids-dependent attenuators. Histidine biosynthetic operons his were predicted to be regulated by histidine-dependent attenuators in Bacillus cereus and Clostridium difficile, and by histidine T-boxes in L. lactis and Streptococcus mutans.
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PMID:Attenuation regulation of amino acid biosynthetic operons in proteobacteria: comparative genomics analysis. 1513 44

It is well known that some amino acids inhibit bacterial growth. L-Serine is known to inhibit the growth of Escherichia coli by inhibition of homoserine dehydrogenase (EC 1.1.1.3). It has been reported that this L-serine inhibition may be prevented by the addition of L-isoleucine or L-threonine to the medium. In our study, however, recovery of the growth inhibition of Escherichia coli by L-serine occurred in the presence of several amino acids, especially L-phenylalanine. In an attempt to further elucidate this inhibition mechanism, different intermediates of aromatic amino acid biosynthesis were added to the growth medium. Recovery from the inhibition did not occur in the presence of prephenate but did occur when phenylpyruvate was added to the medium. The specific activity of prephenate dehydratase decreased in cells grown in the presence of L-serine. However. L-serine did not inhibit in vitro prephenate dehydratase activity, and the expression of pheA, which encodes the prephenate dehydratase, was not depressed by L-serine. We suggest that L-serine acts via another inhibition mechanism. Although this inhibition mechanism has not been fully elucidated, our results suggest that the addition of L-serine to the growth medium inhibits prephenate dehydratase synthesis and thus affects L-phenylalanine biosynthesis.
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PMID:Effect of L-serine on the biosynthesis of aromatic amino acids in Escherichia coli. 1708 51


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