Large Alpha Subunit Research Articles

Immunotoxin Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemic Cells

Cell surface molecules aberrantly or overexpressed by myeloid leukemic cells represent potential disease-specific therapeutic targets for antibodies. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a target. MUC1 is cell surface molecule with extracellular, transmembrane, and cytoplasmic domains; the cytoplasmic tail participates in intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit bound in strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic MUC1 alpha chain. Because the MUC1 alpha chain binds the cell only intermittently in an ‘on-and-off’ manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on leukemic cells by flow cytometry. DMB-5F3 was found to strongly bind not all AML, but rather a specific set of AML subtypes: Chronic Myelomonocytic Leukemia (CMML); Acute Myelomonocytic and Monocytic Leukemia (AML-M4 and AML-M5), and Juvenile Myelomonocytic Leukemia (JMML). Other AML subtypes did not express MUC1. To test the cytotoxic ability of anti-MUC1 antibodies against MUC-expressing AML, we generated a humanized, dimeric single-chain DMB-5F3 scFv fragments fused to recombinant gelonin, a type I ribosome-inactivating toxin which itself is incapable of cell binding or internalization into mammalian cells to which a 6xHis-tag was added. The anti-MUC1 scFv-toxin cytometrically identified using an anti-6xHis-tag was found to bind MUC1 positive cell line. We examined the cytotoxic effects of anti MUC1 scFV-toxin using the MUC-1 positive AML cell lines MV411 and MOLM14 (monocytic leukemic subtype). MUC1 scFV-toxin was found to be highly cytotoxic on MUC1+ AML cell lines, resulting in direct cell death. Our data strongly support the use of an anti-MUC 1 antibody-drug conjugate to selectively target and inhibit the growth of MUC1-expressing AML. DisclosuresNo relevant conflicts of interest to declare.

Targeting Cell-Bound MUC1 on Myelomonocytic and Monocytic Leukemias and Leukemic Stem Cells: Therapeutic Implications

Monocytic neoplasms comprise a heterogeneous group of hematologic malignancies including chronic myelomonocytic leukemia (CMML), juvenile myelomonocytic leukemia (JMML), acute myelomonocytic and monocytic leukemia (AML-M4 and AML-M5), and monocytic sarcoma. Monocytic or granulomonocytic hyperplasia is a finding frequently-if not invariably-shared by these different entities, as is a poor therapeutic outcome in the absence of hematopoietic stem cell transplantation. Cell surface molecules aberrantly expressed or overexpressed by leukemic cells represent potential disease-specific therapeutic targets. MUC1, a polymorphic type I high molecular weight glycoprotein represents such a molecule. MUC1 consists of an extracellular domain containing 20 to 125 tandem repeats of a 20 amino acid-long sequence, followed by a transmembrane domain and a short cytoplasmic tail leading to intracellular signaling. Cleavage of MUC1 yields two unequal chains: a large extracellular alpha subunit containing the tandem repeat array bound in a strong non-covalent interaction to a smaller beta subunit containing the transmembrane and cytoplasmic domains. Essentially all anti-MUC1 antibodies reported to date target the highly immunogenic tandem repeat of the MUC1 alpha chain. Because the alpha chain binds the cell-bound domains of MUC1 only intermittently in an 'on-and-off' manner, agents directed against the alpha chain will not effectively target MUC1+ cells. In contrast, the MUC1 SEA domain represents a stable structure fixed to the cell surface at all times. We therefore generated mAbs that specifically recognize the cell-bound MUC1 SEA domain. One of them, a partially humanized murine mAb termed DMB-5F3 was used to examine the expression of MUC1 on AML cells by flow cytometry. A series of twenty-two AML samples (blood-derived n=12; bone marrow-derived n=10; AML0=2, AML1=2, AML2=10, AML4=1, AML5=5, AML6=2) collected either at the time of diagnosis or at relapse were analysed for MUC1 expression by flow cytometry. A murine mammary tumor cell line stably transfected with human MUC1 DNA served as control. Blasts cells from 5 AML samples highly expressed MUC1, and significantly, all were of monocytic or myelomonocytic lineage (AML4=1, AML5=4). Leukemic stem cells (CD34pos or CD34neg linneg) from the MUC1+ AMLs were examined and likewise found to express MUC1. In addition, AML cell lines MV411, MOLM14, and SHI-1 derived from monocytic leukemic lineage clearly expressed cell surface MUC1, while non- monocytic leukemic cell lines U937, K562, and HL60 had little or no expression. Normal monocytes and monocytes derived from patients with activated monocytosis were also found to express MUC1. Based on these findings we examined MUC1 expression in a series of myelomonocytic leukemia (CMML and JMML). In fifteen CMML samples examined (type 1 n=11, type 2 n=4) (blood n=7, BM n=7) 92%-100% (median 99.7%) of CD14+CD56+ CMML cells bound mAb DMB-5F3 to cell-surface MUC1. CD14+CD16+CD56+ blast cells from 2 pts with JMML were also found to express MUC1 (between 64% and 71 % positive). Based on these findings we conclude that expression of MUC1 is restricted to monocytic and myelomonocytic leukemias and that MUC1 represents an effective target for leukemic immunotherapy. Significantly, anti-MUC1 mAb also targets monocytic leukemic stem cells, reinforcing its therapeutic potential. The fact that the anti-MUC1 antibody DMB-5F3 can enter cells and thereby ferry Ab-bound toxin opens the way for us to demonstrate leukemic cell killing with anti-MUC1 mAb-immunotoxin conjugates. DisclosuresMoreau:Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen-Cilag: Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees; Millennium: Honoraria, Membership on an entity's Board of Directors or advisory committees.

