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X Y He

Publications and source records attributed to X Y He.

At least 37 records · Page 2Linked to original sources
PubMed1999

Induction of specific tolerance to allografts in rats by therapy with non-mitogenic, non-depleting anti-CD3 monoclonal antibody: association with TH2 cytokines not anergy.

BACKGROUND: Anti-CD3 monoclonal antibodies (mAb) are potent immunosuppressives in transplantation but most do not induce tolerance. They induce anergy in Th1 cells but, if they bind to Fc receptors on antigen presenting cells, they activate T cells to release cytokines. METHODS: This study examined the mechanisms of transplant tolerance induction to PVG fully allogeneic grafts in dark agouti rats by G4.18, a mouse immunoglobulinG3 anti-rat CD3 mAb that does not bind rat Fc receptors. Evidence of T cell activation was assayed by flow cytometry, reverse transcription (RT)-polymerase chain reaction (PCR) for cytokine mRNA, and responsiveness in mixed lymphocyte culture. RESULTS: G4.18 treatment modulated T cell receptor/CD3 and CD2 and depleted T cells by <20% but did not induce activation surface markers. mRNA for interleukin (IL)-2, interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, and IL-4 in the lymph node, spleen, and thymus was not increased, and IFN-gamma mRNA was reduced. G4.18-treated and naive rat cells had similar proliferation and expression of IL-2, IFN-gamma, and IL-4 in vitro. G4.18-treated allograft recipients had no induction of mRNA for IL-2, IFN-gamma, TNF-alpha, TNF-beta, IL-4, IL-5, IL-10, perforin, and granzyme A & B in the spleen or grafts, with levels similar to those in isografts. The IL-4 and IL-5 mRNA levels in the spleen but not the graft of G4.18-treated recipients were higher than in rejecting and naive animals. Cells from G4.18-treated graft recipients proliferated more rapidly to the donor than to the third party and had increased IL-4 expression. CONCLUSIONS: G4.18 induced transplant tolerance by a combination of modulation and blocking of the TCR/CD3, associated with increased Th2 cytokines, without depletion, induction of anergy, or nonspecific activation of T cells.

Animals↗
PubMed1999

Identity of heart and liver L-3-hydroxyacyl coenzyme A dehydrogenase.

Rat heart and liver cDNAs for precursor of L-3-hydroxyacyl-CoA dehydrogenase have been cloned and sequenced. The results indicate that these different rat organs express identical dehydrogenases. Furthermore, pig heart mRNA for L-3-hydroxyacyl-CoA dehydrogenase precursor was amplified by reverse transcription-polymerase chain reaction, and all the cDNA clones were found to encode a precursor of liver L-3-hydroxyacyl-CoA dehydrogenase (X.-Y. He, S.-Y. Yang, Biochim. Biophys. Acta 1392 (1998) 119-126) but not the well-documented heart form of the dehydrogenase (K.G. Bitar et al., FEBS Lett. 116 (1980) 196-198). Sequencing data and other evidence establish that the pig, like the rat, has the same dehydrogenase in heart and liver. Since the size and structure of pig heart L-3-hydroxyacyl-CoA dehydrogenase are identical to the pig liver dehydrogenase, reports that relied on the published sequence of the pig heart dehydrogenase need to be re-evaluated. For example, the signature pattern of the L-3-hydroxyacyl-CoA dehydrogenase family is HXFXPX3MXLXE. Furthermore, the published crystal structure of the pig heart dehydrogenase that substantiated each subunit comprising 307 residues with a mercury-binding residue at position 204 (J.J. Birktoft et al., Proc. Natl. Acad. Sci. U.S.A. 84 (1987) 8262-8266) must be re-examined in accordance with this revelation.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1998

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↗
PubMed1998

Treatment with interleukin-4 prolongs allogeneic neonatal heart graft survival by inducing T helper 2 responses.

