Expression and characterization of catalytic and regulatory domains of rat tyrosine hydroxylase.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. Comparison of the amino acid sequences of these three proteins shows that the C-terminal two-thirds are homologous, while the N-terminal thirds are not. This is consistent with a model in which the C-terminal two-thirds constitute a conserved catalytic domain to which has been appended discrete regulatory domains. To test such a model, two mutant proteins have been constructed, expressed in Escherichia coli, purified, and characterized. One protein contains the first 158 amino acids of rat tyrosine hydroxylase. The second lacks the first 155 amino acid residues of this enzyme. The spectral properties of the two domains suggest that their three-dimensional structures are changed only slightly from intact tyrosine hydroxylase. The N-terminal domain mutant binds to heparin and is phosphorylated by cAMP-dependent protein kinase at the same rate as the holoenzyme but lacks any catalytic activity. The C-terminal domain mutant is fully active, with Vmax and Km values identical to the holoenzyme; these results establish that all of the catalytic residues of tyrosine hydroxylase are located in the C-terminal 330 amino acids. The results with the two mutant proteins are consistent with these two segments of tyrosine hydroxylase being two separate domains, one regulatory and one catalytic.     

Dominant C-terminal deletions of FtsZ that affect its ability to localize in Caulobacter and its interaction with FtsA

The cell division protein FtsZ is composed of three regions based on sequence similarity: a highly conserved N-terminal region of ≈320 amino acids; a variable spacer region; and a conserved C-terminal region of eight amino acids. We show that FtsZ mutants missing different C-terminal fragments have dominant lethal effects because they block cell division in Caulobacter crescentus by two different mechanisms. Removal of the C-terminal conserved region, the linker, and 40 amino acids from the end of the N-terminal conserved region (FtsZΔC281) prevents the localization or the polymerization of FtsZ. Because two-hybrid analysis indicates that FtsZΔC281 does not interact with FtsZ, we hypothesize that FtsZΔC281 blocks cell division by competing with a factor required for FtsZ localization or that it titrates a factor required for the stability of the FtsZ ring. The removal of 24 amino acids from the C-terminus of FtsZ (FtsZΔC485) causes a punctate pattern of FtsZ localization and affects its interaction with FtsA. This suggests that the conserved C-terminal region of FtsZ is required for proper polymerization of FtsZ in Caulobacter and for its interaction with FtsA.     

Novel inositol polyphosphate 5-phosphatase localizes at membrane ruffles

We have cloned a novel inositol polyphosphate 5-phosphatase from the rat brain cDNA library. It contains two highly conserved 5-phosphatase motifs, both of which are essential for its enzymatic activity. Interestingly, the proline content of this protein is high and concentrated in its N- and C-terminal regions. One putative SH3-binding motif and six 14-3-3 zeta-binding motifs were found in the amino acid sequence. This enzyme hydrolyzed phosphate at the D-5 position of inositol 1,4,5-trisphosphate, inositol 1,3,4, 5-tetrakisphosphate, and phosphatidylinositol 4,5-bisphosphate, consistent with the substrate specificity of type II 5-phosphatase, OCRL, synaptojanin and synaptojanin 2, already characterized 5-phosphatases. When the Myc-epitope-tagged enzyme was expressed in COS-7 cells and stained with anti-Myc polyclonal antibody, a signal was observed at ruffling membranes and in the cytoplasm. We prepared several deletion mutants and demonstrated that the 123 N-terminal amino acids (311-433) and a C-terminal proline-rich region containing 277 amino acids (725-1001) were essential for its localization to ruffling membranes. This enzyme might regulate the level of inositol and phosphatidylinositol polyphosphates at membrane ruffles.     

Deletion mutants of tyrosine hydroxylase identify a region critical for heparin binding.

Phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase constitute a family of tetrahydropterin-dependent aromatic amino acid hydroxylases. It has been proposed that each hydroxylase is composed of a conserved C-terminal catalytic domain and an unrelated N-terminal regulatory domain. Of the three, only tyrosine hydroxylase is activated by heparin and binds to heparin-Sepharose. A series of N-terminal deletion mutants of tyrosine hydroxylase has been expressed in Escherichia coli to identify the heparin-binding site. The mutants lacking the first 32 or 68 amino acids bind to heparin-Sepharose. The mutant lacking 76 amino acids binds somewhat to heparin-Sepharose and the proteins lacking 88 or 128 do not bind at all. Therefore, an important segment of the heparin-binding site must be composed of the region from residues 76 to 90. All of the deletion mutants are active, and the Michaelis constants for pterins and tyrosine are similar among all the mutant and wild-type enzymes.     

Cloning and sequencing of cDNA coding for rabbit alpha-1-antiproteinase F: amino acid sequence comparison of alpha-1-antiproteinases of six mammals

Rabbit liver cDNA coding for alpha-1-antiproteinase F has been isolated and sequenced. The protein sequence deduced from the nucleotide sequence consists of a 24 amino acid signal peptide and 389 amino acids of the mature polypeptide. Rabbit alpha-1-antiproteinase F showed 74 and 64% homology to human alpha-1-antiproteinase at the nucleotide and amino acid levels, respectively, but the N-terminal five amino acids are lacking in the rabbit protein. The sequences of alpha-1-antiproteinase F of rabbit, human, baboon, sheep, rat, and mouse show about 40% identity, and the reactive site (Met-Ser) is conserved. On the other hand, variable regions are located in the second half to the C-terminal as well as in the N-terminal region.     

Hexokinase-binding properties of the mitochondrial VDAC protein: Inhibition by DCCD and location of putative DCCD-binding sites

The outer mitochondrial membrane receptor for hexokinase binding has been identified as the VDAC protein, also known as mitochondrial porin. The ability of the receptor to bind hexokinase is inhibited by pretreatment with dicyclohexylcarbodiimide (DCCD). At low concentrations, DCCD inhibits hexokinase binding by covalently labeling the VDAC protein, with no apparent effect on VDAC channel-forming activity. The stoichiometry of [¹⁴C]-DCCD labeling is consistent with one to two high-affinity DCCD-binding sites per VDAC monomer. A comparison between the sequence of yeast VDAC and a conserved sequence found at DCCD-binding sites of several membrane proteins showed two sites where the yeast VDAC amino acid sequence appears to be very similar to the conserved DCCD-binding sequence. Both of these sites are located near the C-terminal end of yeast VDAC (residues 257–265 and 275–283). These results are consistent with a model in which the C-terminal end of VDAC is involved in binding to the N-terminal end of hexokinase.     

Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase

Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. γ-butyrobetaine hydroxylase (BBH; EC 1.14.11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of γ-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed γ-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.     

Molecular cloning and characterization of the cDNA encoding the rat liver gamma-butyrobetaine hydroxylase.

Carnitine biosynthesis from lysine and methionine involves five enzymatic reactions. gamma-butyrobetaine hydroxylase (BBH; EC 1.14. 11.1) is the last enzyme of this pathway. It catalyzes the reaction of hydroxylation of gamma-butyrobetaine to carnitine. The cDNA encoding this enzyme has been isolated and characterized. The cDNA contained an open reading frame of 1161 bp encoding a protein of 387 amino acids with a deduced molecular weight of 44.5 kDa. The sequence of the cDNA showed an important homology with the human cDNA recently isolated. Northern analysis showed gamma-butyrobetaine hydroxylase expression in the liver and in some extend in the testis and the epididymis. During this study, it also appeared that BBH mRNA expression was undetectable by Northern analysis during the perinatal period. During the development of the rat, the amount of BBH mRNA appeared after the weaning of the young rat and reached a maximal expression at the adult stage.     

Nucleotide sequence of a full-length complementary DNA clone and amino acid sequence of human phenylalanine hydroxylase

A full-length human phenylalanine hydroxylase complementary DNA (cDNA) clone was isolated from a human liver cDNA library, and the nucleotide sequence encoding the entire enzyme was determined. The cDNA clone contains an inserted DNA fragment of 2448 base pairs, including 19 base pairs of poly(A) at the 3' end. The first methionine codon occurs at nucleotide position 223, followed by an open reading frame of 1353 base pairs, encoding 451 amino acids. Translation of the nucleotide sequence in the open reading frame predicts the amino acid sequence of human phenylalanine hydroxylase. The human protein shows a 96% amino acid sequence homology with the corresponding rat enzyme. The determination of the complete primary structure for phenylalanine hydroxylase represents the first among mixed-function oxidases.     