The Glucoamylase Inhibitor Acarbose Is a Direct Activator of Phosphorylase Kinase

Phosphorylase kinase (PhK), an (alphabetagammadelta)(4) complex, stimulates energy production from glycogen in the cascade activation of glycogenolysis. Its large homologous alpha and beta subunits regulate the activity of the catalytic gamma subunit and account for 81% of PhK's mass. Both subunits are thought to be multidomain structures, and recent predictions based on their sequences suggest the presence of potentially functional glucoamylase (GH15)-like domains near their amino termini. We present the first experimental evidence of such a domain in PhK by demonstrating that the glucoamylase inhibitor acarbose binds PhK, perturbs its structure, and stimulates its kinase activity.

Subunit Structure of Benzylsuccinate Synthase

Benzylsuccinate synthase is a member of the glycyl radical family of enzymes. It catalyzes the addition of toluene to fumarate to form benzylsuccinate as the first step in the anaerobic pathway of toluene fermentation. The enzyme comprises three subunits, alpha, beta, and gamma, that in Thauera aromatica strain T1 are encoded by the tutD, tutG, and tutF genes, respectively. The large alpha-subunit contains the essential glycine and cysteine residues that are conserved in all glycyl radical enzymes. However, the function of the small beta- and gamma-subunits has remained unclear. We have overexpressed all three subunits of benzylsuccinate synthase in Escherichia coli, both individually and in combination. Coexpression of the gamma-subunit (but not the beta-subunit) is essential for efficient expression of the alpha-subunit. The benzylsuccinate synthase complex lacking the glycyl radical could be purified as an alpha(2)beta(2)gamma(2) hexamer by nickel affinity chromatography through a "His(6)" affinity tag engineered onto the C-terminus of the alpha-subunit. Unexpectedly, BSS was found to contain two iron-sulfur clusters, one associated with the beta-subunit and the other with the gamma-subunit that appear to be necessary for the structural integrity of the complex. The spectroscopic properties of these clusters suggest that they are most likely [4Fe-4S] clusters. Removal of iron with chelating agents results in dissociation of the complex; similarly, a mutant gamma-subunit lacking the [4Fe-4S] cluster is unable to stabilize the alpha-subunit when the proteins are coexpressed.

