来源于蓝藻、铜绿微囊藻的尿苷二磷酸葡萄糖脱氢酶基因的克隆和鉴定

以已知的尿苷二磷酸葡萄糖脱氢酶基因的保守区为基础,自行设计一对简并引物,该对引物从形成水华的蓝藻(Synechocystis PCC6803)铜绿微囊藻FACHB 905株(Microcystis aeruginosa FACHB 905)的基因组DNA中扩增到一个476 bp的DNA片段.通过TAIL-PCR和连接介导的PCR两种方法分离该片段的侧翼序列,最后得到大小约2.5 kb的DNA片段.序列分析揭示其中有一个编码462个氨基酸的开放阅读框,我们将此开放阅读框对应的蛋白命名为Mud.该Mud蛋白的氨基酸序列与蓝藻(73%相同,87%相似)和细菌(Bacillus subtilis)(51%相同,67%相似)的尿苷二磷酸葡萄糖脱氢酶氨基酸序列表现高度的同源性.将该mud基因克隆于p-GEX-4T-1融合表达载体并在大肠杆菌中表达GST-Mud融合蛋白,经过酶活力测定发现,GST-Mud蛋白具有一定的尿苷二磷酸葡萄糖脱氢酶活性.用抗GST-Mud蛋白的多抗对M.aeruginosa FACHB 905的胞质蛋白组分进行Western印迹分析,结果显示一条分子量大小约49 kD的专一条带,这个分子量与从基因推断出的蛋白分子量大小基本一致.综上所述,我们认为从微囊藻克隆到的Mud蛋白基因是尿苷二磷酸葡萄糖脱氢酶基因,该酶在其他生物如植物和细菌中参与多糖合成,是多糖合成的关键酶之一,而在藻类中对尿苷二磷酸葡萄糖脱氢酶开展研究却是首次报道.     

Aldehyde dehydrogenases: widespread structural and functional diversity within a shared framework.

Sequences of 16 NAD and/or NADP-linked aldehyde oxidoreductases are aligned, including representative examples of all aldehyde dehydrogenase forms with wide substrate preferences as well as additional types with distinct specificities for certain metabolic aldehyde intermediates, particularly semialdehydes, yielding pairwise identities from 15 to 83%. Eleven of 23 invariant residues are glycine and three are proline, indicating evolutionary restraint against alteration of peptide chain-bending points. Additionally, another 66 positions show high conservation of residue type, mostly hydrophobic residues. Ten of these occur in predicted beta-strands, suggesting important interior-packing interactions. A single invariant cysteine residue is found, further supporting its catalytic role. A previously identified essential glutamic acid residue is conserved in all but methyl malonyl semialdehyde dehydrogenase, which may relate to formation by that enzyme of a CoA ester as a product rather than a free carboxylate species. Earlier, similarity to a GXGXXG segment expected in the NAD-binding site was noted from alignments with fewer sequences. The same region continues to be indicated, although now only the first glycine residue is strictly conserved and the second (usually threonine) is not present at all, suggesting greater variance in coenzyme-binding interactions.     

Relationships within the aldehyde dehydrogenase extended family

One hundred-forty-five full-length aldehyde dehydrogenase-related sequences were aligned to determine relationships within the aldehyde dehydrogenase (ALDH) extended family. The alignment reveals only four invariant residues: two glycines, a phenylalanine involved in NAD binding, and a glutamic acid that coordinates the nicotinamide ribose in certain E-NAD binary complex crystal structures, but which may also serve as a general base for the catalytic reaction. The cysteine that provides the catalytic thiol and its closest neighbor in space, an asparagine residue, are conserved in all ALDHs with demonstrated dehydrogenase activity. Sixteen residues are conserved in at least 95% of the sequences; 12 of these cluster into seven sequence motifs conserved in almost all ALDHs. These motifs cluster around the active site of the enzyme. Phylogenetic analysis of these ALDHs indicates at least 13 ALDH families, most of which have previously been identified but not grouped separately by alignment. ALDHs cluster into two main trunks of the phylogenetic tree. The largest, the "Class 3" trunk, contains mostly substrate-specific ALDH families, as well as the class 3 ALDH family itself. The other trunk, the "Class 1/2" trunk, contains mostly variable substrate ALDH families, including the class 1 and 2 ALDH families. Divergence of the substrate-specific ALDHs occurred earlier than the division between ALDHs with broad substrate specificities. A site on the World Wide Web has also been devoted to this alignment project.     