BACKGROUND: The T helper (Th) 2 cytokine interleukin (IL)-4 has been implicated as a major regulatory cytokine for the induction of transplant tolerance, but few studies have examined the capacity of IL-4 to induce tolerance. The effect of IL-4 therapy alone or with low doses of anti-CD4 monoclonal antibody (mAb) therapy on survival of fully allogeneic PVG neonatal heart graft in adult DA rats was examined. METHODS: Rat recombinant (r) IL-4 was given at 30 microg (10(4) U)/kg daily intraperitoneally for 10 days and MRC OX35 (anti-CD4, nondepleting) or MRC OX81 (anti-IL-4) was given intraperitoneally on days 0, 3, 7, and 10. Semiquantitative reverse transcriptase-polymerase chain reaction was used to assay mRNA for cytokine in the graft, regional node and spleen and fluorescence-activated cell sorting was used to assay alloantibody Ig isotypes. RESULTS: Grafts in rIL-4-treated rats survived a median period of 39 days (range, 28-52 days), significantly longer than in both untreated and nontransfected Chinese hamster ovary-K1 supernatant-treated controls (median, 14 days; range, 10-16 days, P=0.009). rIL-4 treatment with a suboptimal dose of anti-CD4 mAb prolonged median survival to 70 days (range, 63-80 days), which was longer than rIL-4 treatment alone or anti-CD4 mAb alone (median, 36 days; range, 30-55 days; P<0.0045). Combining MRC OX81 with MRC OX35 therapy led to earlier rejection at a median period of 26 days (range, 20-28 days); MRC OX81 alone had no effect on graft survival. Alloantibody titers, especially IgG1, were higher in rIL-4-treated animals and lower in anti-CD4 mAb-treated animals than in animals with normal rejection (P<0.05). IL-4 mRNA was increased in regional lymph nodes and spleen of the rIL-4-treated groups compared with all other groups, but there were no differences for IL-2, interferon-gamma, or IL-10. CONCLUSIONS: rIL-4 therapy markedly prolonged neonatal cardiac allograft survival, and, with anti-CD4 therapy, it further prolonged survival. It induced IL-4 mRNA in lymphoid tissues and enhanced alloantibody production, especially IgG1, which demonstrated enhanced Th2 responses, but did not affect Th1 cytokines.

Animals↗
PubMed1998

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↗
PubMed1998

Humanization and pharmacokinetics of a monoclonal antibody with specificity for both E- and P-selectin.

E- and P-selectin (CD62E and CD62P) are cell adhesion molecules that mediate leukocyte-endothelial cell and leukocyte-platelet interactions and are involved in leukocyte recruitment during inflammation. We previously developed a murine mAb, EP-5C7 (or mEP-5C7), that binds and blocks both E- and P-selectin. When used in humans, murine mAbs have short circulating half-lives and generally induce potent human anti-mouse Ab responses. We therefore engineered a humanized, complementarity determining region-grafted version of mEP-5C7 incorporating human gamma4 heavy and kappa light chain constant regions (HuEP5C7.g4). HuEP5C7.g4 retains the specificity and avidity of mEP-5C7, binding to human E- and P-selectin but not to human L-selectin, and blocking E- and P-selectin-mediated adhesion. Surprisingly, when administered to rhesus monkeys, HuEP5C7.g4 was eliminated from the circulation very rapidly, even faster than the original murine Ab. To isolate the cause of the short serum half-life of HuEP5C7.g4, several Ab variants were constructed. A chimeric IgG4 Ab was made by replacing the humanized V regions with murine V regions. A humanized IgG2 Ab, HuEP5C7.g2, was also made by replacing the human gamma4 with a gamma2 constant region. Results from pharmacokinetic studies in rhesus monkeys demonstrated that the chimeric IgG4 is also rapidly eliminated rapidly from serum, similar to the humanized IgG4 Ab, while the humanized IgG2 Ab displays a long circulation half-life, typical of human Abs.

Amino Acid Sequence↗
PubMed1998

In situ hybridization of hepatitis B DNA in hepatitis B-associated glomerulonephritis.

