A familiar motif in a new context: the catalytic mechanism of hydroxyisourate hydrolase

Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway in soybeans. Its role is to catalyze the hydrolysis of 5-hydroxyisourate, the product of the urate oxidase reaction. There is extensive sequence homology between hydroxyisourate hydrolase and retaining glycosidases; in particular, the conserved active site glutamate residues found in retaining glycosidases are present in hydroxyisourate hydrolase as Glu 199 and Glu 408. However, experimental investigation of their roles, as well as the catalytic mechanism of the enzyme, have been precluded by the instability of 5-hydroxyisourate. Here, we report that diaminouracil serves as a slow, alternative substrate and can be used to investigate catalysis by hydroxyisourate hydrolase. The activity of the E199A protein was reduced 400-fold relative to wild-type, and no activity could be detected with the E408A mutant. Steady-state kinetic studies of the wild-type protein revealed that the pH-dependence of V(max) and V/K describe bell-shaped curves, consistent with the hypothesis that catalysis requires two ionizable groups in opposite protonation states. Addition of 100 mM azide accelerated the reaction catalyzed by the wild-type enzyme 8-fold and the E199A mutant 20-fold but had no effect on the E408A mutant. These data suggest that Glu 408 acts as a nucleophile toward the substrate forming a covalent anhydride intermediate, and Glu 199 facilitates formation of the intermediate by serving as a general acid and then activates water for hydrolysis of the intermediate. Thus, the mechanism of hydroxyisourate hydrolase is strikingly similar to that of retaining glycosidases, even though it catalyzes hydrolysis of an amide bond.     

一种新的乙酰高丝氨酸内酯水解酶基因的克隆和表达

目的 获得新的降解革兰阴性细菌数量阈值感应信号分子乙酰高丝氨酸内酯类化合物(AHL)的水解酶基因。方法 选择性富集和培养土壤中耐热细菌,抽取细菌总DNA作为模板,特异性聚合酶链反应扩增乙酰高丝氨酸内酯水解酶基因,进行克隆和DNA序列分析及原核表达。结果 得到1个新的AHL水解酶基因,该基因与已知基因的核苷酸序列和对应的氨基酸序列同源性最高分别为87％和94％。该基因在原核表达系统中表达,得到了与预期相对分子质鲢(Mr)一致的蛋白质。结论 证实乙酰高丝氨酸内酯水解酶广泛存在于环境微生物中。为进一步研究提供条件。     

Cloning and expression of a Staphylococcus aureus gene encoding a peptidoglycan hydrolase activity.

A gene of Staphylococcus aureus PS47 encoding lytic activity was cloned and expressed in Escherichia coli. Deletion analysis of a recombinant plasmid carrying a 7.4-kilobase-pair fragment (kbp) of S. aureus DNA suggested that the gene was located within a 2.5-kbp EcoRI-XbaI fragment. Analysis of extracts of E. coli harboring recombinant plasmids on denaturing polyacrylamide gels containing purified cell walls of S. aureus showed a clearing zone by a polypeptide of apparent Mr 23,000. The release of dinitrophenylalanine but not reducing groups from purified cell walls by a cell extract of recombinant E. coli suggested that we had cloned an N-acetylmuramyl-L-alanine amidase.     

Probing the evolution of hydroxyisourate hydrolase into transthyretin through active-site redesign

5-Hydroxyisourate hydrolase (HIUase) and transthyretin (TTR) are closely related phylogenetically and structurally, while performing quite different functions. The former catalyzes the hydrolysis of 5-hydroxyisourate within the urate degradation pathway, and the latter is a carrier protein involved in the extracellular transport of thyroid hormones and in the cotransport of retinol. The evolution of HIUase into TTR represents a remarkable example of adaptation of a new function by active-site modification of an enzyme. On the basis of phylogenetic reconstructions and structural comparison of HIUase and TTR, two mutations (Y116T and I16A) were likely to be crucial events in order to induce, after a gene duplication event, the conversion of the enzyme into a binding protein. By rational reshaping of the active sites of HIUase and functional analyses of its mutant forms, we have provided insights into how its neofunctionalization could be achieved. We show here that the two mutations at the active sites of HIUase open up the two ends of the channel that transverses the entire tetrameric protein, generating two cavities accessible to the thyroxine molecule and abrogating, at the same time, the enzymatic activity. Our data indicate that a small number of critical mutations affecting the active site of an enzyme may be sufficient to generate a drastically different function, while a large number of additional mutations may be required for the fine-tuning of the structural and functional features of new proteins.     