Novel characteristics of UDP-glucose dehydrogenase activities in maize: non-involvement of alcohol dehydrogenases in cell wall polysaccharide biosynthesis

UDP-glucose dehydrogenase (UDPGDH) activity was detected in extracts of maize cell-cultures and developing leaves. The reaction product was confirmed as UDP-glucuronate. Leaf extracts from null mutants defective in one or both of the ethanol dehydrogenase genes, ADH1 and ADH2, had similar UDPGDH activities to wild-type, showing that UDPGDH activity is not primarily due to ADH proteins. The mutants showed no defect in their wall matrix pentose:galactose ratios, or matrix:cellulose ratio, showing that ADHs were not required for normal wall biosynthesis. The majority of maize leaf UDPGDH activity had K _m (for UDP-glucose) 0.5–1.0 mM; there was also a minor activity with an unusually high K _m of >50 mM. In extracts of cultured cells, kinetic data indicated at least three UDPGDHs, with K _m values (for UDP-glucose) of roughly 0.027, 2.8 and >50 mM (designated enzymes E_L, E_M and E_H respectively). E_M was the single major contributor to extractable UDPGDH activity when assayed at 0.6–9.0 mM UDP-Glc. Most studies, in other plant species, had reported only E_L-like isoforms. Ethanol (100 mM) partially inhibited UDPGDH activity assayed at low, but not high, UDP-glucose concentrations, supporting the conclusion that at least E_H activity is not due to ADH. At 30 μM UDP-glucose, 20–150 μM UDP-xylose inhibited UDPGDH activity, whereas 5–15 μM UDP-xylose promoted it. In conclusion, several very different UDPGDH isoenzymes contribute to UDP-glucuronate and hence wall matrix biosynthesis in maize, but ADHs are not responsible for these activities.     

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity. The coenzyme specificity of malate dehydrogenase may be modulated by substitution of a single residue, as can the substrate specificity. The mechanism of catalysis of malate dehydrogenase is similar to that of lactate dehydrogenase, an enzyme with which it shares a similar 3-dimensional structure. Substitution of a single amino acid residue of a lactate dehydrogenase changes the enzyme specificity to that of a malate dehydrogenase, but a similar substitution in a malate dehydrogenase resulted in relaxation of the high degree of specificity for oxaloacetate. Knowledge of the 3-dimensional structures of malate and lactate dehydrogenases allows the redesign of enzymes by rational rather than random mutation and may have important commercial implications.     

Cloning of an enzyme that synthesizes a key nucleotide-sugar precursor of hemicellulose biosynthesis from soybean: UDP-glucose dehydrogenase.

Hemicellulose is a major component of primary plant cell walls. Many of the glycosyl residues found in hemicellulose are derived from the sugar precursor UDP-glucuronic acid, which can be converted into UDP-arabinose, UDP-apiose, UDP-galacturonic acid, and UDP-xylose. The enzyme controlling the biosynthesis of UDP-glucuronic acid, UDP-glucose dehydrogenase (EC 1.1.1.22), was cloned from soybean (Glycine max [L.] Merr.) by an antibody screening procedure. This enzyme is surprisingly homologous to the bovine sequence, which is the only other eukaryotic UDP-glucose dehydrogenase sequence known. The characteristic motifs of the enzyme, the catalytic center, a NAD-binding site, and two proline residues for main chain bends, are conserved within the prokaryotic and eukaryotic sequences. The soybean full-length cDNA clone encodes a protein of 480 amino acids with a predicted size of 52.9 kD. The enzyme is highly expressed in young roots, but lower expression levels were observed in expanding tissues of the epicotyl and in young leaves. The expression pattern of the enzyme in different developmental stages strengthens the argument that UDP-glucose dehydrogenase is a key regulator for the availability of hemicellulose precursors.     

链霉菌UDP-葡萄糖脱氢酸编码基因Ste6的克隆表达和性质研究

链霉菌139能够产生一种全新的胞外多糖——依博素（139A）,该多糖体内具有显著抗类风湿性关节炎活性。其生物合成基因簇（GenBank Accession Number：AYl31229）已被鉴定约31．3kb,包含22个开放阅读框（ste1—ste22）。以pET-30a为载体,克隆并在大肠杆菌BL21（DE3）中进行了ste6基因的表达,对该基因的克隆、表达与性质进行了研究。亲和层析法证实,纯化后重组蛋白具有催化UDP-葡萄糖脱氢变成UDP-葡萄糖醛酸的活性。这表明ste6编码产物是葡萄糖脱氢酶。为了证实ste6基因与依博素生物合成的关系,采用单交换基因破坏策略构建了ste6基因阻断突变株。结果初步显示ste6和依博素生物合成相关。     

Characterization of human UDP-glucose dehydrogenase. CYS-276 is required for the second of two successive oxidations

