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1.
C. Sautter 《Planta》1986,167(4):491-503
Microbody transition during the greening of watermelon cotyledons (Citrullus vulgaris Schrad.) was studied by double immunocytochemical labeling of the glyoxysomal marker enzyme isocitrate lyase and the peroxisomal marker enzyme hydroxypyruvate reductase. In order to analyze the immunocytochemistry, developmental stages representing the glyoxysomal, microbodytransition and peroxisomal stages were chosen, taking into account the time course of enzyme activity and the amounts of the respective antigens. It was shown that during microbody transition, between 83 and 91% of all the tested microbodies contained isocitrate lyase as well as hydroxypyruvate reductase, which was significantly higher than in the glyoxysomal and peroxisomal stages of development. Comprehensive controls precluded labeling artifacts. Our results support the one-population hypothesis first proposed by Trelease et al. (1971, Plant Physiol. 48, 461–465).Abbreviations ICJ
isocitrate lyase
- HPR
hydroxypyruvate reductase
- pAg
small protein A-gold complex
- pAG
large protein A-gold complex 相似文献
2.
Import of in-vitro-synthesized glyoxysomal malate dehydrogenase into isolated watermelon glyoxysomes
Glyoxysomal malate dehydrogenase (gMDH; EC 1.1.1.37) is synthesized by a reticulocyte system in the presence of watermelon mRNA (Citrullus vulgaris Schrad., var. Kleckey's Sweet No 6) as a cytosolic, higher-molecular-weight precursor (41 kdalton). We now show that this precursor is posttranslationally sequestered by a crude glyoxysomal fraction or by glyoxysomes purified on a PercollR gradient to a proteolytically protected form (60 min proteinase-K treatment at 4° C) with the size of the gMDH subunit (33 kdalton). In the presence of buffer instead of organelles a complete degradation of the precursor is obtained. The in-vitro organelle import, however, depends upon the presence of proteases such as proteinase K or trypsin. After short proteolytic treatments (e.g. 10 min proteinase K at 4° C), the correct processing of the MDH precursor is obtained even in the absence of organelles. This product, however, is not sequestered in vitro to a protease-resistant form by glyoxysomes. The possibility is discussed that under in-vivo conditions pre-gMDH is processed on the outside of the glyoxysomal membrane and transferred immediately after processing into the organelle presumably as a gMDH monomer followed by refolding and dimerization.Abbreviations gMDH
glyoxysomal malate dehydrogenase
- PMSF
phenylmethylsulfonyl fluoride
- SDS
sodium dodecyl sulfate
- TPCK-trypsin
trypsin treated with l-1-tosylamide-2-phenylethyl chloromethyl ketone
Dedicated to Professor Dr. Hubert Ziegler on the occasion of his 60th birthday 相似文献
3.
A heterologous in-vitro system is described for the import of the precursor to glyoxysomal malate dehydrogenase from watermelon (Citrullus vulgaris Schrad., cv. Kleckey's Sweet No. 6) cotyledons into glyoxysomes from castor-bean (Ricinus communis L.) endosperm. The 41-kDa precursor is posttranslationally sequestered and correctly processed to the mature 33-kDa subunit by a crude glyoxysomal fraction or by glyoxysomes purified on a sucrose gradient. The import and the cleavage of the extrasequence is not inhibited by metal chelators such as 1,10-phenanthroline and ethylenediaminetetraacetic acid. Uncouplers (carbonylcyanide m-chlorophenylhydrazone), ionophores (valinomycin), or inhibitors of oxidative phosphorylation (oligomycin) and ATP-ADP translocation (carboxyatractyloside) do not interfere, thus indicating the independence of the process of import by the organelle from the energization of the glyoxysomal membrane.Abbreviations CCCP
carbonylcyanide m-chlorophenylhydrazone
- EDTA
ethylenediaminetetraacctic acid
- gMDH
glyoxysomal malate dehydrogenase
- PMSF
phenylmethylsulfonyl fluoride 相似文献
4.