3D mapping of glycogenosis-causing mutations in the large regulatory alpha subunit of phosphorylase kinase

Mutations in the liver isoform of the Phosphorylase Kinase (PhK) α subunit (PHKA2 gene) cause X-linked liver glycogenosis (XLG), the most frequent type of PhK deficiency (glycogen-storage disease type IX). XLG patients can be divided in two subgroups, with similar clinical features but different activity of PhK (decreased in liver and blood cells for XLG-I and low in liver but normal or enhanced in blood cells for XLG-II). Here, we show that the PHKA2 missense mutations and small in-frame deletions/insertions are concentrated into two domains of the protein, which were recently described. In the N-terminal glucoamylase domain, mutations (principally leading to XLG-II) are clustered within the predicted glycoside-binding site, suggesting that they may have a direct impact on a possible hydrolytic activity of the PhK α subunit, which remains to be demonstrated. In the C-terminal calcineurin B-like domain (domain D), mutations (principally leading to XLG-I) are clustered in a region predicted to interact with the regulatory region of the PhK catalytic subunit and in a region covering this interaction site. Altogether, these results show that PHKA2 missense mutations or small in-frame deletions/insertions may have a direct impact on the PhK α functions and provide a framework for further experimental investigation.

Protein engineering on biphenyl dioxygenase for conferring activity to convert 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin)

A central part (amino-acid position 268-397 of 458 amino-acid residues) of the biphenyl dioxygenase large (alpha) subunit, BphA1, from Pseudomonas pseudoalcaligenes strain KF707 was exchanged with the corresponding part of BphA1 from another biphenyl-degrading bacterium, Pseudomonas putida strain KF715, to construct hybrid BphA1, BphA1 (715-707). When expressed in Escherichia coli together with the bphA2A3A4BC genes from strain KF707, this enzyme was shown to possess activity for degrading both 1-phenylnaphthalene and 2-phenylnaphthalene. Between central parts of BphA1 from strains KF707 and KF715, the difference of amino-acid residues resided only in position 324-325. An attempt was made to improve the substrate preference of BphA1 by applying random amino-acid substitutions at these positions to BphA1 (715-707). After screening the mutant library to bioconvert several flavonoids, BphA1 (1-22; T324A and I325L) and BphA1 (2-2; T324L and I325I) were selected. When expressed in E. coli together with bphA2A3A4B from strain KF707, both BphA1 (1-22) and BphA1 (2-2) bioconverted the refractory flavonoids, 7-hydroxyflavone and 5,7-dihydroxyflavone (chrysin), which were hardly converted by any unmodified and artificially-modified shuffled biphenyl dioxygeneses, into their vicinal diol forms, i.e., 2-(2,3-dihydroxyphenyl)-7-hydroxy-chromen-4-one and 2-(2,3-dihydroxyphenyl)-5,7-dihydroxy-chromen-4-one, respectively. In addition, trans-chalcone was converted into 3-(2,3-dihydroxyphenyl)-1-phenylpropan-1-one and further into 1,3-bis-(2,3-dihydroxyphenyl)-propan-1-one. The antioxidative activity of these generated compounds was markedly higher than that of the original substrates used.

Calcineurin B‐like domains in the large regulatory α/β subunits of phosphorylase kinase