Renal tissues from 43 of 49 children with hepatitis B virus-associated glomerulonephritis (HBV-GN) were examined for HBV DNA by in situ hybridization (ISH) assay within the last 10 years. HBV DNA was identified in 41 of the 43 cases (95.3%). HBV DNA was distributed generally in the nucleus and cytoplasm of epithelial cells and mesangial cells of glomeruli, and epithelial cells of renal tubules. HBV DNA also existed simultaneously in renal interstitial tissues in some of these cases. The positive results from HBV DNA ISH correlated well with HBV antigen assays. The analyses implied that the more extensive the existence of HBV DNA in the nephron unit and interstitial tissue, the more severe the clinical manifestation. The duration of proteinuria in cases with HBV DNA in renal tubules was much longer than in those with no HBV DNA in renal tubules. The persistence of the HBV genome or genes in the kidney could lead to the expression of viral antigens in renal tissues and might cause cellular pathological alteration. This would support utilization of antiviral therapy, such as cytokines, in the treatment of HBV-GN.

Biomarkers↗
PubMed1997

Induction of tolerance with nondepleting anti-CD4 monoclonal antibodies is associated with down-regulation of TH2 cytokines.

BACKGROUND: Induction of tolerance with anti-CD4 has mainly focused on monoclonal antibodies (mAbs) that deplete CD4+ T cells. In this study, the mechanisms by which nondepleting anti-CD4 mAbs induce tolerance in the Dark Agouti to PVG rat heart graft model were examined. METHODS: Five anti-CD4 mAbs were tested. Immunohistology and cytokine mRNA profiles were analyzed within grafts. Effects of combining anti-CD4 therapy with alloantibody (alloAb), interleukin (IL)-4, and anti-IL-4 mAb were also examined. RESULTS: All mAbs tested induced indefinite graft survival (>150 days), with blocking of alloAb production. Exogenous alloAb did not restore rejection. Similar T cell receptor alphabeta+, CD8+, IL-2 receptor+ T cell, macrophage, and natural killer cell infiltration and comparable MHC II and intercellular adhesion molecule-1 levels were seen in rejecting and tolerant grafts. mRNA for IL-2, interferon-gamma, lymphotoxin, tumor necrosis factor-alpha, transforming growth factor-beta, cytolysin, and granzyme-A/B was comparable, although inducible nitric oxide synthase was slightly reduced in tolerant grafts. IL-4 and IL-5 were significantly reduced in tolerant grafts, although IL-6, IL-10, and IL-13 levels were similar; this was consistent with partial T helper (Th)2 response inhibition, which was also manifested by inhibited alloAb. The combination of alloAb, IL-4, or anti-IL-4 mAb with anti-CD4 did not prevent tolerance induction. CONCLUSIONS: This study demonstrated that anti-CD4 mAb therapy did not inhibit activation and infiltration of Th1 and CD8+ effector T cells. Preferential induction of Th2 responses, especially IL-4, was not essential for the induction of tolerance. Our studies also found no evidence to support induction of anergy or transforming growth factor-beta as mechanisms of tolerance induction. These results question whether IL-4 is required for induction of transplantation tolerance.

Animals↗
PubMed1997

MIC-1, a novel macrophage inhibitory cytokine, is a divergent member of the TGF-beta superfamily.