Methylobacterium sp. MB200 capable of producing glyoxylate from methanol was obtained by enrichment culture using a medium containing methanol
as the sole carbon source. A hpr gene that encodes a hydroxypyruvate reductase (HPR) was cloned from this strain and was ligated into the vector pLAFR3 to
obtain the recombinant plasmid pLAFRh, which was transferred into M. sp. MB200 to generate an recombinant strain MB201. Homologous expression of hpr under the control of the lacZ promoter led to the enhanced glyoxylate accumulation in cultures of Methylobacterium sp MB201. The yield of glyoxylate reached 14.38 mg/mL, representing nearly a twofold increase when compared with the wild-type
strain. 相似文献
5.
The development of glyoxysomal marker enzyme activities and concomitant ultrastructural evidence for the ontogeny of glyoxysomes has been studied in cotyledons of dark-grown watermelon seedlings (Citrullus vulgaris Schrad., var. Florida Giant). Catalase (CAT, EC 1.11.1.6) was stained in glyoxysomal structures with the 3,3-diaminobenzidine procedure. Serial sections and high-voltage electron microscopy were used to analyze the three-dimensional structure of the glyoxysomal population. With early germination CAT was localized in three distinct cell structures: spherical microbodies already present in freshly imbibed cotyledons; in appendices on lipid bodies; and in small membrane vesicles between the lipid bodies. Due to their ribosome-binding capacity, both appendices and small vesicles were identified as derivatives of the endoplasmic reticulum (ER). In the following period, glyoxysome formation and lipid body degradation were found to be inseparable processes. The small CAT-containing vesicles attach to a lipid body on a restricted area. Both lipid body appendices and attached cisternae enlarge around and between tightly packed lipid bodies and eventually become pleomorphic glyoxysomes with lipid bodies entrapped into cavities. The close contact between lipid body and glyoxysomes is maintained until the lipid body is digested and the glyoxysomal cavity becomes filled with cytoplasm. During the entire period of increase in glyoxysomal enzyme activities, no evidence was obtained for destruction of glyoxysomes, but small CAT-containing vesicles were observed from day 2 through day 6 after imbibition, indicating a continuous de novo formation of glyoxysomes. This study does not substantiate the hypothesis that glyoxysomes bud directly from the ER. Rather, ER-derivatives, e.g., lipid body appendices or cisternae attached to lipid bodies are interpreted as being glyoxysomal precursors that grow in close contact with lipid bodies both in volume and surface membrane area.Abbreviations CAT
catalase
- DAB
3,3 diaminobenzidine tetrahydrochloride
- ER
endoplasmic reticulum
- GOX
glycolate oxidase
- HPR
hydroxypyruvate reductase
- HVEM
high-voltage electron microscopy
- ICL
isocitrate lyase
- MS
malate synthase
- RER
rough endoplasmic reticulum
In the figures bars represent 0.1 m (if not stated otherwise) 相似文献
6.
Tairo Hagishita Toyokazu Yoshida Yoshikazu Izumi Toshio Mitsunaga 《FEMS microbiology letters》1996,142(1):49-52
Abstract Immunological characterization of serine-glyoxylate aminotransferase and hydroxypyruvate reductase, key enzymes for the assimilation of one-carbon compounds in methylotrophs, was performed using antibodies raised against these enzymes purified from Hyphomicrobium methylovorum GM2. Immunodiffusion studies indicated that serine-glyoxylate aminotransferase and hydroxypyruvate reductase of all seven Hyphomicrobium strains tested were immunochemically similar. In immunotitration experiments and Western blot analyses of both enzymes in the genera Hyphomicrobium and Methylobacterium , the serineglyoxylate aminotransferase of the genus Methylobacterium exhibited low similarity to that of the genus Hyphomicrobium . For hydroxypyruvate reductase, no immunological relationship was observed between the genera Hyphomicrobium and Methylobacterium , which was in agreement with the differences in primary structure and enzymological properties. 相似文献
7.