UDP-glucose dehydrogenase (UGDH) catalyzes two oxidations of UDP-glucose to yield UDP-glucuronic acid. Pathological overproduction of extracellular matrix components may be linked to the availability of UDP-glucuronic acid; therefore UGDH is an intriguing therapeutic target. Specific inhibition of human UGDH requires detailed knowledge of its catalytic mechanism, which has not been characterized. In this report, we have cloned, expressed, and affinity-purified the human enzyme and determined its steady state kinetic parameters. The human enzyme is active as a hexamer with values for Km and Vmax that agree well with those reported for a bovine homolog. We used crystal coordinates for Streptococcus pyogenes UGDH in complex with NAD+ cofactor and UDP-glucose substrate to generate a model of the enzyme active site. Based on this model, we selected Cys-276 and Lys-279 as likely catalytic residues and converted them to serine and alanine, respectively. Enzymatic activity of C276S and K279A point mutants was not measurable under normal assay conditions. Rate constants measured over several hours demonstrated that K279A continued to turn over, although 250-fold more slowly than wild type enzyme. C276S, however, performed only a single round of oxidation, indicating that it is essential for the second oxidation. This result is consistent with the postulated role of Cys-276 as a catalytic residue and supports its position in the reaction mechanism for the human enzyme. Lys-279 is likely to have a role in positioning active site residues and in maintaining the hexameric quaternary structure.     

The first structure of UDP-glucose dehydrogenase reveals the catalytic residues necessary for the two-fold oxidation

Bacterial UDP-glucose dehydrogenase (UDPGlcDH) is essential for formation of the antiphagocytic capsule that protects many virulent bacteria such as Streptococcus pyogenes andStreptococcus pneumoniae type 3 from the host's immune system. We have determined the X-ray structures of both native and Cys260Ser UDPGlcDH from S. pyogenes (74% similarity to S. pneumoniae) in ternary complexes with UDP-xylose/NAD(+) and UDP-glucuronic acid/NAD(H), respectively. The 402 residue homodimeric UDPGlcDH is composed of an N-terminal NAD(+) dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long (48 A) central alpha-helix. The first 290 residues of UDPGlcDH share structural homology with 6-phosphogluconate dehydrogenase, including conservation of an active site lysine and asparagine that are implicated in the enzyme mechanism. Also proposed to participate in the catalytic mechanism are a threonine and a glutamate that hydrogen bond to a conserved active site water molecule suitably positioned for general acid/base catalysis.     

兽疫链球菌透明质酸合成相关基因hasB的克隆以及性质研究

透明质酸是链球菌荚膜的主要组成部分,有着重要的生理功能。UDP-葡萄糖脱氢酶(HasB)是透明质酸合成中的一个关键酶,而C类链球菌的UDP-葡萄糖脱氢酶编码基因(hasB)尚未被克隆。通过hasB基因的上下游序列设计引物从兽疫链球茵的基因组中克隆出一段序列,测序结果显示其包含一个由1206个碱基组成的开放阅读框,所编码的蛋白序列同化脓链球菌和乳链球菌的UDP-葡萄糖脱氢酶蛋白序列分别有63．1％和70．6％的相似性。将这段基因置于T7启动子下,并在大肠杆菌中进行表达,能够得到一个约47kDa的蛋白,酶活测定显示其具有UDP-葡萄糖脱氢酶活性。这些结果表明所克隆的基因是兽疫链球菌的UDP-葡萄糖脱氢酶编码基因。     

The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii. Molecular cloning and sequence analysis

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8-kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined. The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N-terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non-homologous regions which are very rich in alanyl and prolyl residues. The N-terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.     

Differential processing of human and rat E1 α precursors of the branched-chain α-keto acid dehydrogenase complex caused by an N-terminal proline in the rat sequence

The N-terminal sequences of the E1 α, E1β and E2 subunits of the human branched-chain α-keto acid dehydrogenase complex have been determined by microsequencing. The N-termini of human E1β and E2 subunits (Val and Gly, respectively) are indentical to those of the corresponding rat and bovine subunits. However, the N-terminus of the human E1 α subunit (Ser) is identical to bovine, but differs from the rat E1 α (Phe0 subunit. Comparison of the N-terminal sequences of human and rat E1 α subunits shows that the serine residue at the + 1 position in the human sequence is replaced by a proline residue in the rat sequence. The presence of the proline residue apparently causes a 5′-shift by one residue in the cleavage site by the mitochondrial processing peptidase in the rat sequence, when compared to the human sequence. The results provide evidence that the mitochondrial processing peptidase cannot cleave an X-pro bond, similar to trypsin, chymotrypsinand microsomal signal peptidases.     

