scholarly journals Kinetic alteration of a human dihydrodiol/3α-hydroxysteroid dehydrogenase isoenzyme, AKR1C4, by replacement of histidine-216 with tyrosine or phenylalanine

2000 ◽  
Vol 352 (3) ◽  
pp. 685-691 ◽  
Author(s):  
Tatuya OHTA ◽  
Syuhei ISHIKURA ◽  
Syunichi SHINTANI ◽  
Noriyuki USAMI ◽  
Akira HARA
Keyword(s):  
Hydroxysteroid Dehydrogenase ◽  
Clofibric Acid ◽  
The Other ◽  
Flufenamic Acid ◽  
Histidine Residue ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Competitive Inhibitors ◽  
Dihydrodiol Dehydrogenase

Human dihydrodiol dehydrogenase with 3α-hydroxysteroid dehydrogenase activity exists in four forms (AKR1C1Ő1C4) that belong to the aldoŐketo reductase (AKR) family. Recent crystallographic studies on the other proteins in this family have indicated a role for a tyrosine residue (corresponding to position 216 in these isoenzymes) in stacking the nicotinamide ring of the coenzyme. This tyrosine residue is conserved in most AKR family members including AKR1C1Ő1C3, but is replaced with histidine in AKR1C4 and phenylalanine in some AKR members. In the present study we prepared mutant enzymes of AKR1C4 in which His-216 was replaced with tyrosine or phenylalanine. The two mutations decreased 3-fold the Km for NADP+ and differently influenced the Km and kcat for substrates depending on their structures. The kinetic constants for bile acids with a 12α-hydroxy group were decreased 1.5Ő7-fold and those for the other substrates were increased 1.3Ő9-fold. The mutation also yielded different changes in sensitivity to competitive inhibitors such as hexoestrol analogues, 17β-oestradiol, phenolphthalein and flufenamic acid and 3,5,3´,5´-tetraiodothyropropionic acid analogues. Furthermore, the mutation decreased the stimulatory effects of the enzyme activity by sulphobromophthalein, clofibric acid and thyroxine, which increased the Km for the coenzyme and substrate of the mutant enzymes more highly than those of the wild-type enzyme. These results indicate the importance of this histidine residue in creating the cavity of the substrate-binding site of AKR1C4 through the orientation of the nicotinamide ring of the coenzyme, as well as its involvement in the conformational change by binding non-essential activators.

scholarly journals Mutation of tyrosine-194 and lysine-198 in the catalytic site of pig 3α/β,20β-hydroxysteroid dehydrogenase

1998 ◽  
Vol 334 (3) ◽  
pp. 553-557 ◽  
Author(s):  
Shizuo NAKAJIN ◽  
Noriko TAKASE ◽  
Shuji OHNO ◽  
Satoshi TOYOSHIMA ◽  
Michael E. BAKER
Keyword(s):  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Carbonyl Reductase ◽  
Lysine Residue ◽  
Short Chain ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Aldehydes And Ketones ◽  
Reduction Of Ketones

Pig 3α/β,20β-hydroxysteroid dehydrogenase is an NADPH-dependent enzyme that catalyses the reduction of ketones on steroids and aldehydes and ketones on various xenobiotics, like its homologue carbonyl reductase. 3α/β,20β-Hydroxysteroid dehydrogenase and carbonyl reductase are members of the short-chain dehydrogenases/reductase family, in which a tyrosine residue and a lysine residue have been identified as catalytically important. In pig 20β-hydroxysteroid dehydrogenase these residues are tyrosine-194 and lysine-198. Here we report the effect on the reduction of two ketone and two aldehyde substrates by pig 3α/β,20β-hydroxysteroid dehydrogenase in which tyrosine-194 has been mutated to phenylalanine and cysteine, and lysine-198 has been mutated to isoleucine and arginine. Mutants with phenylalanine-194 or isoleucine-198 are inactive. Depending on the substrate, the mutant with cysteine-194 has a catalytic efficiency of 0.4–1% and the mutant with arginine-198 has a catalytic efficiency of 4–23% of the wild-type enzyme. We also mutated tyrosine-81 and tyrosine-253 to phenylalanine. Although both tyrosines are conserved in 3α/β,20β-hydroxysteroid dehydrogenase and carbonyl reductase, depending on the substrate, the mutant enzymes are as active as, or more active than, wild-type enzyme.