Phosphorylase kinase (PhK) is a large hexadecameric complex that catalyzes the phosphorylation and activation of glycogen phosphorylase (GP). It consists in four copies each of a catalytic subunit (gamma) and three regulatory subunits (alpha beta delta). Delta corresponds to endogenous calmodulin, whereas little is known on the molecular architecture of the large alpha and beta subunits, which probably arose from gene duplication. Here, using sensitive methods of sequence analysis, we show that the C-terminal domain (named domain D) of these alpha and beta subunits can be significantly related to calcineurin B-like (CBL) proteins. CBL are members of the EF-hand family that are involved in the regulation of plant-specific kinases of the CIPK/PKS family, and relieve autoinhibition of their target kinases by binding to their regulatory region. The relationship highlighted here suggests that PhK alpha and/or beta domain D may be involved in a similar regulation mechanism, a hypothesis which is supported by the experimental observation of a direct interaction between domain D of PhKalpha and the regulatory region of the Gamma subunit. This finding, together the identification of significant similarities of domain D with the preceding domain C, may help to understand the molecular mechanism by which PhK alpha and/or beta domain D might regulate PhK activity.

KCNMA1 gene amplification promotes tumor cell proliferation in human prostate cancer

Molecular mechanisms of prostate cancer progression are poorly understood. Here, we studied gene amplification of the large conductance calcium-activated potassium channel alpha subunit (KCNMA1), which is located at the chromosomal region 10q22. Fluorescence in situ hybridization (FISH) revealed KCNMA1 amplification in 16% of 119 late-stage human prostate cancers and in the hormone-insensitive prostate cancer cell line PC-3. In contrast, KCNMA1 amplification was absent in 33 benign controls, 32 precursor lesions and in 105 clinically organ-confined prostate cancers. Amplification was associated with mRNA and protein overexpression as well as increased density of BK channel protein and beta-estradiol-insensitive BK currents in PC-3 cells as compared to non-amplified control cell lines. Specific blockade of BK channels by iberiotoxin or RNA(i) significantly inhibited K(+) currents and growth of PC-3 cells. The data demonstrate that 10q22 amplification drives KCNMA1 expression and cell proliferation. Thus, KCNMA1 qualifies as a promising diagnostic and therapeutic target in patients with prostate cancer.

Stimulation of the XPB ATP-dependent helicase by the beta subunit of TFIIE.

TFIIE and TFIIH are essential for the promoter opening and escape that occurs as RNA polymerase II transits into early elongation. XPB, a subunit of TFIIH, contains an ATP-dependent helicase activity that is used in both of these processes. Here, we show that the smaller beta subunit of TFIIE stimulates the XPB helicase and ATPase activities. The larger alpha subunit can use its known inhibitory activity to moderate the stimulation by the beta subunit. Regions of TFIIE beta required for the helicase stimulation were identified. Mutants were constructed that are defective in stimulating the XPB helicase but still allow intact TFIIE to bind and recruit XPB and TFIIH to form the pre-initiation complex. In a test for the functional significance of the stimulatory effect of TFIIE beta, these mutant forms of TFIIE were shown to be defective in a transcription assay on linear DNA. The data suggest that the beta subunit of TFIIE is an ATPase and helicase co-factor that can assist the XPB subunit of TFIIH during transcription initiation and the transition to early elongation, enhancing the potential diversity of regulatory targets.

Expression and distribution of voltage-gated sodium channels in the cerebellum.

In order to understand the effects of sodium channels on synaptic signaling and response in the cerebellum, it is essential to know for each class of neuron what sodium channel isoforms are present, and the properties and distribution of each. Sodium channels are heteromultimeric membrane proteins, consisting of a large alpha subunit that forms the pore, and one or more beta subunits. Ten genes encode an alpha subunit in mammals, and of these, four are expressed in the cerebellum: Nav1.1, Nav1.2, Nav1.3 and Nav1.6. Three genes encode beta subunits (Nabeta1-3), and all three are expressed in the cerebellum. However, Nav1.3 and Nabeta3 have been found only in the developing cerebellum. All sodium channels recorded in the cerebellum are TTX-sensitive with similar kinetics, making it difficult to identify the isoforms electrically. Thus, most of the expression studies have relied on techniques that allow visualization of sodium channel subtypes at the level of mRNA and protein. In situ hybridization and immunolocalization studies demonstrated that granule cells predominantly express Nav1.2, Nav1.6, Nabeta1, and Nabeta2. Protein for Nav1.2 and Nav1.6 is localized primarily in granule cell parallel fibers. Purkinje cells express Nav1.1, Nav1.6, Nabeta1 and Nabeta2. The somato-dendritic localization of Nav1.1 and Nav1.6 in Purkinje cells suggests that these isoforms are involved in the integration of synaptic input. Deep cerebellar nuclei neurons expressed Nav1.1 and Nav1.6 as well as Nabeta1. Bergmann glia expressed Nav1.6, but not granule cell layer astrocytes. Some sodium channel isoforms that are not expressed normally in the adult cerebellum are expressed in animals with mutations or disease. Electrophysiological studies suggest that Nav1.6 is responsible for spontaneous firing and bursting features in Purkinje cells, but the specialized functions of the other subunits in the cerebellum remain unknown.