Import and orientation of the MWFE protein in mitochondrial NADH-ubiquinone oxidoreductase

The MWFE subunit of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) is a small, essential membrane protein of 70 amino acids that is made in the cytosol, imported into mitochondria, and assembled without further proteolytic processing. The experiments identify the first approximately 30 amino acids as a minimal mitochondrial targeting sequence, and establish its orientation in the inner membrane and in complex I. This sequence has a highly conserved glutamate at position 4, which is not typical of a mitochondrial targeting signal. However, it is not essential for MWFE function. Within this sequence there is also a 'stop-transfer' signal. The membrane anchor cannot be replaced by that from another subunit within complex I.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

Substitutions in the C-terminal portion of the catalytic domain partially reverse assembly defects introduced by mutations in the N-terminal linker sequence of cytochrome P450 2C2.

Mutations in a 7-amino acid linker segment, immediately following the N-terminal signal anchor sequence of cytochrome P450 2C2, have been shown to affect proper assembly of hemoprotein and decrease activity of the mutants expressed in COS cells. In contrast, C2pmBalC1, in which cytochrome P450 2C1 residues were substituted for those of cytochrome P450 2C2 in the C-terminal region, exhibited increased activity when expressed in COS-1 cells. To examine further the basis for the increased activity of C2pmBalC1 in COS-1 cells, the protein was expressed in insect cells and Escherichia coli. The amounts of the functional P450 species of C2pmBalC1 expressed in these systems and the ratios of P450 to P420 were greater than those of cytochrome P450 2C2, indicating that more efficient assembly underlies the increased activity of C2pmBalC1. To determine whether the C-terminal substitutions could compensate for the decreased assembly mediated by the N-terminal linker mutations, the linker mutations were introduced into C2pmBalC1. If all 7 amino acids in the linker were deleted, no enzymatically active cytochrome P450 2C2 or C2pmBalC1 was detected in COS-1, insect, or bacterial cells expressing the mutants. The mutant C2A2, in which two alanines were substituted for the linker, had no detectable laurate hydroxylase activity in COS-1 cells, and minor amounts of hemoprotein for this mutant were expressed in E. coli and insect cells. In contrast, the same mutation in C2pmBalC1 reduced activity only 50% in COS-1 cells and markedly elevated levels of P450 expression in bacteria and insect cells. The A2 mutation did not affect the enzymatic activity of either cytochrome P450 2C2 or C2pmBalC1 assayed in whole cell lysates of insect cells but reduced the activity of partially purified enzymes assayed in a reconstituted assay system. These findings indicate that mutations introduced into the C-terminal region of P450 2C2 can facilitate assembly of the proteins and partially reverse the decreased assembly resulting from the N-terminal mutations.     

Characterization of endo-beta-N-acetylglucosaminidase from alkaliphilic Bacillus halodurans C-125

The genome sequencing project on alkaliphilic Bacillus halodurans C-125 revealed a putative endo-beta-N-acetylglucosaminidase (Endo-BH), which consists of a signal peptide of 24 amino acids, a catalytic region of 634 amino acids exhibiting 50.1% identity with the endo-beta-N-acetylglucosaminidase from Arthrobacter protophormiae (Endo-A), and a C-terminal tail of 220 amino acids. Transformed Escherichia coli cells carrying the Endo-BH gene exhibited endo-beta-N-acetylglucosaminidase activity. Recombinant Endo-BH hydrolyzed high-mannose type oligosaccharides and hybrid type oligosaccharides, and showed transglycosylation activity. On deletion of 219 C-terminal amino acid residues of Endo-BH, the wild type level of activity was retained, whereas with deletions of the Endo-A homolog domain, the proteins were expressed as inclusion bodies and these activities were reduced. These results suggest that the enzymatic properties of Endo-BH are similar to those of Endo-A, and that the C-terminal tail does not affect the enzyme activity. Although the C-terminal tail region is not essential for enzyme activity, the sequence is also conserved among endo-beta-N-acetylglucosaminidases of various origins.     