Localization, implications for function, and gene expression of chymotrypsin-like proteinases of cytotoxic RNK-16 lymphocytes

Rat RNK-16 leukemia cells kill YAC-1, which are the cells lysed by rodent natural killer lymphocytes. We found chymotrypsin-like proteinase ('chymase') activity in the RNK-16 dense granules that also contain cytolytic activity. The chymase activity hydrolyzed the thiobenzyl peptide substrate Suc-Phe-Leu-Phe-SBzl and, in comparison to RNK-16 tryptase activity, was selectively inhibited by three different types of serine proteinase inhibitors. The selective inhibitors were the fungal aldehyde chymostatin, the chloromethylketone Z-Gly-Leu-Phe-CH2Cl, and the mechanism-based or 'suicide' inhibitor 7-amino-4-chloro-3-(2-phenylethoxy)isocoumarin. These proteinase inhibitors also blocked RNK-16 granule-mediated cytolysis. Chymostatin, a reversible inhibitor, delayed granule-mediated cytolysis, whereas the irreversible chloromethylketone and isocoumarin proteinase inhibitors completely abrogated granule-mediated cytolysis. The two irreversible inhibitors displayed biphasic inhibition of the chymase activity, indicating that at least two chymases are present in the granules. By Northern blot analysis, we found that RNK-16 mRNA hybridized strongly with a cDNA probe of CCPI, a mouse cytotoxic T lymphocyte serine proteinase gene. These data imply that chymase activity in the cytotoxic granules is important for cytolytic function and is likely to belong to a new subfamily of serine proteinases.     

Cloning and expression of the soybean DapA gene encoding dihydrodipicolinate synthase

The rate-limiting step in the pathway for lysine synthesis in plants is catalyzed by the enzyme dihydrodipicolinate synthase (DS). We have cloned the portion of the soybean (Glycine max cv. Century) DapA cDNA that encodes the mature DS protein. Expression of the cloned soybean cDNA as a lacZ fusion protein was selected in a dapA ^- Escherichia coli auxotroph. The DS activity of the fusion protein was characterized in E. coli extracts. The DS activity of the fusion protein was inhibited by lysine concentrations that also inhibited native soybean DS, while E. coli DS activity was much less sensitive to inhibition by lysine.     

Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme

We report the identification and purification of a novel enzyme from soybean root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reaction is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase. The enzyme was purified from crude extracts of soybean root nodules approximately 100-fold to apparent homogeneity with a final specific activity of 10 micromol/min/mg. The enzyme exhibited a native molecular mass of approximately 68 kDa by gel filtration chromatography and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 +/- 2 kDa. The purified enzyme obeyed normal Michaelis-Menten kinetics, and the K(m) for 5-hydroxyisourate was determined to be 15 microM. The amino-terminal end of the purified protein was sequenced, and the resulting sequence was not found in any available data bases, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin.     

Cloning and expression of bile salt hydrolase gene from Lactobacillus plantarum JPP2

通过PCR方法从植物乳杆菌JPP2中扩增出胆盐水解酶(BSH)相关基因bsh3,利用中间克隆载体pMD19-T将其构建于表达载体pET-28b上,并转化入表达宿主菌E.coli BL21 (DE3),成功构建重组BSH的工程菌.核苷酸及推导的氨基酸序列分析表明,正确克隆出目的基因.诱导表达后,SDS-PAGE电泳结果显示出特异性蛋白质条带,其分子量约为38 kDa.此单克隆体系的构建为进一步研究BSH的功能奠定基础.     

植物乳杆菌JPP2胆盐水解酶相关基因克隆和表达

通过PCR方法从植物乳杆菌JPP2中扩增出胆盐水解酶（BSH）相关基因bsh3,利用中间克隆载体pMD19-T将其构建于表达载体pET-28b上,并转化入表达宿主菌E．coli BL21（DE3）,成功构建重组BSH的工程菌。核苷酸及推导的氨基酸序列分析表明,正确克隆出目的基因。诱导表达后,SDS-PAGE电泳结果显示出特异性蛋白质条带,其分子量约为38kDa。此单克隆体系的构建为进一步研究BSH的功能奠定基础。     

Cloning and sequencing of an epoxide hydrolase gene from Rhodosporidium paludigenum.

Epoxide hydrolase (EH) activity was recently described in yeasts and highly selective hydrolysis of epoxides was observed during whole cell biotransformations. To expand the available molecular data regarding yeast EHs, the EH encoding gene from Rhodosporidium paludigenum (CBS 6565) was isolated, cloned and sequenced. The genomic EH sequence revealed a 1600 bp sequence interrupted by six introns. cDNA sequence analysis revealed an open reading frame of 1236 bp with a deduced polypeptide length of 411 amino acids. The deduced amino acid sequence revealed a relative high degree of sequence homology compared to the amino acid sequence of the EH from Rhodotorula glutinis.     