Macrophages play a key role in both normal and pathological processes involving immune and inflammatory responses, to a large extent through their capacity to secrete a wide range of biologically active molecules. To identify some of these as yet not characterized molecules, we have used a subtraction cloning approach designed to identify genes expressed in association with macrophage activation. One of these genes, designated macrophage inhibitory cytokine 1 (MIC-1), encodes a protein that bears the structural characteristics of a transforming growth factor beta (TGF-beta) superfamily cytokine. Although it belongs to this superfamily, it has no strong homology to existing families, indicating that it is a divergent member that may represent the first of a new family within this grouping. Expression of MIC-1 mRNA in monocytoid cells is up-regulated by a variety of stimuli associated with activation, including interleukin 1beta, tumor necrosis factor alpha (TNF-alpha), interleukin 2, and macrophage colony-stimulating factor but not interferon gamma, or lipopolysaccharide (LPS). Its expression is also increased by TGF-beta. Expression of MIC-1 in CHO cells results in the proteolytic cleavage of the propeptide and secretion of a cysteine-rich dimeric protein of Mr 25 kDa. Purified recombinant MIC-1 is able to inhibit lipopolysaccharide -induced macrophage TNF-alpha production, suggesting that MIC-1 acts in macrophages as an autocrine regulatory molecule. Its production in response to secreted proinflammatory cytokines and TGF-beta may serve to limit the later phases of macrophage activation.

Amino Acid Sequence↗
PubMed1997

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↗
PubMed1997

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↗
PubMed1997

Importance of the gamma-carboxyl group of glutamate-462 of the large alpha-subunit for the catalytic function and the stability of the multienzyme complex of fatty acid oxidation from Escherichia coli.

His450 of the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli was recently identified as an essential catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase [He, X-Y., & Yang, S.-Y. (1996) Biochemistry 35, 9625-9630]. To explore the roles of acidic residues in the dehydrogenase catalysis, every conserved acidic residue in the dehydrogenase functional domain except for those in the NAD-binding motif was replaced with alanine. The resulting mutant complexes were overproduced and characterized. Their component enzymes other than the dehydrogenase were affected very slightly. Removal of the beta-carboxyl group of Asp524 and Asp542 caused only a 3- and 4-fold, respectively, decrease in the catalytic efficiency of the dehydrogenase, thereby showing that their involvement in the dehydrogenase catalysis was limited. In contrast, the alpha/Glu462-->Ala mutant complex showed a greater than 160-fold reduction in the kcat of the dehydrogenase in the forward direction without a significant change of the k(m) for the substrate. The catalytic properties of the alpha/Glu462-->Gln mutant complex were found to be similar to those of the alpha/Glu462-->Ala mutant complex except that the kcat of the dehydrogenase in the backward direction was about 4-fold lower and the Km for the substrate of the thiolase was 6-fold higher. It is concluded that the negative charge of the gamma-carboxyl group of Glu462, but not its ability to form a hydrogen bond, is critical for its interaction with His450, thereby assisting in the catalysis of the dehydrogenase. The pKa of His450 in the E.NADH binary complex was virtually unchanged by the replacement of Glu462 with Ala or Gln. It seems that the binding of substrate is necessary for forming a strong interaction between His450 and Glu462 with the result that the electroneutrality in the active site is maintained and the activation energy of the reaction is lowered. Additionally, the negative charge of Glu462 increases the thermostability of the multienzyme complex.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1996

Histidine-450 is the catalytic residue of L-3-hydroxyacyl coenzyme A dehydrogenase associated with the large alpha-subunit of the multienzyme complex of fatty acid oxidation from Escherichia coli.