Primary structure of xylene monooxygenase: similarities to and differences from the alkane hydroxylation system.

Xylene monooxygenase, encoded by the TOL plasmid of Pseudomonas putida, catalyzes the oxidation of toluene and xylenes and consists of two different subunits encoded by xylA and xylM. In this study, the complete nucleotide sequences of these genes were determined and the amino acid sequences of the xylA and xylM products were deduced. The XylM sequence had a 25% homology with alkane hydroxylase, which catalyzes the omega-hydroxylation of fatty acids and the terminal hydroxylation of alkanes. The sequence of the first 90 amino acids of XylA exhibited a strong similarity to the sequence of chloroplast-type ferredoxins, whereas the rest of the XylA sequence resembled that of ferredoxin-NADP+ reductases. Based on this information, the structure and function of xylene monooxygenase were deduced. XylM may be a catalytic component for the hydroxylation of the carbon side chain of toluene and xylenes and, as is the alkane hydroxylase protein, may be a membrane-bound protein containing ferrous ion as a prosthetic group. XylA may have two domains consisting of an N-terminal region similar to chloroplast-type ferredoxins and a C-terminal region similar to ferredoxin-NADP+ reductases. The ferredoxin portion of XylA may contain a [2Fe-2S] cluster and reduce the oxidized form of the XylM hydroxylase. The activity determined by the C-terminal region of the XylA sequence may be the reduction of the oxidized form of ferredoxin by concomitant oxidation of NADH.     

天蚕卵黄原蛋白cDNA的克隆及序列分析

天蚕卵黄原蛋白cDNA的全碱基序列由5 720个碱基构成,一个开读框有5 334个碱基序列,编码了包括由15个氨基酸组成的信号肽在内的共1 778个氨基酸的蛋白质前体。天蚕和柞蚕卵黄原蛋白的同源性非常高,氨基酸序列达到92.6%,碱基序列达到94%。天蚕、柞蚕和家蚕的卵黄原蛋白的糖链附加位点(N-linkedg-lycosylationsite):柞蚕有4个,家蚕有5个,天蚕和家蚕相同保存着5个。在N-末端区域,天蚕和家蚕有多聚丝氨酸区域和可被胰蛋白酶识别的RSRR部位;柞蚕虽然也具有多聚丝氨酸区域,但没有可被胰蛋白酶识别的RSRR部位。在C-末端区域里,大多数昆虫从G ICG功能部位至C-末端结尾具有7个~10个半胱氨酸(Cys)。鳞翅目的4种昆虫柞蚕、天蚕、家蚕和舞毒蛾(A.pernyi,A.yam am ai,B.m ori,L.dispar)都是7个半胱氨酸。     

In vitro and in vivo function of the C-terminus of Escherichia coli single-stranded DNA binding protein.

We constructed several deletion mutants of Escherichia coli single-stranded DNA binding protein (EcoSSB) lacking different parts of the C-terminal region. This region of EcoSSB is composed of two parts: a glycine and proline-rich sequence of approximately 60 amino acids followed by an acidic region of the last 10 amino acids which is highly conserved among the bacterial SSB proteins. The single-stranded DNA binding protein of human mitochondria (HsmtSSB) lacks a region homologous to the C-terminal third of EcoSSB. Therefore, we also investigated a chimeric protein consisting of the complete sequence of the human mitochondrial single-stranded DNA binding protein (HsmtSSB) and the C-terminal third of EcoSSB. Fluorescence titrations and DNA-melting curves showed that the C-terminal third of EcoSSB is not essential for DNA-binding in vitro. The affinity for single-stranded DNA and RNA is even increased by the removal of the last 10 amino acids. Consequently, the nucleic acid binding affinity of HsmtSSB is reduced by the addition of the C-terminus of EcoSSB. All mutant proteins lacking the last 10 amino acids are unable to substitute wild-type EcoSSB in vivo. Thus, while the nucleic acid binding properties do not depend on an intact C-terminus, this region is essential for in vivo function. Although the DNA binding properties of HsmtSSB and EcoSSB are quite similar, HsmtSSB does not function in E.coli. This failure cannot be overcome by fusing the C-terminal third of EcoSSB to HsmtSSB. Thus differences in the N-terminal parts of both proteins must be responsible for this incompatibility. None of the mutants was defective in tetramerization. However, mixed tetramers could only be formed by proteins containing the same N-terminal part. This reflects structural differences between the N-terminal parts of HsmtSSB and EcoSSB. These results indicate that the region of the last 10 amino acids, which is highly conserved among bacterial SSB proteins, is involved in essential protein-protein interactions in the E.coli cell.     

