芳香族氨基酸羟化酶家族结构与催化功能

芳香族氨基酸羟化酶(AAAH）家族是一类单加氢酶,包括苯丙氨酸羟化酶(PAH)、酪氨酸羟化酶(TH)和色氨酸羟化酶(TPH). 在辅因子四氢生物蝶呤、铁原子及氧存在下,分别催化苯丙氨酸、酪氨酸、色氨酸的羟化反应. 多种疾病如苯丙酮尿症、帕金森氏病以及神经相关疾病的发病机制均与这类酶有关. 本文综述近年来对芳香族氨基酸羟化酶家族蛋白结构功能、底物特异性、催化机制等方面的研究进展,为该类酶的定向进化及功能应用提供新思路.     

苯丙酸尿症和高苯丙氨酸血症是一回事吗?

<正>答:苯丙酮尿症(phenylketouria,PKU)是一种氨基酸代谢异常疾病。经典型PKU是由于苯丙氨酸羟化酶基因(PAH)的突变导致该酶活性降低或丧失,致使血中苯丙氨酸代谢受阻,不能转化为酪氨酸,表现为高苯丙氨酸血症和高苯丙酮酸尿症。非经典型PKU分别由DHPR,GTP-CH,6-PTS基因的突变所引起,导致生物喋呤代谢缺陷,影响苯丙氨酸羟     

紫色色杆菌苯丙氨酸羟化酶的异源表达及重组酶学性质研究

来源于紫色色杆菌(Chromobacterium violaceum)的苯丙氨酸羟化酶结构简单,性质更接近于人的苯丙氨酸羟化酶,具有潜在的医药应用价值。从紫色色杆菌基因组中克隆得到苯丙氨酸羟化酶基因pah。构建重组表达载体pET24a-pah,并在Escherichia coli BL21(DE3)中实现高效表达。离子层析纯化后,重组蛋白比酶活高达503.2 U/mg。酶学性质研究显示,该重组酶的最适温度为40℃左右,50℃时PAH的半衰期为15 min;最适pH在7.5左右,在pH6-8范围内较稳定。37℃,pH7.5条件下,Km值为1.5 mmol/L,Vmax为0.5 mmol/min,kcat为5.05/s,催化效率kcat/Km为3.37 L/mmol·s。     

水牛CD14 cDNA的克隆、表达及鉴定

目的:克隆水牛白细胞分化抗原14(buffalo cluster of differentiation antigen14,bCD14)基因,表达bCD14蛋白,并进行Western Blot鉴定.方法:采用RT-PCR方法从水牛外周血白细胞中扩增bCD14基因,构建重组质粒pET28a-bCD14,转化入E coli BL21,IPTG诱导表达,对表达蛋白进行可溶性分析及Western blot鉴定.结果:bCD14基因含有一个1 122bp的开放阅读框,编码373个氨基酸;与印度水牛、挪威大鼠和人CD14的cDNA序列同源性分别为97.95%、68.78%、78.60%,氨基酸同源性分别为96.78%、61.27%、72.34%;主要以包涵体形式表达,表达蛋白经Western Blot鉴定,得到了一条约46 kD的特异性条带.结论:该文成功克隆了bCD14基因,表达了bCD14蛋白,为进一步揭示水牛抵抗革兰氏阴性菌感染的免疫机制奠定了基础.     

山羊Klf4基因克隆、原核表达和His-Klf4融合蛋白纯化北大核心CSCD

旨在克隆山羊Klf4基因,构建重组质粒pET30a-Klf4,通过原核表达技术获得纯化的His-Klf4融合蛋白。从山羊的生殖脊中提取总RNA,用RT-PCR的方法扩增Klf4基因编码区序列,通过TA克隆构建pMD18-T-Klf4重组质粒。酶切鉴定与测序分析后将Klf4的cDNA亚克隆至pET-30a载体,构建重组质粒pET30a-Klf4。酶切鉴定后将其导入大肠杆菌BL21中,用IPTG诱导表达,SDS-PAGE和Western blotting检测确认,镍离子金属螯合亲和层析法纯化His-Klf4融合蛋白。结果表明:(1)从山羊生殖脊中克隆了Klf4基因,其开放阅读框由1 434个核苷酸组成,编码478个氨基酸,而且该序列与绵羊的同源性最高,达到98.5%;(2)经过SignalP 4.0在线分析,Klf4的编码蛋白不存在信号肽;(3)经SDS-PAGE和Western blotting分析表明,重组质粒pET30a-Klf4在大肠杆菌中得以表达;在变性条件下纯化,获得了His-Klf4融合蛋白。     