scholarly journals Structural and Biochemical Studies on the Regulation of Biotin Carboxylase by Substrate Inhibition and Dimerization

2011 ◽  
Vol 286 (27) ◽  
pp. 24417-24425 ◽  
Author(s):  
Chi-Yuan Chou ◽  
Liang Tong
Keyword(s):  
Binding Sites ◽  
Analytical Ultracentrifugation ◽  
Substrate Inhibition ◽  
Kinetic Studies ◽  
The Other ◽  
Biotin Carboxylase ◽  
Binding Modes ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Dimer Interface

Biotin carboxylase (BC) activity is shared among biotin-dependent carboxylases and catalyzes the Mg-ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. BC has been studied extensively over the years by structural, kinetic, and mutagenesis analyses. Here we report three new crystal structures of Escherichia coli BC at up to 1.9 Å resolution, complexed with different ligands. Two structures are wild-type BC in complex with two ADP molecules and two Ca2+ ions or two ADP molecules and one Mg2+ ion. One ADP molecule is in the position normally taken by the ATP substrate, whereas the other ADP molecule occupies the binding sites of bicarbonate and biotin. One Ca2+ ion and the Mg2+ ion are associated with the ADP molecule in the active site, and the other Ca2+ ion is coordinated by Glu-87, Glu-288, and Asn-290. Our kinetic studies confirm that ATP shows substrate inhibition and that this inhibition is competitive against bicarbonate. The third structure is on the R16E mutant in complex with bicarbonate and Mg-ADP. Arg-16 is located near the dimer interface. The R16E mutant has only a 2-fold loss in catalytic activity compared with the wild-type enzyme. Analytical ultracentrifugation experiments showed that the mutation significantly destabilized the dimer, although the presence of substrates can induce dimer formation. The binding modes of bicarbonate and Mg-ADP are essentially the same as those to the wild-type enzyme. However, the mutation greatly disrupted the dimer interface and caused a large re-organization of the dimer. The structures of these new complexes have implications for the catalysis by BC.

scholarly journals Mutational Replacement of Leu-293 in the Class C Enterobacter cloacae P99 β-Lactamase Confers Increased MIC of Cefepime

2002 ◽  
Vol 46 (6) ◽  
pp. 1966-1970 ◽  
Author(s):  
Sergei B. Vakulenko ◽  
Dasantila Golemi ◽  
Bruce Geryk ◽  
Maxim Suvorov ◽  
James R. Knox ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Broad Spectrum ◽  
Random Mutagenesis ◽  
Enterobacter Cloacae ◽  
The Other ◽  
Mutant Enzyme ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Wide Range ◽  
Class C

ABSTRACT The class C β-lactamase from Enterobacter cloacae P99 confers resistance to a wide range of broad-spectrum β-lactams but not to the newer cephalosporin cefepime. Using PCR mutagenesis of the E. cloacae P99 ampC gene, we obtained a Leu-293-Pro mutant of the P99 β-lactamase conferring a higher MIC of cefepime (MIC, 8 μg/ml, compared with 0.5 μg/ml conferred by the wild-type enzyme). In addition, the mutant enzyme produced higher resistance to ceftazidime but not to the other β-lactams tested. Mutants with 15 other replacements of Leu-293 were prepared by site-directed random mutagenesis. None of these mutant enzymes conferred MICs of cefepime higher than that conferred by Leu-293-Pro. We determined the kinetic parameters of the purified E. cloacae P99 β-lactamase and the Leu-293-Pro mutant enzyme. The catalytic efficiencies (k cat/Km ) of the Leu-293-Pro mutant β-lactamase for cefepime and ceftazidime were increased relative to the respective catalytic efficiencies of the wild-type P99 β-lactamase. These differences likely contribute to the higher MICs of cefepime and ceftazidime conferred by this mutant β-lactamase.

scholarly journals The engineered, cytosolic form of human type I 3beta-hydroxysteroid dehydrogenase/isomerase: purification, characterization and crystallization

