Molecular mechanisms of fatty acid beta-oxidation enzyme catalysis.
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Acyl Coenzyme A↗
Biomedical subjects
S Y Yang
Publications and source records attributed to S Y Yang.
Clinical results of the transoral operation for lesions of the craniovertebral junction and its abnormalities.
BACKGROUND: We treated 20 cases of craniovertebral junction lesions via a transoral approach. Developmental abnormalities of the craniovertebral junction accounted for 15 cases; there were 3 cases of tumor, 1 case of osteomyelitis, and 1 case of rheumatoid arthritis. METHODS: The transoral transpharyngeal approach was used in all cases. In 17 non-tumoral patients the anterior margin of the atlas and the odontoid process were resected. In one patient with a ventral clivus chordoma, both a transoral and a transnasal transsphenoidal approach was used for partial resection of the tumor mass. In two cases the median transpharyngeal approach was combined with a jaw-facial incision. In one case a metastatic adenocarcinoma, and in another a neurinoma of the accessory nerve straddling the posterior fossa and the pharyngeal region were removed. RESULTS: After operation four cases developed craniovertebral joint instability and required posterior cervical fusion or external fixation with a halo brace. Follow-up ranged from 2 to 44 months. In most patients neurologic function slowly improved. One worsened, and one died of respiratory failure after operation. Two patients with malignant tumors died during the follow-up period. CONCLUSION: In patients with developmental malformations, transoral decompression will result in some neurologic improvement and arrest the progress of symptoms. Patients with tumors are likely to show a good neurologic outcome when transoral surgery is performed in the early stage of the condition.
Adolescent↗
Determination and clinical significance of plasma levels of prostaglandins in patients with acute brain injury.
BACKGROUND: Models of brain injury in experimental animals have shown that the level of prostaglandins (PGs) is increased in damage brain tissue, and that PGs play an important role in secondary brain damage. Almost all previous studies of the relationship between PGs and brain injury have been carried out in animals. In the present study we show that the PGs change in humans with brain injury. METHODS: The plasma levels of thromboxane B2, 6-Keto-PGF2alpha, prostaglandin F2alpha, and prostaglandin E2 were measured by radioimmunoassay on the 1st, 3rd, 7th, and 14th days after brain injury. The same measurements were made on a control group of 26 healthy volunteers. RESULTS: The levels of all four PGs were elevated, most markedly in the first week, with levels remaining high in the second week in the severely injured. On the first day levels were, on average, three times those found in the controls, with a seven-fold rise in some of the severely injured patients. Dividing the patients into three groups according to outcome, it was found that if the PGs were markedly increased to begin with and remained high, death or permanent disability was likely. In the group with good outcome, the levels dropped steadily from the initial high levels. The T/K ratio was studied. It related closely to the severity of injury, being higher in more severe injuries and decreasing with recovery. In patients who did not recover, the ratio increased steadily to its highest value on the 7th day, and remained high at the 14th. CONCLUSION: Changes in PGs levels were closely related to the brain injury severity and its outcome, and there was a marked disturbance of the levels of PGs, which therefore appears to be an important indicator of secondary brain damage.
Acute Disease↗
HLA-B22 diversity including a novel B54 variant (B*5507) in the Korean population.
Diversity in the HLA-B22 group was investigated in the Korean population using PCR-SSOP and DNA sequencing analyses. Allelic typing of the B22 gene was performed by gene amplification of the polymorphic exons 2 and 3 of the HLA-B genes from 91 B22 positive individuals followed by a hybridization assay using 63 digoxigenin-labelled probes. Five different SSOP patterns including an unexpected pattern were identified and correlated well with the observed serologic types and with data obtained from DNA sequencing analyses. Novel allele, B*5507, was identified from two unrelated individuals who exhibited standard B54 serologic reactivity but an unexpected SSOP pattern. The DNA sequence of B*5507 is identical to B*5502 in exons 2 and 3 except for a single nucleotide substitution at codon 45 (GAG-->GGG) altering glutamic acid to glycine. Among the already known B molecules, this substitution has been observed only in the B54 molecule encoded by B*5401 allele. This is the evidence that Gly-45 is a crucial site forming the B54 serologic epitope. Interestingly, both alleles (B*5401 and B*5507) exhibit strong association with Cw*0102. Along with previous data, B22 appears to be a very diverse group in the Korean population consisting of at least seven different alleles. B*5401, B*5502, and B*5601 are the most frequent alleles. B*5507, B*5501, B*5504, and B*5604 appear at lower frequencies. Data obtained from this study will be useful in hematopoietic stem cell donor searches as well as in determination of a typing strategy for the HLA-B22 types in this population.