苯丙酮尿症分子遗传学研究进展

苯丙酮尿症是由于苯丙氨酸羟化酶基因突变引起的常染色体隐性遗传病。文章综述了苯丙酮尿症中的苯丙氨酸羟化酶基因的定位、结构、突变、调控以及突变基因的体外表达和苯丙氨酸羟化酶的三维结构特点等分子遗传学进展,阐述了苯丙氨酸羟化酶基因的突变对苯丙氨酸羟化酶的体外表达及其三维结构的影响, 以及部分基因型与表型相关的分子机制。 Abstract: Phenylketonuria(PKU) is one kinds of autusomal recessive disease caused by phenylalanine hydroxylase(PAH) gene mutation. This article reviews the recent molecular heredity progress on the phenylalanine hydroxylase gene’s orientation、structureand gene mutation and gene regulation. At same time, mutation gene in vitro expression and the character of 3D structure of PAH in PKU are involved. In this paper, also discussed the inflence of vitro expression and 3D protein structure by gene mutations and the molecular mechanism of the relationship between genotype and phenotype in PKU patient.     

棕色棉F3'H-羟化酶基因的克隆与生物信息学分析

根据葡萄的类黄酮3′-羟化酶(F3'H)基因全长cDNA序列Blast所得棉花的EST序列设计引物,以开花后16 d(DPA16)的新彩棉5号(xC-5)纤维为材料,利用RACE和RT-PCR技术分离得到了2个类黄酮3′-羟化酶基因cDNA序列,此2个序列编码区完全相同,仅在3'UTR区存在片段长短的差异,推测可能是基因转录后加工方式不同所造成.克隆所获得的棉花F3'H基因编码区全长1 533 bp,编码510个氨基酸,氨基酸序列分析预测表明,该基因所编码蛋白含有一个跨膜结构域,是一种分泌蛋白,定位于内质网上,并含有一段与细胞色素P450功能区相匹配的保守功能域;序列比对结果表明,棉花F3'H基因与其他多个物种的F3'H基因在氨基酸序列上有较高的同源性;聚类分析结果表明,棉花F3'H蛋白与双子叶植物大豆的F3'H亲缘关系较为接近,而与单子叶植物高梁等作物则较远.     

利玛原甲藻中聚酮合酶基因克隆与分析

为探讨聚酮合酶 (polyketide synthase, PKS)基因与藻毒素合成的关系,揭示PKS基因在赤潮毒素合成中的作用,采用兼并引物,通过PCR技术获得利玛原甲藻(Prorocentrum lima)可能存在的I型PKS基因;并对所获得PKS基因的同源性进行了分析,构建了基于PKS氨基酸序列的系统进化树;采用RT-PCR技术分析了PKS基因在利玛原甲藻中的表达状况;并通过多聚腺苷酸RNA的扩增、细菌的分离鉴定、限制性内切酶酶切、Southern blotting等技术对PKS基因进行了分析.结果表明,利玛原甲藻中PKS基因与海洋原甲藻聚为一支,在利玛原甲藻中有显著表达;以Oligo(T)引物进行RT-PCR扩增时,可出现18S rRNA和PKS基因相应条带;限制性内切酶酶切和Southern blotting结果显示,该基因中存在明显的甲基化;16S rRNA基因序列分析显示,从利玛原甲藻培养液中分离到的细菌与海洋放线菌假诺卡氏菌属(Pseudonocardia)基因序列同源性达到99%,该菌株中并不存在PKS基因.结果显示,所获得的PKS基因是利玛原甲藻聚酮合酶基因,基因序列已提交GenBank (EF521601);PKS可能在腹泻性贝毒合成中起着关键作用.     