2001 ◽  
Vol 27 (1) ◽  
pp. 77-83 ◽  
Author(s):  
JL Thomas ◽  
JI Mason ◽  
G Blanco ◽  
ML Veisaga
Keyword(s):  
Molecular Mass ◽  
Hydroxysteroid Dehydrogenase ◽  
Size Exclusion ◽  
Sf9 Cells ◽  
Type I ◽  
Wild Type ◽  
Enzyme Protein ◽  
Wild Type Enzyme ◽  
Human Type ◽  
Type K

Human type I 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD/isomerase) is an integral membrane protein of human placental trophoblast and of insect Sf9 cells transfected with recombinant baculovirus containing the cDNA encoding the enzyme. Purified native or wild-type enzyme remains in solution only in the presence of detergent that may prevent crystallization. The membrane-spanning domain (residues 283-310) of the enzyme protein was deleted in the cDNA using PCR-based mutagenesis. The modified enzyme was expressed by baculovirus in the cytosol instead of in the microsomes and mitochondria of the Sf9 cells. The cytosolic form of 3beta-HSD/isomerase was purified using affinity chromatography with Cibacron Blue 1000. The NAD(+) and NaCl used to elute the enzyme were removed by size-exclusion centrifugation. Hydroxylapatite chromatography yielded a 26-fold purification of the enzyme. SDS-PAGE revealed a single protein band for the purified cytosolic enzyme (monomeric molecular mass 38.8 kDa) that migrated just below the wild-type enzyme (monomeric molecular mass 42.0 kDa). Michaelis-Menten constants measured for 3beta-HSD substrate (dehydroepiandrosterone) utilization by the purified cytosolic enzyme (K(m)=4.5 microM, V(max)=53 nmol/min per mg) and the pure wild-type enzyme (K(m)=3.7 microM, V(max)=43 nmol/min per mg), for isomerase substrate (5-androstene-3,17-dione) conversion by the purified cytosolic (K(m)=25 microM, V(max)=576 nmol/min per mg) and wild-type (K(m)=28 microM, V(max)=598 nmol/min per mg) enzymes, and for NAD(+) reduction by the 3beta-HSD activities of the cytosolic (K(m)=35 microM, V(max)=51 nmol/min per mg) and wild-type (K(m)=34 microM, V(max)=46 nmol/min per mg) enzymes are nearly identical. The isomerase activity of the cytosolic enzyme requires allosteric activation by NADH (K(m)=4.6 microM, V(max)=538 nmol/min per mg) just like the wild-type enzyme (K(m)=4.6 microM, V(max)=536 nmol/min per mg). Crystals of the purified, cytosolic enzyme protein have been obtained. The inability to crystallize the detergent-solubilized, wild-type microsomal enzyme has been overcome by engineering a cytosolic form of this protein. Determining the tertiary structure of 3beta-HSD/isomerase will clarify the mechanistic roles of potentially critical amino acids (His(261), Tyr(253)) that have been identified in the primary structure.

scholarly journals The negative charge of glutamic acid-820 in the gastric H+,K+-ATPase α-subunit is essential for K+ activation of the enzyme activity

1998 ◽  
Vol 331 (2) ◽  
pp. 465-472 ◽  
Author(s):  
Harm P. H. HERMSEN ◽  
Herman G. P. SWARTS ◽  
Jan B. KOENDERINK ◽  
Jan Joep H. H. M. De PONT
Keyword(s):  
Atpase Activity ◽  
Transmembrane Domain ◽  
The Other ◽  
Lower Sensitivity ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Α Subunit ◽  
Activity Maximum ◽  
Activity Measurements ◽  
Atpase Subunits