Multienzyme complexes of fatty acid oxidation from Escherichia coli with Gln or Ala substituting for His450 or with Ala in place of Gly322 in the large alpha-subunit have been purified and characterized. The alpha/Gly322-->Ala mutation did not significantly affect the catalytic efficiencies (kcat/k(m)) of different component enzymes except for a 6.1-fold decrease in the kcat/k(m) of L-3-hydroxyacyl-CoA dehydrogenase and a 10-fold increase in the k(m) for NADH. This observation confirms the prediction [Yang, X.-Y. H., Schulz, H., Elzinga, M., & Yang, S.-Y. (1991) Biochemistry 30, 6788-6795] that the E. coli dehydrogenase has an NAD-binding site at its amino-terminal domain and structurally resembles the pig heart dehydrogenase. The pH dependence of the kcat/k(m) of the E. coli dehydrogenase suggested the catalytic involvement of an amino acid residue with a pKa of 6, which is presumably a histidine residue as proposed previously on the basis of chemical modifications. Since His450 of the E. coli multifunctional protein is the only histidine conserved in all known L-3-hydroxyacyl-CoA dehydrogenases, and since its counterpart in pig heart enzyme appeared to be close to the 3-keto group of the fatty acyl moiety of the substrate, His450 was replaced by either Gln or Ala. The catalytic properties of 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, and delta 3-cis-delta 2-trans-enoyl-CoA isomerase of the alpha/His450-->Gln mutant complex were virtually unchanged except for a small decrease in the kcat values of the latter two enzymes. In contrast, the dehydrogenase of this mutant complex was almost inactive due to a greater than 3000-fold decrease in its kcat and a 6-fold increase in the k(m) for NADH. The alpha/His450-->Ala mutant complex showed similar catalytic behaviors. Taken together, several lines of evidence lead to the conclusion that His450 is the catalytic residue of L-3-hydroxyacyl-CoA dehydrogenase of the E. coli multifunctional fatty acid oxidation protein.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1995

Peroxisomes contain delta 3,5,delta 2,4-dienoyl-CoA isomerase and thus possess all enzymes required for the beta-oxidation of unsaturated fatty acids by a novel reductase-dependent pathway.

The presence of delta 3,5,delta 2,4-dienoyl-CoA isomerase in peroxisomes was demonstrated by determining the subcellular distribution of this enzyme in rat liver. The peroxisomal and mitochondrial forms of the isomerase exhibit similar chain length specificities and they are homologous as indicated by the recognition of the peroxisomal 66-kDa enzyme by an antiserum raised against the mitochondrial 32-kDa isomerase. This report demonstrates that peroxisomes contain all enzymes required for the beta oxidation of unsaturated fatty acids with odd-numbered double bonds by a novel pathway in which double bonds are reductively removed by the NADPH-dependent 2,4-dienoyl-CoA reductase.

Animals↗
PubMed1995

Glutamate 139 of the large alpha-subunit is the catalytic base in the dehydration of both D- and L-3-hydroxyacyl-coenzyme A but not in the isomerization of delta 3, delta 2-enoyl-coenzyme A catalyzed by the multienzyme complex of fatty acid oxidation from Escherichia coli.

Multienzyme complexes of fatty acid oxidation from Escherichia coli with either an alpha/Glu139-->Gln or an alpha/Arg134-->Gln mutation in the large alpha-subunit have been overproduced and characterized. The catalytic properties of the five different component enzymes of the alpha/Arg134-->Gln mutant complex showed no significant changes as compared with those of the wild type complex. In contrast, the 3-hydroxyacyl-coenzyme A (CoA) epimerase activity of the alpha/Glu139-->Gln mutant complex was not detected, and this mutant complex has lost almost all of the enoyl-CoA hydratase activity due to a greater than 3000-fold decrease in the kcat of the enoyl-CoA hydratase without a significant change in the Km value. The catalytic properties of 3-ketoacyl-CoA thiolase and L-3-hydroxyacyl-CoA dehydrogenase were virtually unaffected by the mutation. Together, these observations lead to the conclusion that the gamma-carboxylic group of Glu139 functions as a catalytic base in the dehydration of both D- and L-3-hydroxyacyl-CoA. These findings also support a dehydration/hydration mechanism for 3-hydroxyacyl-CoA epimerase but do not agree with an epimerase activity independent of enoyl-CoA hydratase as proposed for the glyoxysomal tetrafunctional protein [Preisig-Müller, R., Gühnemann-Schäfer, K., & Kindl, H. (1994) J. Biol. Chem. 269, 20475-20481]. Since this mutation caused the kcat of delta 3-cis-delta 2-trans-enoyl-CoA isomerase to decrease by only 60%, even though the Km value was significantly increased, it seems that Glu139 of the E. coli multifunctional protein does not function as a catalytic residue in the isomerization reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Hydroxyacyl CoA Dehydrogenases↗