Alleles↗
HLA-A*30 alleles and associated haplotypes in the Korean population.
HLA-A30 is one of the serologically characterized specificities and has been known to exhibit different haplotypic associations in different ethnic groups. This study reveals the presence of two HLA-A*30 alleles, A*3001 (31/48; 64.6%) and A*3004 (17/48; 35.4%), with each allele existing as a distinct haplotype in the Korean population. One haplotype, A*3001-Cw*0602-B*1302-DRB1*0701-DQA1*02-DQB1*02, found in 77.4% of A*3001-positive individuals is likely identical to the haplotype previously identified by serology in Asians and European Caucasians. A second haplo-type, A*3004-Cw*0802-B*1401-DRB1*0404-DQA1*03-DQB1*0402, found in 64.7% of A*3004-positive individuals has not previously been described. Conserved and distinct haplotypic associations of the two A*30 alleles imply that the two haplotypes may have evolved separately and are relatively new in this population.
Alleles↗
Cytochrome P450 1A1 genetic polymorphisms and risk of hepatocellular carcinoma among chronic hepatitis B carriers.
Cigarette smoking has been associated with increased risk of hepatocellular carcinoma (HCC) in some epidemiological studies. Cytochrome P450 1A1 (CYP1A1) is involved in the biotransformation of tobacco-derived polycyclic aromatic hydrocarbons (PAHs) into carcinogenic metabolites. The aim of this study was to determine whether CYP1A1 polymorphisms were related to HCC risk among chronic hepatitis B virus (HBV) carriers. Genotypic variants of CYP1A1 were determined using polymerase chain reaction in 81 incident cases of HCC and 409 controls nested in a cohort study of 4841 male chronic HBV carriers. No overall association between CYP1A1 genotypes and HCC was observed. The presence of the Mspl (odds ratio (OR) 3.15, P = 0.0196) or Ile-Val (OR 1.99, P = 0.0855) variant allele of CYP1A1 increased HCC risk among smokers, but posed no increased risk among non-smokers. The smoking-related HCC risk was most pronounced among those who had a susceptible allele of the CYP1A1 and a deficient genotype of glutathione S-transferase M1, which detoxifies PAH electrophilic metabolites produced by CYP1A1. In the absence of the Ile-Val variant allele, the Mspl polymorphism was still associated with smoking-related HCC. This study suggests that tobacco-derived PAHs play a role in HCC risk among chronic HBV carriers, and CYP1A1 polymorphism is an important modulator of the hepatocarcinogenic effect of PAHs. The Mspl and Ile-Val polymorphisms of CYP1A1 may have different mechanisms for increasing susceptibility to smoking-related HCC.
Adult↗
HLA-C disparity between patients and unrelated donors matched for HLA-A, -B, and -DRB1 alleles: impact of serological vs. DNA typing for HLA-A and -B loci.