大鼠sFcγRIIb基因原核表达及活性鉴定

旨在克隆大鼠FcγRIIb基因,构建s FcγRIIb原核表达体系,制备重组大鼠s FcγRIIb蛋白。采用RT-PCR技术从RBL-2H3细胞中克隆FcγRIIb胞外区基因,构建重组表达质粒转化大肠杆菌诱导表达,镍柱亲和层析纯化重组蛋白,复性后利用Western blotting、ELISA进行鉴定。结果显示,成功克隆大鼠s FcγRIIb基因,构建原核表达载体s FcγRIIb-p ET17b,转化大肠杆菌BL21(DE3)。通过对表达体系进行优化,确定IPTG浓度为1.0 mmol/L,诱导时间为4 h时蛋白表达效率最高,重组蛋白主要以包涵体形式存在。包涵体经8 mol/L尿素溶解,利用Ni-NTA柱亲和层析获得了较高纯度的重组蛋白。采用梯度透析复性法对s FcγRIIb进行复性后,经Western blotting、ELISA与竞争性ELISA鉴定,重组蛋白s FcγRIIb可被特异性抗体所识别且与Ig G具有结合能力。成功建立了大鼠FcγRIIb基因原核表达体系,制备了具有生物学功能的重组蛋白。     

大肠杆菌ppsA基因的克隆表达

L-苯丙氨酸是人体内8种必需氨基酸之一,是医药大输液的基本成分.苯丙氨酸和门冬氨酸组成的二肽甲酯是一种新型甜味剂,市场需求正在不断增长,因此构建高产酸的苯丙氨酸基因工程菌已成为形势所趋.苯丙氨酸工程菌的构建是基于芳香族氨基酸的生化合成途径进行的,包括单基因表达工程菌和多基因表达工程菌.单基因表达主要集中在芳香族转氨酶tyrB基因上,国内外有不少报道[1～3],苯丙酮酸转化为苯丙氨酸的转化率约为91%.在多基因表达方面,Ikeda等[4]克隆了3个芳香族氨基酸合成的有关基因,并进行多基因表达,苯丙氨酸产量为28g/L.磷酸烯醇式丙酮酸(P…     

Sequence and expression of the Drosophila phenylalanine hydroxylase mRNA

We report the cloning, nucleotide (nt) sequence and expression of the cDNA (pah) encoding phenylalanine hydroxylase (PAH) of Drosophila melanogaster. The strong hybridization signals observed in genomic blots when D. melanogaster DNA was probed with 32P-labeled human pah cDNA, indicated the existence of a high degree of sequence similarity between the pah genes of both species. The length of the pah genomic fragment is about 30 to 40 kb. The cDNA contains 84 bp of the 5'-untranslated region, 1359 bp of the protein-coding region and 87 bp of the 3' region, with only one polyadenylation signal. The isolated cDNA is probably full-length, since the size of the D. melanogaster PAH mRNA is 1.5 kb. At the nt level, the similarity of the D. melanogaster cDNA with human and rat pah cDNAs is 57.9% and 58.1%, respectively. The highest similarities are restricted to the nt sequence coding for the presumed hydroxylation domain. There is no nt sequence similarity between the first three exons of the human pah gene and an equivalent fraction of the D. melanogaster pah gene. At the amino acid (aa) level, the similarity in the presumed hydroxylation domain is 88.5%, in which two motifs of the structure AGLLSSXXXL are found, where X represents any aa. It was interesting to notice the conservation of aa 408, 311 and 280, where mutations are associated with phenylketonuria in humans. We observed, moreover, that, as it occurs in humans and rats, the expression of the D. melanogaster pah gene is tissue-specific and temporally regulated.     

Expression of calpastatin, minopontin, NIPSNAP1, rabaptin-5 and neuronatin in the phenylketonuria (PKU) mouse brain: possible role on cognitive defect seen in PKU

Phenylketonuria (PKU) is an inborn error of amino acid metabolism. Phenylalanine hydroxylase (PAH) deficiency results in accumulation of phenylalanine (Phe) in the brain and leads to pathophysiological abnormalities including cognitive defect, if Phe diet is not restricted. Neuronatin and 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1) reportedly have role in memory. Therefore, gene expression was examined in the brain of mouse model for PKU. Microarray expression analysis revealed reduced expression of calpastatin, NIPSNAP 1, rabaptin-5 and minopontin genes and overexpression of neuronatin gene in the PKU mouse brain. Altered expression of these genes was further confirmed by one-step real time RT-PCR analysis. Western blot analysis of the mouse brain showed reduced levels of calpastatin and rabaptin-5 and higher amount of neuronatin in PKU compared to the wild type. These observations in the PKU mouse brain suggest that altered expression of these genes resulting in abnormal proteome. These changes in the PKU mouse brain are likely to contribute cognitive impairment seen in the PKU mouse, if documented also in patients with PKU.     