Francisco J. Corpas Marina Leterrier Juan C. Begara-Morales Raquel Valderrama Mounira Chaki Javier López-Jaramillo Francisco Luque José M. Palma María N. Padilla Beatriz Sánchez-Calvo Capilla Mata-Pérez Juan B. Barroso 《Biochimica et Biophysica Acta (BBA)/General Subjects》2013
Background
Protein tyrosine nitration is a post-translational modification (PTM) mediated by nitric oxide-derived molecules. Peroxisomes are oxidative organelles in which the presence of nitric oxide (NO) has been reported.Methods
We studied peroxisomal nitroproteome of pea leaves by high-performance liquid chromatography with tandem mass spectrometry (LC–MS/MS) and proteomic approaches.Results
Proteomic analysis of peroxisomes from pea leaves detected a total of four nitro-tyrosine immunopositive proteins by using an antibody against nitrotyrosine. One of these proteins was found to be the NADH-dependent hydroxypyruvate reductase (HPR). The in vitro nitration of peroxisomal samples caused a 65% inhibition of HPR activity. Analysis of recombinant peroxisomal NADH-dependent HPR1 activity from Arabidopsis in the presence of H2O2, NO, GSH and peroxynitrite showed that the ONOO− molecule caused the highest inhibition of activity (51% at 5 mM SIN-1), with 5 mM H2O2 having no inhibitory effect. Mass spectrometric analysis of the nitrated recombinant HPR1 enabled us to determine that, among the eleven tyrosine present in this enzyme, only Tyr-97, Tyr-108 and Tyr-198 were exclusively nitrated to 3-nitrotyrosine by peroxynitrite. Site-directed mutagenesis confirmed Tyr198 as the primary site of nitration responsible for the inhibition on the enzymatic activity by peroxynitrite.Conclusion
These findings suggest that peroxisomal HPR is a target of peroxynitrite which provokes a loss of function.General significance
This is the first report demonstrating the peroxisomal NADH-dependent HPR activity involved in the photorespiration pathway is regulated by tyrosine nitration, indicating that peroxisomal NO metabolism may contribute to the regulation of physiological processes under no-stress conditions. 相似文献8.
Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k
i=2.6 mmol · l-1 for gCS, k
i=0.33 mmol · l-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)+RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)+RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)+RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB
5,5-dithiobis-(2-nitrobenzoic acid)
- gCS
glyoxysomal citrate synthase
- gMDH
glyoxysomal malate dehydrogenase
-
k
i
inhibitor constant
- mCS
mitochondrial citrate synthase
- OAA
oxaloacetate
- poly(A)+RNA
polyadenylated RNA
- SDS-PAGE
sodium dodecyl sulfate-polyacrylamide gel electrophoresis 相似文献
9.
Nitrite reductase (EC 1.6.6.4) prepared from pea roots was found to be immunologically indistinguishable from pea leaf nitrite reductase. Comparisons of the pea root enzyme with nitrite reductase from leaf sources showed a close similarity in inhibition properties, light absorption spectrum, and electron paramagnetic resonance signals. The resemblances indicate that the root nitrite reductase is a sirohaem enzyme and that it functions in the same manner as the leaf enzyme in spite of the difference in reductant supply implicit in its location in a non-photosynthetic tissue.Abbreviations DEAE
diethylaminoethyl
- EPR
electron paramagnetic resonance
- NIR
nitrite reductase
- SDS-PAGE
sodium dodecyl sulphate-polyacrylamide gel electrophoresis 相似文献
10.
The activities of nitrite reductase (EC 1.7.7.1) are 60–70% of wild-type activity in pigment-deficient leaves of the chloroplast-ribosomedeficient mutants albostrians (Hordeum vulgare) and iojap (Zea mays). The activity and apoprotein of nitrate reductase (EC 1.6.6.1.) are lacking in the barley mutant. Only very low activities of nitrate reductase can be extracted from leaves of the maize mutant. The molybdenum cofactor of nitrate reductase and xanthine dehydrogenase (EC 1.2.3.2) is present in maize and barley mutant plants. However, it is not inducible by nitrate in pigment-deficient leaves of albostrians. From these results we conclude: (i) Nitrite reductase (a chloroplast enzyme) is synthesized in the cytoplasm and does not need the presence of nitrate reductase for the induction and maintenance if its activity. (ii) The loss or low activity of nitrate reductase is a consequence of the inability of the mutants to accumulate the apoprotein of this enzyme. (iii) The chloroplasts influence the accumulation (i.e. most probably the synthesis) of the nonchloroplast enzyme, nitrate reductase. The accumulation of nitrate reductase needs a chloroplast factor which is not provided by mutant plastids blocked at an early stage of their development.Abbreviations CRM
cross-reacting material
- Mo-co
molybdenum cofactor
- NiR
nitrite reductase
- NR
nitrate reductase 相似文献
11.