High incidences of graft failure, graft-vs.-host disease (GVHD), and serious infections following unrelated donor (URD) marrow transplantation, despite apparent human leukocyte antigen (HLA) identity, may reflect the presence of molecular disparities, including those for HLA-C alleles between the patient and the URD. The level of these disparities could be significant, because as many as 42 alleles are currently known for HLA-C locus. We studied 84 patients and 251 potential URDs to evaluate 1) the extent of HLA-C disparity between the patient and the URD identified by serology for HLA-A and -B and by DNA typing for -DRB1 and 2) the level of HLA-C disparity between patients and URDs matched by high-resolution DNA typing for HLA-A, -B, and -DRB1. The DNA typing was performed at the Memorial Sloan Kettering Cancer Center, and the serotyping was provided by the registries. Of 251 URDs matched by HLA-A and -B serology and -DRB1 (sA_sB_dnaDRB1 ); 94, 75, and 82 were 6/6, 5/6, and 4/6 matches, respectively. Of 94 sA_sB_dnaDRB1 6/6 URDs, 51 (54.3%) were matched for both HLA-C alleles. In contrast, 31 (41.3%) 5/6 (p=0.12) and 15 (18.3%) 4/6 (p < 0.01) sA_sB_dnaDRB1 URDs were matched for both HLA-C alleles. Following DNA typing for HLA-A and -B, 52 (55.3%) of 94 6/6, 30 (40%) of 75 5/6, and 25 (30.5%) of 82 4/6 sA_sB_dnaDRB1 URDs remained 6/6, 5/6, and 4/6 matches at the DNA level (dnaA_B_DRB1). HLA-C disparities continued to exist in the dnaA_B_DRB1 URD group. Of 54 dnaA_B_DRB1 6/6 URDs, 41 (75.9%) were matched for both HLA-C alleles. Only 45.3% of the 5/6 (p=0.01) and 22.2% of the 4/6 (p < 0.01) dnaA_B_DRB1 URDs were matched for both HLA-C alleles. In the 6/6 category, the frequency of HLA-C matching improved (75.9 vs. 54.3%; p=0.01) following DNA matching for HLA-A and -B. In comparison to mismatching for HLA-B locus, mismatching for either HLA-DRB1 or -A resulted in a lower odds ratio for HLA-C disparity. The presence of a common haplotype in the sA_sB_dnaDRBl (p=0.06) URD category improved the level of HLA-C matching. We identified alleles that are associated with high (B*1501, B*4402, B*5101, DRB1*0101, A*0201, A*1101, A*2301, and A*3201) or low (B*0702, B*0801, B*1302, B*3502, DRB1*0301, DRB1*1104, A*0101, A*3001, and A*6801) probability of HLA-C disparity. Overall, sA_sB_dnaDRB1 as well as dnaA_B_DRB1 matched URDs for non-Caucasian patients were more likely to have HLA-C disparity in comparison to the matched URDs of Caucasian patients. However, a high incidence of HLA-C disparities was identified even in the URDs for Caucasian patients. Whether the disparities demonstrated by this study contribute to the higher immunological complications noted following URD bone marrow transplantation is unclear. Outcome analysis and studies aimed at understanding the functional role of HLA-C may provide an answer.
Bone Marrow Transplantation↗
Polyspecific self-reactive antibodies in individuals infected with human immunodeficiency virus facilitate T cell deletion and inhibit costimulatory accessory cell function.
Self-reactive polyspecific IgG antibodies (PSAs) arise in human immunodeficiency virus (HIV)-seropositive subjects before they develop AIDS. Self-reactive PSA levels correlate with the destruction of CD8 T cells in HIV-infected individuals and mediate the antibody-dependent cellular toxicity-based destruction of human T cells in tissue culture. PSAs react across the species barrier and bind to T cell antigens in mice. Such reactivity with mouse lymphocytes was not detected in normal human serum. Injection of human PSA IgG causes massive T cell depletion in the spleen, lymph nodes, and thymus in mice: evidence that PSA IgG facilitates T cell destruction in vivo. In addition to facilitating macrophage cytotoxicity, self-reactive PSA IgG inhibits the macrophage-mediated activation of T cells with antigen receptor-specific monoclonal antibody or with antigen. Exogenous costimulatory stimuli or interleukin (IL)-12 can reverse the inhibition. In contrast, exogenous IL-10 mimics this inhibition. These data implicate PSA IgG as a pathogenic factor in the development of HIV disease.