Isolation and sequence of a cDNA clone which contains the complete coding region of rat phenylalanine hydroxylase. Structural homology with tyrosine hydroxylase, glucocorticoid regulation, and use of alternate polyadenylation sites

Screening of a rat liver cDNA expression library constructed in the vector lambda gt11 with an affinity purified antiserum to rat phenylalanine hydroxylase has resulted in the isolation of two clones which contain the complete coding region (1362 base pairs) of phenylalanine hydroxylase and the entire 3'-untranslated region (562 base pairs). From the nucleotide sequence we deduced the amino acid sequence of the enzyme. The molecular weight is 51,632 (452 amino acids). The rat enzyme is highly homologous to human phenylalanine hydroxylase. The two proteins differ in only 36 amino acids (92% homology), many of which are conservative changes. A dot matrix computer program was used to analyze regions of homology with the amino acid sequence of rat tyrosine hydroxylase. Considerable homology was detected from amino acid 140 in the rat enzyme to the C terminus, but little or no homology was apparent in the N-terminal region. The cDNA clone was used to determine the levels of phenylalanine hydroxylase mRNA in rat tissues using RNA blot hybridization. Two mRNA species were detected, with approximate lengths of 2,000 and 2,400 nucleotides, which appear to derive from use of alternate polyadenylation signals. No difference in mRNA size was found in rats which have different phenylalanine hydroxylase alleles. The kidney was found to contain about 10% of the mRNA found in the liver, and no phenylalanine hydroxylase mRNA was detected in rat brain. Reuber H4 hepatoma cells were also analyzed for phenylalanine hydroxylase mRNA. The parental cells contained mRNA species of the same sizes as in rat liver. Incubation in 10(-6) M hydrocortisone for 24 h resulted in an 18-fold increase in the mRNA level. Mutant hepatoma cells which express very little phenylalanine hydroxylase contained less than 5% of the parental mRNA, but the gene still responded to hydrocortisone.     

Tetrahydrobiopterin and Biogenic Amine Metabolism in the hph-1 Mouse

Abstract: hph-1 mice, which have defective tetrahydrobiopterin biosynthesis due to decreased GTP cyclohydrolase I activity, have been used to investigate the effects of tetrahydrobiopterin deficiency on aromatic l -amino acid monooxygenases and brain monoamine metabolism. Liver tetrahydrobiopterin levels were decreased, and tetrahydrobiopterin deficiency and reduced levels of dopamine, norepinephrine, serotonin, and their metabolites in the brain occurred both pre- and postnatally. Chronic subcutaneous tetrahydrobiopterin elevated brain levels to values higher than those seen in controls but had no effect on monoamine metabolism. In vivo activities of tyrosine hydroxylase and tryptophan hydroxylase were significantly decreased. There was a 30% decrease in the in vitro activity of striatal tyrosine hydroxylase and 50% decrease in liver phenylalanine hydroxylase. Western blotting demonstrated that the lower monooxygenase activities resulted from a reduced absolute amount of tyrosine hydroxylase and phenylalanine hydroxylase protein. The findings suggest involvement of tetrahydrobiopterin in the control of the steady-state concentration of the aromatic l -amino acid monooxygenases. In addition, demonstration of central monoamine changes in the hph-1 mouse make it a possible model system for the investigation of the neuropathological mechanisms in Dopa-responsive dystonia, which has recently been linked with mutations in the gene for GTP cyclohydrolase I.     

Structural similarities among enzyme pterin binding sites as demonstrated by a monoclonal anti-idiotypic antibody

BALB/c mice were immunized with a synthetic co-factor of the aromatic amino acid hydroxylases, 6,7-dimethyl-5,6,7,8-tetrahydropterin, conjugated to albumin. Hybridoma cell lines isolated from the immunized mice secreted monoclonal antibodies reacting specifically with the pterin molecule and monoclonal antibodies which were found to bind phenylalanine hydroxylase. Several lines of evidence were consistent with the anti-phenylalanine hydroxylase antibodies being anti-idiotype antibodies mimicking the pterin molecule and binding to the pterin binding site of phenylalanine hydroxylase. (a) An anti-idiotype monoclonal antibody, NS7, when reimmunized into mice produced anti-pterin antibodies consistent with NS7 being an internal image anti-idiotypic antibody. (b) NS7 antibody was prevented from binding to phenylalanine hydroxylase when a competitive inhibitor of phenylalanine hydroxylase enzyme activity, 6,7-dimethyl-7,8-dihydropterin, was bound to phenylalanine hydroxylase. (c) NS7 antibody was shown to bind to a wide range of pterin-requiring enzymes: phenylalanine, tyrosine and tryptophan hydroxylases, dihydropteridine reductase, dihydrofolate reductase, and sepiapterin reductase. Thus the NS7 antibody has successfully mimicked a common portion of the pterin cofactors utilized by these enzymes and demonstrated structure homology in their pterin binding sites despite their diverse function and little amino acid sequence homology except among the three aromatic amino acid hydroxylases.     