We have subjected peas (Pisum sativum L.) to four different oxidative stresses: cold conditions (4 °C) in conjunction with light, treatment with paraquat, fumigation with ozone, and illumination of etiolated seedlings (greening). In crude extracts of leaves from stressed plants, an increase (up to twofold) in activity of glutathione reductase (GR) was observed which was consistent with previous reports from several laboratories. In all cases, except for ozone fumigation, the increase in activity was not due to an elevation in the steady-state levels of GR protein. None of the applied stresses had any effect on steady-state levels of GR mRNA. In contrast to the small increase in GR activity, the K
m of GR for glutathione disulphide showed a marked decrease when determined for extracts of stressed leaves, compared with that from unstressed plants. This indicates that GR from stressed plants has an increased affinity for glutathione disulphide. The profile of GR activity bands fractionated on non-denaturing acrylamide gels varied for extracts from differently stressed leaves and when compared with GR from unstressed plants. The changes in GR-band profiles and the alteration in the kinetic properties are best explained as changes in the isoform population of pea GR in response to stress.Abbreviations GR
glutathione reductase
- GSSG
glutathione disulphide
- Rubisco
Ribulose-1,5-bisphosphate carboxylase-oxygenase
- RNase A/T1
ribonucleases A and T1
We are grateful to Prof. Alan Wellburn and Dr. Phil Beckett (Division of Biological Sciences, University of Lancaster, UK) for providing ozone-fumigated material and Dr. Jeremy Harbinson for providing material grown at 4° C. This work was supported by a grant-in-aid to the John Innes Institute from the Agricultural and Food Research Council. E.A.E. and C.E. gratefully acknowledge the support of a John Innes Foundation studentship and a European Molecular Biology Organisation Fellowship respectively. 相似文献
12.
Latent nitrate reductase activity is associated with the plasma membrane of corn roots 总被引:9,自引:0,他引:9
Latent nitrate reductase activity (NRA) was detected in corn (Zea mays L., Golden Jubilee) root microsome fractions. Microsome-associated NRA was stimulated up to 20-fold by Triton X-100 (octylphenoxy polyethoxyethanol) whereas soluble NRA was only increased up to 1.2-fold. Microsome-associated NRA represented up to 19% of the total root NRA. Analysis of microsomal fractions by aqueous two-phase partitioning showed that the membrane-associated NRA was localized in the second upper phase (U2). Analysis with marker enzymes indicated that the U2 fraction was plasma membrane (PM). The PM-associated NRA was not removed by washing vesicles with up to 1.0 M NACl but was solubilized from the PM with 0.05% Triton X-100. In contrast, vanadate-sensitive ATPase activity was not solubilized from the PM by treatment with 0.1% Triton X-100. The results show that a protein capable of reducing nitrate is embedded in the hydrophobic region of the PM of corn roots.Abbreviations L1
first lower phase
- NR
nitrate reductase
- NRA
nitrate-reductase activity
- PM
plasma membrane
- T:p
Triton X-100 (octylphenoxy polyethoxyethanol) to protein ratio
- U2
second upper phase 相似文献
13.
Ascrobate free-radical reductase (EC 1.6.5.4) from potato tubers was purified to apparent homogencity by a method which included ammonium-sulfate precipitation, gel filtration and chromatography on diethylaminoethyl cellulose and hydroxylapatite. Gel filtration and gel electrophoresis showed that the purified enzyme was monomeric with a molecular weight of about 42 000. Enzyme activity was heat lable and severely inhibited by thiol reagents. The Km values for enzyme substrates were estimated.Abbreviations AFR
ascorbate free radical
- AsA
ascorbic acid
- DE-32(52)
diethylaminoethyl cellulose
- Tricine
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine 相似文献
14.