Acquired Immunodeficiency Syndrome↗
Intracoronary propofol attenuates myocardial but not coronary endothelial dysfunction after brief ischaemia and reperfusion in dogs.
We have investigated the effects of propofol on recovery of regional mechanical and coronary endothelial function and on lipid peroxidation in post-ischaemic myocardium in dogs. The animals were assessed for 180 min during reperfusion after 15-min of occlusion of the left anterior descending coronary artery (LAD). They were treated with intracoronary (i.c.) propofol 5 or 20 micrograms/ml of coronary flow or vehicle (control group) for 60 min, beginning 30 min before LAD occlusion. Propofol significantly enhanced recovery of regional contractile function (70% and 81% of baseline segment shortening in the propofol 5 and 20 micrograms ml-1 groups, respectively, compared with 51% in controls at 3 h of reperfusion). However, LAD flow responses to i.c. acetylcholine were similarly attenuated regardless of treatment with propofol throughout reperfusion. The increase in malondialdehyde induced by ischaemia-reperfusion was significantly suppressed by both doses of propofol. These results demonstrated that in vivo, propofol ameliorated dysfunction of the myocardium but not of the coronary endothelium resulting from brief ischaemia and reperfusion; the protection may be related, at least in part, to its ability to reduce lipid peroxidation.
Anesthetics, Intravenous↗
Hormone-refractory prostate cancer cells express functional follicle-stimulating hormone receptor (FSHR).
PURPOSE: Understanding growth regulation in hormone-refractory prostate cancer may provide avenues for novel treatment interventions. This study was conducted to characterize the expression of the receptor (FSHR) for follicle-stimulating hormone (FSH) in androgen-independent prostate cancer cell lines and in human malignant prostate tissues. MATERIALS AND METHODS: Western blotting, immunohistochemistry (IHC), and flow cytometric analysis were used to study the expression of FSHR. The effect of FSH on cell growth and clonogenicity was studied using proliferation and clonogenic assays. RESULTS: Immunohistochemistry revealed expression of FSH in PC3 and Du145 cells. FSHR was identified in PC3 and Du145 cells, as well as in human adenocarcinoma of the prostate. The specificity of the FSHR detected on prostate cancer tissues or cells by IHC and Western blotting was confirmed by preabsorbing the antibodies with the immunizing antigens. Stimulation of these hormone-refractory cells with FSH triggered a proliferative response in vitro, suggesting that the receptor is biologically active. CONCLUSION: Hormone-refractory prostate cancer cells express FSH and biologically active FSHR. Our results suggest that FSHR and its ligand may play a role in the regulation of the growth of hormone-refractory prostate cancers.
Androgen Antagonists↗
Molecular cloning, expression in Escherichia coli, and characterization of a novel L-3-hydroxyacyl coenzyme A dehydrogenase from pig liver.
A novel L-3-hydroxyacyl-CoA dehydrogenase from pig liver has been cloned, expressed, purified, and characterized. This enzyme is a homodimer with a molecular mass of 65.6 kDa, and is distinguished from the dehydrogenase of pig heart by its structural features and catalytic properties. Its subunit, consisting of 302 amino acid residues, has two additional residues in a key region of the active center while it lacks a sequence of seven residues in the NAD+-binding domain, when compared with the subunit of pig heart enzyme. In addition, there are substitutions of four single residues. The catalytic efficiency of pig liver dehydrogenase was significantly greater than that of the heart enzyme for short-chain substrate, but its catalytic rates declined with an increase in substrate chain-lengths. The distinction between pig liver and heart dehydrogenases cannot be attributed to a species difference, and thus it is concluded that there exist different isoforms of monofunctional L-3-hydroxyacyl-CoA dehydrogenases in pig. High level expression of mitochondrial L-3-hydroxyacyl-CoA dehydrogenase in Escherichia coli has provided a very convenient way to purify this important beta-oxidation enzyme. There is substantial homology between pig liver dehydrogenase and various multifunctional beta-oxidation enzymes in the active center of these enzymes; a consensus sequence, HX3PX1-3MXLXE, is proposed as the signature sequence of l-3-hydroxyacyl-CoA dehydrogenases.