BeWo choriocarcinoma cells produce laminin 10.

BeWo is a choriocarcinoma cell line that generates an extracellular matrix (ECM) rich in laminin and is a useful model for human trophoblast. Immunofluorescence with monoclonal antibodies demonstrates that BeWo ECM contains laminin subunits beta1 and gamma1. Immunoprecipitation from conditioned medium shows that the cells secrete two distinct laminin trimers both containing beta1 and gamma1 but with alpha subunits of approx. 400 and 450 kDa. The culture medium also contains a species thought to be beta1 gamma1 dimer. Immunoprecipitation with monoclonal antibody 4C7, previously thought to recognize the alpha1 subunit, isolates complexes containing only the smaller alpha subunit. A second complex containing the larger alpha subunit along with beta1, gamma1 and a 150 kDa polypeptide is precipitated from 4C7-depleted medium with an anti-(laminin 1) polyclonal antibody. Peptide sequencing demonstrates that the 4C7-reactive species is alpha5, which is present as two similarly sized polypeptides. mRNA species encoding laminin subunits alpha1, alpha5, beta1, beta2 and gamma1 are all present in the cells. These results demonstrate the secretion of a novel laminin isoform, laminin 10, the subunit composition of which is alpha5 beta1 gamma1. Laminin 1 is also produced. No evidence for the secretion of beta2-containing laminin isoforms could be derived despite the presence of beta2 mRNA. Analysis with reverse transcriptase-mediated PCR also showed the presence of laminin alpha5 in first-trimester placenta and decidua.

Localization and development of chromogranin A and luteinizing hormone immunoreactivities in the secretory-specific cells of the hypophyseal pars tuberalis of the chicken.

The pars tuberalis mainly consists of the secretory cells specific to this portion of the pituitary. We examined the localization and development of luteinizing hormone (LH) and chromogranin A in the chicken pars tuberalis by immunohistochemistry. The vast majority of the chicken pars tuberalis was occupied by cells immunoreactive for both LH and chromogranin A. Furthermore, immunoblot analysis of chicken pars tuberalis extracts with LH antiserum demonstrated that two bands, the large alpha-subunit and small beta-subunit of the LH molecule, were expressed in this tissue as well as in the pars distalis. A band for chromogranin A was also detected in pars tuberalis extracts with chromogranin A antiserum. In contrast to the cells of mammalian species that contain only a few small secretory granules, the specific cells of the chicken pars tuberalis were characterized by the presence of many secretory granules ranging from 90 to 400 nm in diameter. Postembedding immunogold labeling showed that gold particles representing immunoreactivity for LH were densely located on all secretory granules of the secretory-specific cells. Many secretory granules, especially the large ones, of the cells were also loaded with immunogold particles for chromogranin A. Double immunogold labeling confirmed that LH and chromogranin A were colocalized on the same secretory granules. During embryonic development, the primordium of the pars tuberalis was first detected at 8 days of incubation as a small group of cells containing LH- and chromogranin-immunoreactive cells. In the pars distalis, the onset of LH and chromogranin expression occurred earlier, at 6 days of incubation. At 10 days of incubation, the pars tuberalis primordium became large cell masses consisting of LH- and chromogranin-immunoreactive cells, which were located close to the median eminence. Subsequently, the primordium extended along the median eminence progressively with age. At 14 days of incubation, it reached to the rostral end and surrounded the median eminence as slender cell cords. These results indicate that specific cells of the chicken pars tuberalis synthesize a glycoprotein hormone related to the LH molecule, which is stored in the secretory granules together with chromogranin A. The pars tuberalis may be involved in the regulation of gonadal function in a different way from that of the pars distalis.