Brain succinic semialdehyde dehydrogenase: identification of reactive lysyl residues labeled with pyridoxal-5'-phosphate

An NAD+ dependent succinic semialdehyde dehydrogenase from bovine brain was inactivated by pyridoxal-5'- phosphate. Spectral evidence is presented to indicate that the inactivation proceeds through formation of a Schiff's base with amino groups of the enzyme. After NaBH(4) reduction of the pyridoxal-5'-phosphate inactivated enzyme, it was observed that 3.8 mol phosphopyridoxyl residues were incorporated/enzyme tetramer. The coenzyme, NAD+, protected the enzyme against inactivation by pyridoxal-5'-phosphate. The absorption spectrum of the reduced and dialyzed pyridoxal-5'-phosphate-inactivated enzyme showed a characteristic peak at 325 nm, which was absent in the spectrum of the native enzyme. The fluorescence spectrum of the pyridoxyl enzyme differs completely from that of the native enzyme. After tryptic digestion of the enzyme modified with pyridoxal-5'-phosphate followed by [3H]NaBH4 reduction, a radioactive peptide absorbing at 210 nm was isolated by reverse-phase HPLC. The sequences of the peptide containing the phosphopyridoxyllysine were clearly identical to sequences of other mammalian succinic semialdehyde dehydrogenase brain species including human. It is suggested that the catalytic function of succinic semialdehyde dehydrogenase is modulated by binding of pyridoxal-5'-phosphate to specific Lys(347) residue at or near the coenzyme-binding site of the protein.     

甘菊BADH基因cDNA的克隆及在盐胁迫下的表达

利用PCR、RT—PCR和PCR—RACE技术,从菊科植物甘菊（Dendranthema lavandulifolium）中克隆到2个甜菜碱醛脱氢酶（betaine aldehyde dehydrogenase,BADH）基因的同源基因,分别命名为DlBADH1和DlBADH2,GenBank登录号分别为DQ011151和DQ011152。DlBADH1的cDNA全长1821bp,其开放阅读框编码503个氨基酸的蛋白质;DlBADH2全长1918bp,编码506个氨基酸的蛋白质。两个基因核苷酸序列的同源性为97％,推导的氨基酸序列的同源性为98％。与已发表的其它植物BADH基因氨基酸序列的同源性在64％以上。在推导的氨基酸序列中,均含有醛脱氢酶所具有的高度保守的十肽（VTLELGGKSP）以及与酶功能有关的半胱氨酸残基（C）。在推导的氨基酸序列的系统关系中,甘菊位于其它双子叶植物和单子叶植物之间,与其植物分类的系统关系相吻合。RT—PCR—Southern半定量表达分析表明,甘菊BADH基因家族中存在表达受盐诱导的成员。     

Characterization of human UDP-glucose dehydrogenase reveals critical catalytic roles for lysine 220 and aspartate 280

Human UDP-glucose dehydrogenase (UGDH) is a homohexameric enzyme that catalyzes two successive oxidations of UDP-glucose to yield UDP-glucuronic acid, an essential precursor for matrix polysaccharide and proteoglycan synthesis. We previously used crystal coordinates for Streptococcus pyogenes UGDH to generate a model of the human enzyme active site. In the studies reported here, we have used this model to identify three putative active site residues: lysine 220, aspartate 280, and lysine 339. Each residue was site-specifically mutagenized to evaluate its importance for catalytic activity and maintenance of hexameric quaternary structure. Alteration of lysine 220 to alanine, histidine, or arginine significantly impaired enzyme function. Assaying activity over longer time courses revealed a plateau after reduction of a single equivalent of NAD+ in the alanine and histidine mutants, whereas turnover continued in the arginine mutant. Thus, one role of this lysine may be to stabilize anionic transition states during substrate conversion. Mutation of aspartate 280 to asparagine was also severely detrimental to catalysis. The relative position of this residue within the active site and dependence of function on acidic character point toward a critical role for aspartate 280 in activation of the substrate and the catalytic cysteine. Finally, changing lysine 339 to alanine yielded the wild-type Vmax, but a 165-fold decrease in affinity for UDP-glucose. Interestingly, gel filtration of this substrate-binding mutant also determined it was a dimer, indicating that hexameric quaternary structure is not critical for catalysis. Collectively, this analysis has provided novel insights into the complex catalytic mechanism of UGDH.     

Computational analysis of the quaternary structural changes induced by point mutations in human UDP-glucose dehydrogenase

UDP-glucose dehydrogenase (UGDH) is an enzyme catalyzing the conversion of UDP-glucose to UDP-glucuronic acid. Site-directed mutagenesis studies have revealed that human UGDH (hUGDH) has distinct oligomeric states that vary with different point mutations. In this study we have investigated how the changes in the oligomer-forming propensity may be involved in the thermal motion of wild-type hUGDH and its mutants, using normal mode analysis (NMA). Our results show that the perturbation caused by the mutation of a residue at a considerably distant location from the oligomeric interfaces is preferentially distributed throughout specific sites, especially the large flexible regions in the hUGDH structure, thereby changing the motional fluctuation pattern at the oligomeric interfaces. A large-magnitude cooperative motion at the oligomeric interfaces is a critical factor in interfering with the hexamer formation of the enzyme. In particular, structural stability at the dimeric interface is necessary to retain the hexameric structure of hUGDH.