3-Hydroxyacyl CoA Dehydrogenases↗
A human brain L-3-hydroxyacyl-coenzyme A dehydrogenase is identical to an amyloid beta-peptide-binding protein involved in Alzheimer's disease.
A novel L-3-hydroxyacyl-CoA dehydrogenase from human brain has been cloned, expressed, purified, and characterized. This enzyme is a homotetramer with a molecular mass of 108 kDa. Its subunit consists of 261 amino acid residues and has structural features characteristic of short chain dehydrogenases. It was found that the amino acid sequence of this human brain enzyme is identical to that of an endoplasmic reticulum amyloid beta-peptide-binding protein (ERAB), which mediates neurotoxicity in Alzheimer's disease (Yan, S. D., Fu, J., Soto, C., Chen, X., Zhu, H., Al-Mohanna, F., Collison, K., Zhu, A., Stern, E., Saido, T., Tohyama, M., Ogawa, S., Roher, A., and Stern, D. (1997) Nature 389, 689-695). The purification of human brain short chain L-3-hydroxyacyl-CoA dehydrogenase made it possible to characterize the structural and catalytic properties of ERAB. This NAD+-dependent dehydrogenase catalyzes the reversible oxidation of L-3-hydroxyacyl-CoAs to form 3-ketoacyl-CoAs, but it does not act on the D-isomers. The catalytic rate constant of the purified enzyme was estimated to be 37 s-1 with apparent Km values of 89 and 20 microM for acetoacetyl-CoA and NADH, respectively. The activity ratio of this enzyme for substrates with chain lengths of C4, C8, and C16 was approximately 1:2:2. The human short chain L-3-hydroxyacyl-CoA dehydrogenase gene is organized into six exons and five introns and maps to chromosome Xp11.2. The amino-terminal NAD-binding region of the dehydrogenase is encoded by the first three exons, whereas the other exons code for the carboxyl-terminal substrate-binding region harboring putative catalytic residues. The results of this study lead to the conclusion that ERAB involved in neuronal dysfunction is encoded by the human short chain L-3-hydroxyacyl-CoA dehydrogenase gene.
3-Hydroxyacyl CoA Dehydrogenases↗
Effect of rebamipide on the glycosaminoglycan content of the ulcerated rat stomach.
To elucidate the mechanism of the anti-ulcer effect of rebamipide (2-(4-chlorobenzoylamino)-3-[2-(1H)-quinolinon-4-yl] propionic acid), changes in glycosaminoglycan (GAG), uronic acid and hexosamine contents of stomach tissue were examined in rats treated with the ulcer-inducing agents and/or rebamipide. Uronic acid and hexosamine contents in acid hydrolysates of stomach tissue were increased after diethyldithiocarbamate (DDC, 800 mg/kg, s.c.) or histamine (300 mg/kg, i.p.) treatment, and similar changes in the GAG, uronic acid, and hexosamine levels were observed in stomach tissue extracts. Pretreatment with rebamipide (60 mg/kg, i.p.) resulted in an additional increase in the contents of the above components after DDC or histamine treatment. However, rebamipide treatment alone did not increase the gastric contents of GAG and GAG components in normal rats. Gel filtration chromatography of extracted GAGs suggested that DDC, histamine and rebamipide treatments do not cause a change in the aggregated forms of gastric GAGs. These results suggest that rebamipide stimulates the GAG synthesis in the ulcerated stomach and that this effect may contribute to the healing process of gastric ulcer.
Alanine↗
Purification of active Na+-K+-ATPase using a new ouabain-affinity column.