Isolation of a human-brain sodium-channel gene encoding two isoforms of the subtype III alpha-subunit.

Voltage-gated sodium channels are members of a multigene family of transmembrane proteins that are important determinants of electrical excitability in cell membranes. These proteins are typically composed of a large alpha-subunit and one or two beta-subunits. The primary structure of alpha-subunits is highly conserved among different subtypes and different species. Based on the conserved sequences and application of the rapid amplification of cDNA ends (RACE) reaction, we have isolated three overlapping clones from human brain. These sequences share highest homology (89%) to the rat brain subtype III gene and cover a 4.2-kb expanse of the transcript. The 5'-most clone has a translation start site located in the same region as other mammalian brain sodium channel genes. A 92-nucleotide insert was found in domain I at a location previously demarcated by published splice sites in rat brain sodium channels IIN/IIA and IIIN/IIIA. It is most likely that this transcript represents the two isoforms (neonatal and adult) of the human brain sodium channel gene, SCN3A (GenBank accession numbers AF035685 and AF035686). As is the case for rat brain sodium channels IIN/IIA and IIIN/IIIA, these isoforms are generated through an alternative splicing mechanism. The conservation of the exon structure suggests that alternative RNA splicing is a common feature for sodium channel mRNA processing and may play an important role in modulating the channel function.

Identification and characterization of genes encoding carbazole 1,9a-dioxygenase in Pseudomonas sp. strain CA10.

Nucleotide sequence analysis of the flanking regions of the carBC genes of Pseudomonas sp. strain CA10 revealed that there were two open reading frames (ORFs) ORF4 and ORF5, in the upstream region of carBC. Similarly, three ORFs, ORF6 to ORF8, were found in the downstream region of carBC. The deduced amino acid sequences of ORF6 and ORF8 showed homologies with ferredoxin and ferredoxin reductase components of bacterial multicomponent dioxygenase systems, respectively. ORF4 and ORF5 had the same sequence and were tandemly linked. Their deduced amino acid sequences showed about 30% homology with large (alpha) subunits of other terminal oxygenase components. Functional analysis using resting cells harboring the deleted plasmids revealed that the products of ORF4 and -5, ORF6, and ORF8 were terminal dioxygenase, ferredoxin, and ferredoxin reductase, respectively, of carbazole 1,9a-dioxygenase (CARDO), which attacks the angular position adjacent to the nitrogen atom of carbazole, and that the product of ORF7 is not indispensable for CARDO activity. Based on the results, ORF4, ORF5, ORF6, and ORF8 were designated carAa, carAa, carAc, and carAd, respectively. The products of carAa, carAd, and ORF7 were shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be polypeptides with molecular masses of 43, 36, and 11 kDa, respectively. However, the product of carAc was not detected in Escherichia coli. CARDO has the ability to oxidize a wide variety of polyaromatic compounds, including dibenzo-p-dioxin, dibenzofuran, biphenyl, and polycyclic aromatic hydrocarbons such as naphthalene and phenanthrene. Since 2,2',3-trihydroxydiphenyl ether and 2,2',3-trihydroxybiphenyl were identified as metabolites of dibenzo-p-dioxin and dibenzofuran, respectively, it was considered that CARDO attacked at the angular position adjacent to the oxygen atom of dibenzo-p-dioxin and dibenzofuran as in the case with carbazole.

Structure and function of cardiac sodium and potassium channels.