Homology between phenylalanine and tyrosine hydroxylases reveals common structural and functional domains

Phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TYH) are mixed-function oxidases that share many characteristic biochemical and immunological properties. The recent cloning and sequencing of full-length cDNAs for both human PAH and rat TYH allow detailed comparison of their primary structures. There is a high degree of homology between PAH and TYH on nucleic acid and amino acid levels. The pattern of homology suggests that these molecules are comprised of a homologous core containing the determinants for enzymatic activity and a nonhomologous region that contributes to substrate specificity and regulation. The degree of homology also suggests that these two proteins evolved from a common ancestor.     

A monoclonal antibody to aromatic amino acid hydroxylases. Identification of the epitope.

PH8 monoclonal antibody has previously been shown to react with all three aromatic amino acid hydroxylases, being particularly useful for immunohistochemical staining of brain tissue [Haan, Jennings, Cuello, Nakata, Chow, Kushinsky, Brittingham & Cotton (1987) Brain Res. 426, 19-27]. Western-blot analysis of liver extracts showed that PH8 reacted with phenylalanine hydroxylase from a wide range of vertebrate species. The epitope for antibody PH8 has been localized to the human phenylalanine hydroxylase sequence between amino acid residues 139 and 155. This highly conserved region of the aromatic amino acid hydroxylases has 11 out of 17 amino acids identical in phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase.     

Modeled ligand-protein complexes elucidate the origin of substrate specificity and provide insight into catalytic mechanisms of phenylalanine hydroxylase and tyrosine hydroxylase.

NMR spectroscopy and X-ray crystallography have provided important insight into structural features of phenylalanine hydroxylase (PAH) and tyrosine hydroxylase (TH). Nevertheless, significant problems such as the substrate specificity of PAH and the different susceptibility of TH to feedback inhibition by l-3,4-dihydroxyphenylalanine (l-DOPA) compared with dopamine (DA) remain unresolved. Based on the crystal structures 5pah for PAH and 2toh for TH (Protein Data Bank), we have used molecular docking to model the binding of 6(R)-l-erythro-5,6,7,8-tetrahydrobiopterin (BH4) and the substrates phenylalanine and tyrosine to the catalytic domains of PAH and TH. The amino acid substrates were placed in positions common to both enzymes. The productive position of tyrosine in TH.BH4 was stabilized by a hydrogen bond with BH4. Despite favorable energy scores, tyrosine in a position trans to PAH residue His290 or TH residue His336 interferes with the access of the essential cofactor dioxygen to the catalytic center, thereby blocking the enzymatic reaction. DA and l-DOPA were directly coordinated to the active site iron via the hydroxyl residues of their catechol groups. Two alternative conformations, rotated 180 degrees around an imaginary iron-catecholamine axis, were found for DA and l-DOPA in PAH and for DA in TH. Electrostatic forces play a key role in hindering the bidentate binding of the immediate reaction product l-DOPA to TH, thereby saving the enzyme from direct feedback inhibition.     

The phylogeny of the aromatic amino acid hydroxylases revisited by characterizing phenylalanine hydroxylase from Dictyostelium discoideum

The social amoeba Dictyostelium discoideum contains only one aromatic amino acid hydroxylase (AAAH) gene compared to at least three in metazoans. As shown in this work this gene codes for a phenylalanine hydroxylase (DictyoPAH) and phylogenetic analysis places this enzyme close to the precursor AAAHs, aiding to define the evolutionary history of the AAAH family. DictyoPAH shows significant similarities to other eukaryote PAH, but it exhibits higher activity with tetrahydrodictyopterin (DH4) than with tetrahydrobiopterin (BH4) as cofactor. DH4 is an abundant tetrahydropterin in D. discoideum while BH4 is the natural cofactor of the AAAHs in mammals. Moreover, DictyoPAH is devoid of the characteristic regulatory mechanisms of mammalian PAH such as positive cooperativity for L-Phe and activation by preincubation with the substrate. Analysis of the few active site substitutions between DictyoPAH and mammalian PAH, including mutant expression analysis, reveals potential structural determinants for allosteric regulation.