Ouabain, a specific inhibitor of Na+-K+-ATPase, was coupled to epoxy agarose via a 13-atom spacer to make an affinity column that specifically binds Na+-K+-ATPase. Na+-K+-ATPase from rat and dog kidney was bound to the column and was eluted as a function of enzyme conformation, altered by adding specific combinations of ligands. Na+-K+-ATPase from both sources bound to the column in the presence of Na + ATP + Mg and in solutions containing 30 mM K. No binding was observed in the presence of Na or Na + ATP. These experiments suggest that Na+-K+-ATPase binds to the column under the same conditions that it binds to untethered ouabain. Na+-K+-ATPase already bound to the column was competitively eluted with excess free Na + ouabain or with Na + ATP. The latter eluted active enzyme. For comparable amounts of bound Na+-K+-ATPase, Na + ouabain and Na + ATP eluted more rat than dog Na+-K+-ATPase, consistent with the lower affinity of the rat Na+-K+-ATPase for ouabain. The ouabain-affinity column was used to purify active Na+-K+-ATPase from rat kidney microsomes and rat adrenal glomerulosa cells. The specific activity of the kidney enzyme was increased from approximately 2 to 15 micromol Pi . mg-1 . min-1. Na+-K+-ATPase purified from glomerulosa cells that were prelabeled with [32P]orthophosphate was phosphorylated on the alpha-subunit, suggesting that these cells contain a kinase that phosphorylates Na+-K+-ATPase.
Adenosine Triphosphate↗
Expression of the basic fibroblast growth factor gene in mild and more severe head injury in the rat.
OBJECT: The goal of this study was to investigate the relationship between basic fibroblast growth factor (bFGF) gene expression and neuropathological changes in the hippocampus after varying degrees of brain injury. METHODS: Mild and severe brain injury in rats was produced by using Marmarou's method. There were 25 animals in each brain injury group and 25 additional animals served as controls. Basic fibroblast growth factor gene expression was investigated by means of RNA hybridization, in situ hybridization, immunohistochemical analysis, and histological analysis using hematoxylin and eosin staining. A 3.7-kb bFGF messenger (m)RNA was detected in the rat hippocampus in both control and injured rats. In the mild injury group its expression was increased at 12 hours after injury and peaked on the 3rd day. Neuronal degeneration in the hippocampal CA2 and CA3 sectors was maximum on that day. In the severe injury group, the expression of the bFGF gene was the same as that in the mild injury group at corresponding times, but the number of surviving neurons in the CA2 and CA3 sectors was much lower than in the mild injury group. In situ hybridization showed that the main cells that expressed bFGF mRNA were pyramidal and granulocytic neurons in all three experimental groups. The number of neurons expressing bFGF mRNA in the severe injury group was less than that in the mild injury group, but the intensity of expression was greater. Immunohistochemical staining showed that the number of neurons expressing the bFGF protein was less in the severe injury group than in the mild injury group. CONCLUSIONS: It is concluded that after mild injury there is a close relationship between the expression of the bFGF gene and the degree of histological change in the hippocampus; this indicates that as one of the growth factors, bFGF may participate in the protection and repair processes of neurons following brain injury. In severe injury there is a reduced expression of bFGF. The reason for this appears to be that more of the cells that have the potential to express bFGF have died, reducing the ability to express the bFGF gene. Conversely, it is possible that there may be an intrinsic insufficiency of expression of the gene, compatible with the known vulnerability of the hippocampus to many pathological conditions. Consideration should be given to supplying exogenous bFGF to protect the brain, particularly the hippocampus, after injury.
Animals↗
[Effect of nitric oxide on the myoelectric activity of sphincter of Oddi and arterial blood pressure of rabbit].
Thirty-two rabbits were used to investigate the effect of nitric oxide (NO) on the myoelectric activity of the sphincter of Oddi (SO) and arterial blood pressure. Intravenous infusion of NG-nitro-L-arginine (L-NNA), an inhibitor of nitric oxide synthase (NOS), caused increase in SO phasic wave amplitude and arterial blood pressure. The increase in SO phasic wave amplitude by infusion of L-NNA was reversed by L-arginine, but arterial blood pressure remained at a high level. It was observed that SO phasic wave amplitude, frequency and arterial blood pressure were significantly decreased by intravenously infusion of sodium nitroprusside (SNP), a substance producing NO. These observations suggest that (1) NO inhibits the resting activity of SO and modulates relaxation of vascular smooth muscle; (2) different tissues show different responses to L-NNA and L-arginine.