The application of patch-clamp and molecular approaches has resulted in an increasingly refined understanding of the molecular entities underlying cardiac sodium and potassium currents. The sodium current results from expression of a single large alpha-subunit, whereas multiple potassium currents and potassium channel alpha-subunits have been identified. Recapitulation of some ion currents in heterologous expression systems requires not only expression of alpha-subunits but also ancillary (beta) subunits. Domains common to functions such as activation, inactivation, and drug block are now being identified in alpha- and beta-gene products. Variability in the expression or function of individual ion-channel genes is an increasingly recognized source of variability in the ion currents recorded in heart cells under physiological conditions (e.g. during development) as well as in disease.

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.

Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: potential mononuclear non-heme iron coordination sites.

The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISP(TOL)) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISP(TOL). The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISP(TOL) activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [14C]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of Cis-[14C]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TWO proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X3-4,-Asp-X2-His-X4-5-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.

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.

Purification and characterization of the oxygenase component of biphenyl 2,3-dioxygenase from Pseudomonas sp. strain LB400.

The iron-sulfur protein of biphenyl 2,3-dioxygenase (ISPBPH) was purified from Pseudomonas sp. strain LB400. The protein is composed of a 1:1 ratio of a large (alpha) subunit with an estimated molecular weight of 53,300 and a small (beta) subunit with an estimated molecular weight of 27,300. The native molecular weight was 209,000, indicating that the protein adopts an alpha 3 beta 3 native conformation. Measurements of iron and acid-labile sulfide gave 2 mol of each per mol of alpha beta heterodimer. The absorbance spectrum showed peaks at 325 and 450 nm with a broad shoulder at 550 nm. The spectrum was bleached upon reduction of the protein with NADPH in the presence of catalytic amounts of ferredoxinBPH and ferredoxinBPH oxidoreductase. The electron paramagnetic resonance spectrum of the reduced protein showed three signals at gx = 1.74, gy = 1.92, and gz = 2.01. These properties are characteristic of proteins that contain a Rieske-type [2Fe-2S] center. Biphenyl was oxidized to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene by ISPBPH in the presence of ferredoxinBPH, ferredoxinBPH oxidoreductase, NADPH, and ferrous iron. Naphthalene was also oxidized to a cis-dihydrodiol, but only 3% was converted to product under the same conditions that gave 92% oxidation of biphenyl. Benzene, toluene, 2,5-dichlorotoluene, carbazole, and dibenzothiophene were not oxidized. ISPBPH is proposed to be the terminal oxygenase component of biphenyl 2,3-dioxygenase where substrate binding and oxidation occur via addition of molecular oxygen and two reducing equivalents.

Modulation of Cardiac Na+ Channel Expression in Xenopus Oocytes by β1 Subunits

Voltage-gated Na+ channels consist of a large alpha subunit of 260 kDa associated with beta 1 and/or beta 2 subunits of 36 and 33 kDa, respectively. alpha subunits of rat cardiac Na+ channels (rH1) are functional when expressed alone in Xenopus oocytes or mammalian cells. beta 1 subunits are present in the heart, and localization of beta 1 subunit mRNA by in situ hybridization shows expression in the perinuclear cytoplasm of cardiac myocytes. Coexpression of beta 1 subunits with rH1 alpha subunits in Xenopus oocytes increases Na+ currents up to 6-fold in a concentration-dependent manner. However, no effects of beta 1 subunit coexpression on the kinetics or voltage dependence of the rH1 Na+ current were detected. Increased expression of Na+ currents is not observed when an equivalent mRNA encoding a nonfunctional mutant beta 1 subunit is coexpressed. Our results show that beta 1 subunits are expressed in cardiac muscle cells and that they interact with alpha subunits to increase the expression of cardiac Na+ channels in Xenopus oocytes, suggesting that beta 1 subunits are important determinants of the level of excitability of cardiac myocytes in vivo.