Hindenori Hayashi 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,585(2):220-228
Urate oxidase and catalase were purified from rat liver peroxisomes, and respective antibodies were prepared from rabbits by the administration of these enzymes. Although urate oxidase generally precipitates in immunoprecipitation-possible pH ranges (pH 4.5–9.5), the enzyme remained soluble in 50 mM glycine buffer (pH 9.5) containing 50% glycerol up to concentration of 0.3 mg/ml. Anti-urate oxidase reacted with purified urate oxidase as well as with the crude preparation.After [3H]leucine was injected to rats, urate oxidase and catalase were purified from rat liver at certain intervals, and further precipitated by respective antibodies. The half-life of the catalase was 39 h and that of urate oxidase, 20 h. When the sonicated light mitochondrial fraction was incubated at 37°C and at pH 7.0 or 5.6, inactivation of catalase did not seem to differ between these pH values, and approximately 80% of the catalase activity remained even after 8 h. Urate oxidase was inactivated very rapidly at pH 5.6; only 30% of its activity survived incubation for 6 h. This inactivation was found to occur by some proteolytic process.From these findings, the turnover rate of urate oxidase was found to be different from that of catalase, and this distinction seemed to be due to different sensitivity to some degradative enzymes. 相似文献
15.
Two isoforms (isoenzymes) of glutathione reductase (NADPH: oxidized glutathione oxidoreductase, EC 1.6.4.2; GR) were clearly resolved when enzyme preparations partially purified from the unicellular alga Chlamydomonas reinhardtii were subjected to column chromatofocusing in the pH range from 8 to 4. One isoform (GR I) exhibited an almost electroneutral isoelectric point (pI, 6.9–7.1) and the other (GR II) was a very acidic protein (pI, 4.7–4.9). Both GRs are, however, homodimeric flavoproteins with similar molecular masses of approx. 127 kDa. Cross-reaction with an antibody against the cyanobacterial GR allowed determination of their subunit molecular masses by Western blotting after polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a value of 66 kDa being estimated in both cases. The two algal GR isoforms showed similar K
m values for the oxidized form of glutathione (approx. 50 M). However, the K
m values for NADPH were different, being 7 M and 28 M for GR I and GR II, respectively. The two isoforms also differed in their optimum pH. Thus, whereas GR I showed a clear maximum at neutral pH, GR II exhibited a broader optimum around pH 8.5 and was more active in the alkaline range. The relative contribution of the two isoforms to the total activity in enzyme preparations of cells disrupted by two different methods indicates that GR I should be a cytoplasmic isoform and GR II a plastidic isoform. The physiological roles of the GR isoenzymes found in Chlamydomonas are discussed and some of their properties compared with those of GRs isolated from other photosynthetic organisms.Abbreviations GSSG
glutathione, oxidized form
- GR
NAD-PH-glutathione reductase (EC 1.6.4.2)
- G3P
glyceraldehyde-3-phosphate
- pI
isoelectric point
- SDS-PAGE
polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate
This work was supported in part by grants NO. PB 87–401, PB 90–99 and BIO 91–1078 of the DGICYT (Ministerio de Educatión y Ciencia, Spain) and the Autonomous Government of Andalusia (Spain). Postdoctoral aid from the Alexander von Humboldt Foundation (Bonn, FRG) to A.S. is also acknowledged. 相似文献
16.
The localization of nitrate reductase (NR; EC 1.6.6.2) in cells of root tissues ofZea mays L. (W64A W182L) was determined using post-embedding immunogold labeling at the electron-microscopy level and using silver enhancement of the colloidal-gold signal for light microscopy. Nitrate reductase is located in the cytoplasm of root epidermal and cortical cells, and in the cells of the parenchyma and pericycle within the vascular cylinder. A weaker signal was also obtained in parenchymal cells of the pith lying next to the xylem. A positive signal for NR protein was seen in the chloroplast fraction of maize leaves and in the plastid fraction of roots. This signal was lost when affinity-purified antibodies were used. Sections of Lowicryl-embedded tissue were found to be suitable for the localization of the non-abundant NR protein when adequate controls and signal-enhancement procedures were used.Abbreviations IgG
immunoglobulin G
- NR
nitrate reductase
- PEPCase
phosphoenolpyruvate carboxylase
This research was funded by Natural Sciences and Engineering Research Council (NSERC) of Canada grants ISE0125461 (AO), OGP0106265 (JSG) and an NSERC Visiting Scientist Award to E.F. 相似文献
17.