Animals↗
Glutamate-119 of the large alpha-subunit is the catalytic base in the hydration of 2-trans-enoyl-coenzyme A catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli.
Glu139 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was identified as the catalytic residue of enoyl-CoA hydratase [Yang, S.-Y., He, X.-Y., & Schulz, H. (1995) Biochemistry 34, 6441-6447]. To determine whether any of the other conserved protic residues is directly involved in the hydratase catalysis, the multienzyme complexes with either an alpha/Asp69 --> Asn or an alpha/Glu119 --> Gln mutation were overproduced and characterized. The catalytic properties of 3-ketoacyl-CoA thiolase and l-3-hydroxyacyl-CoA dehydrogenase of the mutant complexes were almost unaffected. The amidation of Asp69 and Glu119 caused a 7.6- and 88-fold decrease, respectively, in the kcat of enoyl-CoA hydratase without a significant change in the Km value of the hydratase as well as a 5.9- and 62-fold increase, respectively, in the Km of Delta3-cis-Delta2-trans-enoyl-CoA isomerase with a very small decrease in the kcat of the latter enzyme. The data suggest that the carboxyl group of Glu119 is particularly important to the catalytic activity of enoyl-CoA hydratase. Furthermore, the wild-type hydratase shows a bell-shaped pH dependence of the kcat/Km with pKa values of 5.9 and 9.2, whereas the Glu119 --> Gln mutant hydratase has only a single pKa of 9.5. A simple explanation for these observations is that a deprotonated Glu119 and a protonated Glu139 are required for the high kcat of the enoyl-CoA hydratase. The results of site-directed mutagenesis studies, together with the structural information about the spatial arrangement of two conserved glutamate residues of rat liver enoyl-CoA hydratase [Engel, C. K., Mathieu, M., Zeelen, J. P., Hiltunen, J. K., and Wierenga, R. K. (1996) EMBO J. 15, 5135-5145] to which Glu119 and Glu139 of the large alpha-subunit correspond, lead to the conclusion that the gamma-carboxyl group of Glu119 serves as the second general acid-base functional group in catalyzing the hydration of 2-trans-enoyl-CoA.
Acyl Coenzyme A↗
Cloning and expression of the fadH gene and characterization of the gene product 2,4-dienoyl coenzyme A reductase from Escherichia coli.
The fadH gene coding for an NADPH-dependent 2.4-dienoyl-CoA reductase from Escherichia coli has been cloned by the polymerase chain reaction. This gene is located at 67.65 min on the E. coli chromosome. The complete open reading frame contains 2019 bp coding for the processed protein of 671 amino acid residues, with a calculated molecular mass of 72.55 kDa, which lacks the N-terminal methionine. Construction and expression of the plasmid pNDH, which contained the fadH gene under the control of the T7 promoter, resulted in a 110-fold increase in the reductase activity above the level detected in E. coli cells containing the control vector. The kinetic parameters of the purified reductase were determined to be 50 microM and 2.3 microM for the Km values of NADPH and 2-trans, 4-trans-decadienoyl-CoA, respectively, and 16 s(-1) for the k(cat) value. Analysis of the kinetic data revealed that the reaction catalyzed by this enzyme proceeds via a ping-pong mechanism. The observed dissimilarity between the E. coli and mammalian 2,4-dienoyl-CoA reductase sequences suggests that they have evolved from distinct ancestral genes. Sequence analysis also suggests that the N-terminal part of the E. coli reductase contains the FAD-binding domain whereas the NADPH-binding domain is located in the C-terminal region of the protein.
Amino Acid Sequence↗