To investigate the role of Glu820, located in transmembrane domain M6 of the α-subunit of gastric H+,K+-ATPase, a number of mutants was prepared and expressed in Sf9 cells using a baculovirus encoding for both H+,K+-ATPase subunits. The wild-type enzyme and the E820D (Glu820 → Asp) mutant showed a similar biphasic activation by K+ on the ATPase activity (maximum at 1 mM). The mutant E820A had a markedly decreased K+ affinity (maximum at 40–100 mM). The other mutants, E820Q, E820N, E820L and E820K, showed no K+-activated ATPase activity at all, whereas all mutants formed a phosphorylated intermediate. After preincubation with K+ before phosphorylation mutant E820D showed a similar K+-sensitivity as the wild-type enzyme. The mutants E820N and E820Q had a 10–20 times lower sensitivity, whereas the other three mutants were hardly sensitive towards K+. Upon preincubation with 3-(cyanomethyl)-2-methyl-8-(phenylmethoxy)imidazo[1,2a] pyridine (SCH 28080), all mutants showed similar sensitivity for this drug as the wild-type enzyme, except mutant E820Q, which could only partly be inhibited, and mutant E820K, which was completely insensitive towards SCH 28080. These experiments suggest that, with a relatively large residue at position 820, the binding of SCH 28080 is obstructed. The various mutants showed a behaviour in K+-stimulated-dephosphorylation experiments similar to that for K+-activated-ATPase-activity measurements. These results indicate that K+ binding, and indirectly the transition to the E2 form, is only fully possible when a negatively charged residue is present at position 820 in the α-subunit.

scholarly journals Sequence of the cDNA of a human dihydrodiol dehydrogenase isoform (AKR1C2) and tissue distribution of its mRNA

1998 ◽  
Vol 334 (2) ◽  
pp. 399-405 ◽  
Author(s):  
Hiroaki SHIRAISHI ◽  
Syuhei ISHIKURA ◽  
Kazuya MATSUURA ◽  
Yoshihiro DEYASHIKI ◽  
Mitsuo NINOMIYA ◽  
...  
Keyword(s):  
Human Liver ◽  
Human Colon ◽  
Hydroxysteroid Dehydrogenase ◽  
The Other ◽  
Specific Primers ◽  
Tissue Samples ◽  
Mrna Species ◽  
Ht29 Cells ◽  
Human Colon Carcinoma ◽  
Dihydrodiol Dehydrogenase

Human liver contains three isoforms (DD1, DD2 and DD4) of dihydrodiol dehydrogenase with 20α- or 3α-hydroxysteroid dehydrogenase activity; the dehydrogenases belong to the aldo–oxo reductase (AKR) superfamily. cDNA species encoding DD1 and DD4 have been identified. However, four cDNA species with more than 99% sequence identity have been cloned and are compatible with a partial amino acid sequence of DD2. In this study we have isolated a cDNA clone encoding DD2, which was confirmed by comparison of the properties of the recombinant and hepatic enzymes. This cDNA showed differences of one, two, four and five nucleotides from the previously reported four cDNA species for a dehydrogenase of human colon carcinoma HT29 cells, human prostatic 3α-hydroxysteroid dehydrogenase, a human liver 3α-hydroxysteroid dehydrogenase-like protein and chlordecone reductase-like protein respectively. Expression of mRNA species for the five similar cDNA species in 20 liver samples and 10 other different tissue samples was examined by reverse transcriptase-mediated PCR with specific primers followed by diagnostic restriction with endonucleases. All the tissues expressed only one mRNA species corresponding to the newly identified cDNA for DD2: mRNA transcripts corresponding to the other cDNA species were not detected. We suggest that the new cDNA is derived from the principal gene for DD2, which has been named AKR1C2 by a new nomenclature for the AKR superfamily. It is possible that some of the other cDNA species previously reported are rare allelic variants of this gene.

 L-allo-Threonine aldolase with an H128Y/S292R mutation fromAeromonas jandaeiDK-39 reveals the structural basis of changes in substrate stereoselectivity

2014 ◽  
Vol 70 (6) ◽  
pp. 1695-1703 ◽  
Author(s):  
Hui-Min Qin ◽  
Fabiana Lica Imai ◽  
Takuya Miyakawa ◽  
Michihiko Kataoka ◽  
Nahoko Kitamura ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Active Site ◽  
Fold Increase ◽  
Saturation Mutagenesis ◽  
Structural Basis ◽  
Tyrosine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Threonine Aldolase

L-allo-Threonine aldolase (LATA), a pyridoxal-5′-phosphate-dependent enzyme fromAeromonas jandaeiDK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123–Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonineversusL-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.

scholarly journals Site-directed mutagenesis of the putative active site of human 17β-hydroxysteroid dehydrogenase type 1