九香虫保幼激素环氧水解酶基因克隆、生物信息学及表达分析

[目的]保幼激素环氧水解酶(Juvenile hormone epoxide hydrolase,JHEH)是昆虫体内保幼激素(Juvenile hormone,JH)的主要降解酶.本文旨在分析九香虫Aspongopus chinensis Dallas保幼激素环氧水解酶基因序列(AcJHEH)信息,探索其在九香虫生长发育过程中的作用.[方法]以九香虫成虫的cDNA为模板,采用RT-PCR技术克隆获得AcJHEH基因序列,并利用生物信息学软件对其编码蛋白的理化特性、结构特征和系统进化进行分析;采用qRT-PCR技术检测并分析九香虫不同龄期和不同组织中AcJHEH的相对表达量.[结果]成功获得了AcJHEH的完整开放阅读框ORF序列,全长均为1 363 bp,共编码453个氨基酸,预测分子量为51.74 ku,等电点(pI)为7.66,分子蛋白式C2414H3683N581O653S14,含有N-端跨膜基序XWG、催化三联体、保守基序HGWP和2个酪氨酸,属于环氧水解酶家族.系统进化树分析表明,AcJHEH与茶翅蝽Halyomorpha halys的JHEH聚为一支,再与同为半翅目的臭虫Cimex lectulariu和绿盲蝽Apolygus lucorum的JHEH聚为一类.荧光定量PCR结果显示,AcJHEH在九香虫不同发育阶段和各组织中均有表达,且脂肪中的表达量极显著高于其它组织(P＜0.001);滞育九香虫成虫表达量最高,其次为4-5龄若虫都极显著高于其它发育阶段(P＜0.001).[结论]九香虫JHEH属于环氧水解酶家族,确定为保幼激素环氧水解酶.依据qPCR结果推测AcJHEH通过改变九香虫体内保幼激素浓度来影响九香虫4-5龄若虫蜕皮发育、成虫生殖系统发育和滞育等.     

Cloning and expression of a parathion hydrolase gene from a soil bacterium, Burkholderia sp. JBA3

A bacterium, Burkholderia sp. JBA3, which can mineralize the pesticide parathion, was isolated from an agricultural soil. The strain JBA3 hydrolyzed parathion to p-nitrophenol, which was further utilized as the carbon and energy sources. The parathion hydrolase was encoded by a gene on a plasmid that strain JBA3 harbored, and it was cloned into pUC19 as a 3.7-kbp Sau3AI fragment. The ORF2 (ophB) in the cloned fragment encoded the parathion hydrolase composed of 526 amino acids, which was expressed in E. coli DH10B. The ophB gene showed no significant sequence similarity to most of other reported parathion hydrolase genes.     

Cloning and expression of a phloretin hydrolase gene from Eubacterium ramulus and characterization of the recombinant enzyme

Phloretin hydrolase catalyzes the hydrolytic C-C cleavage of phloretin to phloroglucinol and 3-(4-hydroxyphenyl)propionic acid during flavonoid degradation in Eubacterium ramulus. The gene encoding the enzyme was cloned by screening a gene library for hydrolase activity. The insert of a clone conferring phloretin hydrolase activity was sequenced. Sequence analysis revealed an open reading frame of 822 bp (phy), a putative promoter region, and a terminating stem-loop structure. The deduced amino acid sequence of phy showed similarities to a putative protein of the 2,4-diacetylphloroglucinol biosynthetic operon from Pseudomonas fluorescens. The phloretin hydrolase was heterologously expressed in Escherichia coli and purified. The molecular mass of the native enzyme was approximately 55 kDa as determined by gel filtration. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the deduced amino acid sequence of phy indicated molecular masses of 30 and 30.8 kDa, respectively, suggesting that the enzyme is a homodimer. The recombinant phloretin hydrolase catalyzed the hydrolysis of phloretin to equimolar amounts of phloroglucinol and 3-(4-hydroxyphenyl)propionic acid. The optimal temperature and pH of the catalyzed reaction mixture were 37 degrees C and 7.0, respectively. The K(m) for phloretin was 13 +/- 3 microM and the k(cat) was 10 +/- 2 s(-1). The enzyme did not transform phloretin-2'-glucoside (phloridzin), neohesperidin dihydrochalcone, 1,3-diphenyl-1,3-propandione, or trans-1,3-diphenyl-2,3-epoxy-propan-1-one. The catalytic activity of the phloretin hydrolase was reduced by N-bromosuccinimide, o-phenanthroline, N-ethylmaleimide, and CuCl(2) to 3, 20, 35, and 85%, respectively. Phloroglucinol and 3-(4-hydroxyphenyl)propionic acid reduced the activity to 54 and 70%, respectively.     