Conserved amino acids at the C-terminus of rat phospholipase D1 are essential for enzymatic activity.

Rat brain phospholipase D1 (rPLD1) has two highly conserved motifs [H(X)K(X)4D, denoted HKD] located at the N-terminal and C-terminal halves, which are required for activity. Association of the two halves is essential for rPLD1 activity, which probably brings the two HKD domains together to form a catalytic center. In the present study, we find that an intact C-terminus is also essential for the catalytic activity of rPLD1. Serial deletion of the last four amino acids, EVWT, which are conserved in all mammalian PLD isoforms, abolished the catalytic activity of rPLD1. This loss of catalytic activity was not due to a lack of association of the N-terminal and C-terminal halves. Mutations of the last three amino acids showed that substitutions with charged or less hydrophobic amino acids all reduced PLD activity. For example, mutations of Thr1036 and Val1034 to Asp or Lys caused marked inactivation, whereas mutation to other amino acids had less effect. Mutation of Trp1035 to Leu, Ala, His or Tyr caused complete inactivation, whereas mutation of Glu1033 to Ala enhanced activity. The size of the amino acids at the C-terminus also affected the catalytic activity of PLD, reduced activity being observed with conservative mutations within the EVWT sequence (such as T/S, V/L or W/F). The enzyme was also inactivated by the addition of Ala or Val to the C-terminus of this sequence. Interestingly, the inactive C-terminal mutants could be complemented by cotransfection with a wild-type C-terminal half to restore PLD activity in vivo. These data demonstrate that the integrity of the C-terminus of rPLD1 is essential for its catalytic activity. Important features are the hydrophobicity, charge and size of the four conserved C-terminal amino acids. It is proposed that these play important roles in maintaining a functional catalytic structure by interacting with a specific domain within rPLD1.     

Cloning of a cDNA Coding for Active Tyrosine Hydroxylase in the Rainbow Trout (Oncorhynchus mykiss): Comparison with Other Hydroxylases and Enzymatic Expression

Abstract: Although catecholamines are of critical importance for neuroendocrine function in teleost fishes, there has been no tool to give access to pretranslational regulation of their synthesis enzymes. In this study, we undertook cloning of the cDNA coding for the tyrosine hydroxylase (TH) of the rainbow trout ( Oncorhynchus mykiss ). First, we looked for a tissue sufficiently rich in TH to make an expression library. The cDNA coding for the rainbow trout TH (rtTH) was then cloned and sequenced. The rtTH sequence encodes a protein of 489 amino acids. Several domains and amino acids required for enzyme activity, like cysteines or phosphorylation sites, are highly conserved between species. Northern blot analysis showed a single rtTH messenger RNA of 4.2 kb expressed in the anteroventral brain. The ability of rtTH to hydroxylate l -tyrosine was analyzed by transient expression of the rtTH cDNA in COS-1 cells. In vitro TH activity, using COS-1 cell extracts, demonstrated that TH activity in transfected cells was 40-fold higher than in untransfected cells. Western blot analysis revealed a single protein of ∼65 kDa in both COS-1 cells and in trout brain. This rtTH cDNA provides us with a tool for further studies on pretranslational regulation of the enzyme in salmonids.