1994 ◽  
Vol 304 (1) ◽  
pp. 289-293 ◽  
Author(s):  
T J Puranen ◽  
M H Poutanen ◽  
H E Peltoketo ◽  
P T Vihko ◽  
R K Vihko
Keyword(s):  
Amino Acid ◽  
Catalytic Properties ◽  
Catalytic Efficiency ◽  
Hydroxysteroid Dehydrogenase ◽  
Site Directed Mutagenesis ◽  
Dimensional Structure ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Wild Type Enzyme

Several amino acid residues (Cys54, Tyr155, His210, His213 and His221) at a putative catalytic site of human 17 beta-hydroxysteroid dehydrogenase type 1 were mutated to Ala. Replacement of His221 by Ala remarkably reduced the catalytic activity, which resulted from a change of both the Km and the Vmax. values of the enzyme. Compared with the wild-type enzyme, the catalytic efficiency of the His221-->Ala mutant was reduced 20-fold for the oxidative reaction and 11-fold for the reductive reaction. With similar mutations at His210 or His213, no notable effects on the catalytic properties of the enzyme were detected. However, a simultaneous mutation of these amino acid residues decreased the Vmax. values of both oxidation and reduction by about 50% from those measured for the wild-type enzyme. Although Cys54 has been localized in the cofactor-binding region of the enzyme, a Cys54-->Ala mutation did not lead to changes in the enzymic activity. The most dramatic effects on the catalytic properties of the enzyme were achieved by mutating Tyr155, which resulted in an almost completely inactivation of the enzyme. The decreased enzymic activities of the Tyr155-->Ala, His210-->Ala + His213-->Ala and His221-->Ala mutations were also reflected in a reduced immunoreactivity of the enzymes. The results thus suggest that the lower catalytic efficiency of the mutant enzymes is due to an exchange of catalytically important amino acid residues and/or remarkable alterations in the three-dimensional structure of the enzyme. The recently detected polymorphisms (Ala237<-->Val and Ser312<-->Gly) were not found to affect either the catalytic or the immunological properties of the type 1 enzyme.

scholarly journals Identification of essential histidine residues in UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferase-T1

1997 ◽  
Vol 328 (1) ◽  
pp. 193-197 ◽  
Author(s):  
Stephanie WRAGG ◽  
K. Fred HAGEN ◽  
A. Lawrence TABAK
Keyword(s):  
Recombinant Proteins ◽  
Initial Rate ◽  
Initial Step ◽  
Enzymic Activity ◽  
Histidine Residue ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Histidine Residues ◽  
Sds Page ◽  
Enzyme Biosynthesis

UDP-N-acetyl-D-galactosamine:polypeptide N-acetylgalactosaminyltransferases (ppGaNTases) catalyse the initial step of mucin-type O-glycosylation. The activity of bovine ppGaNTase-T1 isoenzyme was inhibited by diethyl pyrocarbonate (DEPC) modification. Activity was partially restored by hydroxylamine treatment, indicating that one of the reactive residues was a histidine. The transferase was protected against DEPC inactivation when UDP-GalNAc and EPO-G, a peptide pseudo-substrate PPDAAGAAPLR, were simultaneously present, while presence of EPO-G alone did not alter DEPC inactivation. However, inclusion of UDP-GalNAc alone potentiated DEPC-inhibition of the enzyme, suggesting that UDP-GalNAc binding changes the accessibility or reactivity of an essential histidine residue. Deletion of the first 56 amino acids (including one hisitidine residue) yielded a fully active secreted form of the bovine ppGaNTase-T1 enzyme. Each of the 14 remaining histidines in the enzyme were mutated to alanine, and the recombinant mutants were recovered from COS7 cells. The mutation of histidine residues His211 → Ala and His344 → Ala resulted in recombinant proteins with no detectable enzymic activity. A significant decrease in the initial rate of GalNAc transfer to the substrate was observed with mutants His125 → Ala and His341 → Ala (1% and 6% of wild-type activity respectively). Mutation of the remaining ten histidine residues yielded mutants that were indistinguishable from the wild-type enzyme. Mutagenesis and SDS/PAGE analysis of all N-glycosylation sequons revealed that positions N-95 and N-552 are occupied by N-linked sugars in COS7 cells. Ablation of either site did not perturb enzyme biosynthesis or enzyme activity.

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