A set of monoclonal antibodies has been raised against NADH-nitrate reductase (NR; EC 1.6.6.1) from spinach (Spinacea oleracea L.) leaves. Antibodies were screened by enzyme-linked immunosorbent assay and by their ability to inhibit various activities of the enzyme. The six monoclonals selected (AFRC MAC 74 to 79) are all gamma globulins; four (MAC 74 to 77) inhibit all terminal donating activities (NADH-NR; flavin mononucleotide, reduced form (FMNH2)-NR; and methyl viologen, reduced form (MV)-NR) and two (MAC 78 and 79) inhibit the acceptor activities (NADH-NR, and NADH-cytochrome c reductase). MAC 74 to 77 inhibit the NADH-NR activity of crude extracts of a variety of species (mono- and dicotyledoneae) while MAC 78 and 79 are effective against spinach and marrow, but not oil-seed rape, cucumber, oats, wheat and barley.Abbreviations Cyt c Rase
cytochrome c reductase
- ELISA
enzyme-linked immunosorbent assay
- FAD(H2)
flavin adenine dinucleotide (reduced form)
- FMN(H2)
flavin mononucleotide (reduced form)
- McAb
monoclonal antibody
- MV
methyl viologen reduced form
- NR
nitrate reductase 相似文献
18.
Maize (Zea mays L.) grown on low (0.8 mM) NO
3
-
, as well as untransformed and transformed Nicotiana plumbaginifolia constitutively expressing nitrate reductase (NR), was used to study the effects of NO
3
-
on the NR activation state. The NR activation state was determined from the relationship of total activity extracted in the presence of ethylenediaminetetracetic acid to that extracted in the presence of Mg2+. Light activation was observed in both maize and tobacco leaves. In the tobacco lines, NO
3
-
did not influence the NR activation state. In excised maize leaves, no correlation was found between the foliar NO
3
-
content and the NR activation state. Similarly, the NR activation state did not respond to NO
3
-
. Since the NR activation state determined from the degree of Mg2+-induced inhibition of NR activity is considered to reflect the phosphorylation state of the NR protein, the protein phosphatase inhibitor microcystin LR was used to test the importance of protein phosphorylation in the NO
3
-
-induced changes in NR activity. In-vivo inhibition of endogenous protein phosphatase activity by microcystin-LR decreased the level of NR activation in the light. This occurred to the same extent in the presence or absence of exogenous NO
3
-
. We conclude that NO
3
-
does not effect the NR activation state, as modulated by protein phosphorylation in either tobacco (a C3 species) or maize (a C4 species). The short-term regulation of NR therefore differs from the NO
3
-
-mediated responses observed for phosphoenolpyruvate carboxylase and sucrose phosphate synthase.Abbreviations Chl
chlorophyll
- MC
microcystin-LR
- PEP-Case
phosphoenolpyruvate carboxylase
- SPS
sucrose-phosphate synthase
We are indebted to Madeleine Provot and Nathalie Hayes for excellent technical assistance. This work was funded by EEC Biotechnology Contract No. BI02 CT93 0400, project of technical priority, Network D — Nitrogen Utilisation and Efficiency. 相似文献
19.
Structural basis of substrate specificity in human glyoxylate reductase/hydroxypyruvate reductase 总被引:3,自引:0,他引:3
Human glyoxylate reductase/hydroxypyruvate reductase (GRHPR) is a D-2-hydroxy-acid dehydrogenase that plays a critical role in the removal of the metabolic by-product glyoxylate from within the liver. Deficiency of this enzyme is the underlying cause of primary hyperoxaluria type 2 (PH2) and leads to increased urinary oxalate levels, formation of kidney stones and renal failure. Here we describe the crystal structure of human GRHPR at 2.2 A resolution. There are four copies of GRHPR in the crystallographic asymmetric unit: in each homodimer, one subunit forms a ternary (enzyme+NADPH+reduced substrate) complex, and the other a binary (enzyme+NADPH) form. The spatial arrangement of the two enzyme domains is the same in binary and ternary forms. This first crystal structure of a true ternary complex of an enzyme from this family demonstrates the relationship of substrate and catalytic residues within the active site, confirming earlier proposals of the mode of substrate binding, stereospecificity and likely catalytic mechanism for these enzymes. GRHPR has an unusual substrate specificity, preferring glyoxylate and hydroxypyruvate, but not pyruvate. A tryptophan residue (Trp141) from the neighbouring subunit of the dimer is projected into the active site region and appears to contribute to the selectivity for hydroxypyruvate. This first crystal structure of a human GRHPR enzyme also explains the deleterious effects of naturally occurring missense mutations of this enzyme that lead to PH2. 相似文献
20.