Cloning and expression of soluble epoxide hydrolase from potato

Five cDNAs encoding a putative soluble epoxide hydrolase (sEH) from potato were isolated and characterized. The cDNAs contained open reading frames encoding 36 kDa polypeptides which were highly homologous to the carboxy terminal region of mammalian sEH. When one of the cDNAs was expressed in a baculovirus system a soluble 38 kDa protein with epoxide hydrolase activity was produced. The recombinant enzyme hydrolyzed a commonly used diagnostic substrate for the soluble form of mammalian EH. Inhibitor profiles of the recombinant potato and mammalian sEH were also similar. The expression of sEH in potato was found to be regulated by both developmental and environmental signals. Levels of mRNA for sEH were higher in meristematic tissue than in mature leaves. This mRNA was also observed to accumulate on wounding and application of exogenous methyl jasmonate.     

Cloning and expression of the Salmonella enterotoxin gene.

This report examines the genetic basis for Salmonella typhimurium Q1 enterotoxin production. A 918-base-pair XbaI-HincII fragment of plasmid pJM17, composed of cholera toxin (CT) coding sequences (ctxAB), was used as a gene probe. With this probe, the S. typhimurium enterotoxin was identified on a 6.3-kilobase EcoRI-PstI fragment of chromosomal DNA from plasmidless strain Q1. We cloned this 6.3-kilobase fragment into Escherichia coli RR1. The genetic map of the cloned Salmonella enterotoxin (stx) gene was similar but not identical to the CT and E. coli heat-labile enterotoxin genes. By using synthetic oligonucleotides derived from the sequences of CT subunits A (ctxA) and B (ctxB), it was revealed that there were some conserved regions of DNA encoding the enterotoxins of strain Q1 and Vibrio cholerae. Expression of the cloned stx gene in minicells and subsequent Western blot (immunoblot) analysis with CT antitoxin demonstrated that the Salmonella enterotoxin had two or more subunits with molecular sizes of 45, 26, and 12 kilodaltons. Crude cell lysates of E. coli RR1(pCHP4), containing the cloned Salmonella enterotoxin gene, elicited fluid secretion in ligated rabbit intestinal loops and firm induration in rabbit skin. Both of these enterotoxic responses were neutralized by antisera specific for CT. Mucosal tissue from positive intestinal loops contained elevated levels of cyclic AMP. These data suggest some evolutionary relatedness between the enterotoxin genes of S. typhimurium and V. cholerae.     

Regulation of gene expression in the adipocyte: implications for obesity and proto-oncogene function

Adipose cells are the principal site for the storage of energy in the form of triglycerides in vertebrates. Marked changes in adipocyte-specific gene expression occur during cell differentiation and in disordered states of systemic energy balance, such as obesity and cachexia (wasting). Proteins related to the products of thefos andjun oncogenes have been implicated astrans-acting factors in the differentiation-dependent control of fat cell gene expression.     

Cloning of a carbofuran hydrolase gene from Achromobacter sp. strain WM111 and its expression in gram-negative bacteria.

A 14-kilobase-pair (kbp) EcoRI DNA fragment that encodes an enzyme capable of rapid hydrolysis of N-methylcarbamate insecticides (carbofuran hydrolase) was cloned from carbofuran-degrading Achromobacter sp. strain WM111. When used to probe Southern blots containing plasmid and total DNAs from WM111, this 14-kbp fragment hybridized strongly to a 14-kbp EcoRI fragment from the greater than 100-kbp plasmid harbored by this strain but weakly to EcoRI-digested total DNA from Achromobacter sp. strain WM111, indicating that the gene for N-methylcarbamate degradation (mcd) is plasmid encoded. Further subcloning localized the mcd gene on a 3-kbp ScaI-ClaI fragment. There was little or no expression of this gene in the alternative gram-negative hosts Pseudomonas putida, Alcaligenes eutrophus, Acinetobacter calcoaceticus, and Achromobacter pestifer. Western blotting (immunoblotting) of the protein products produced by low-level expression in P. putida confirmed that this 3-kbp fragment encodes the two 70+-kilodalton protein products seen in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified carbofuran hydrolase.     

Cloning of a cDNA for a constitutive NRT1 transporter from soybean and comparison of gene expression of soybean NRT1 transporters

We have isolated a cDNA for a putative transporter, named GmNRT1-3, in the NRT1 family from soybean. It was predicted to have a similar topological structure not only to both GmNRT1-1 and GmNRT1-2 reported previously, but also to other members of the family. Two other cDNAs isolated have parts of the sequence for putative NRT1 transporters, GmNRT1-4 and GmNRT1-5, suggesting that at least five NRT1 transporters occur in soybean. These GmNRT1 genes and the GmNRT2 gene, encoding a soybean NRT2 nitrate transporter, showed different expression patterns to each other under various nitrogen conditions. Specifically, GmNRT1-3 was constitutively expressed in both roots and leaves, while GmNRT1-2 was gradually expressed as the roots developed in the presence of ammonium as a nitrogen source, but not in the presence of both ammonium and nitrate. Based on these results, we discussed the possible regulation in the expression and role of these transporters in nitrate uptake.