Mass spectrometric analysis of the reactions catalyzed by -2-haloacid dehalogenase mutants and implications for the roles of the catalytic amino acid residues

-2-Haloacid dehalogenase catalyzes the hydrolytic dehalogenation of -2-haloalkanoic acids to produce the corresponding -2-hydroxyalkanoic acids. Asp10 of -2-haloacid dehalogenase from Pseudomonas sp. YL nucleophilically attacks the α-carbon atom of the substrate to form an ester intermediate, which is subsequently hydrolyzed by an activated water molecule. We previously showed that the replacement of Thr14, Arg41, Ser118, Lys151, Tyr157, Ser175, Asn177, and Asp180 causes significant loss in the enzyme activity, indicating the involvement of these residues in catalysis. In the present study, we tried to determine which process these residues are involved in by monitoring the formation of the ester intermediate by measuring the molecular masses of the mutant enzymes using ionspray mass spectrometry. When the wild-type enzyme and the T14A, S118D, K151R, Y157F, S175A, and N177D mutant enzymes were mixed with the substrate, the ester intermediate was immediately produced. In contrast, the R41K, D180N, and D180A mutants formed the intermediate much more slowly than the wild-type enzyme, indicating that Arg41 and Asp180 participate in the formation of the ester intermediate. This study presents a new method to analyze the roles of amino acid residues in catalysis.     

X-ray structure and mechanism of ZgHAD,a l-2-haloacid dehalogenase from the marine Flavobacterium Zobellia galactanivorans

Haloacid dehalogenases are potentially involved in bioremediation of contaminated environments and few have been biochemically characterized from marine organisms. The l -2-haloacid dehalogenase (l -2-HAD) from the marine Bacteroidetes Zobellia galactanivorans Dsij^T (ZgHAD) has been shown to catalyze the dehalogenation of C2 and C3 short-chain l -2-haloalkanoic acids. To better understand its catalytic properties, its enzymatic stability, active site, and 3D structure were analyzed. ZgHAD demonstrates high stability to solvents and a conserved catalytic activity when heated up to 60°C, its melting temperature being at 65°C. The X-ray structure of the recombinant enzyme was solved by molecular replacement. The enzyme folds as a homodimer and its active site is very similar to DehRhb, the other known l -2-HAD from a marine Rhodobacteraceae. Marked differences are present in the putative substrate entrance sites of the two enzymes. The H179 amino acid potentially involved in the activation of a catalytic water molecule was confirmed as catalytic amino acid through the production of two inactive site-directed mutants. The crystal packing of 13 dimers in the asymmetric unit of an active-site mutant, ZgHAD-H179N, reveals domain movements of the monomeric subunits relative to each other. The involvement of a catalytic His/Glu dyad and substrate binding amino acids was further confirmed by computational docking. All together our results give new insights into the catalytic mechanism of the group of marine l -2-HAD.     

Crystallization and preliminary X-ray analysis of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10.

Haloacid dehalogenases are enzymes that cleave carbon-chlorine or carbon-bromine bonds of 2-haloalkanoates. X-ray-quality crystals of L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8-7.0, using macroseeding techniques. The crystals, which diffract in the X-ray beam up to 2.0 A resolution, belong to the spacegroup C2221. Cell parameters are a = 58.8 A, b = 93.1 A, c = 84.2 A. A native data set to 2.3 A has been collected, with a completeness of 97% and an Rsym of 6.0%.     

Sec-dependent and Sec-independent translocation of haloacid dehalogenase Chd1 of Burkholderia cepacia MBA4 in Escherichia coli

2-Haloacid dehalogenases are hydrolytic enzymes that cleave the halogen-carbon bond(s) in haloalkanoic acids. We have previously isolated a cryptic haloacid dehalogenase gene from Burkholderia cepacia MBA4 and expressed it in Escherichia coli. This recombinant protein is unusual in having a long leader sequence, a property of periplasmic enzymes. In this paper, we report the functional role of this leader sequence. Western blot analyses showed that Chd1 is translocated to the periplasm. The results on the expression of Chd1 in the presence of sodium azide suggested the cleavage of the leader to be Sec-dependent. Chimeras of Chd1 and green fluorescent protein demonstrated that the leader sequence is fully functional in translocating the fusion protein to the periplasm. The expression of the chimeras in Sec mutants supported the Sec-dependent translocation. Surprisingly, recombinant Chd1 and a chimera with no leader sequence were also found in the periplasm.     

Crystallization and preliminary X-ray crystallographic studies of L-2-haloacid dehalogenase from Pseudomonas sp. YL

The dimeric L -2-haloacid dehalogenase from Pseudomonas sp. YL, (subunit mass, 26179 Da), has been crystallized by vapor diffusion, supplemented by repetitive seeding, against a 50 mM potassium dihydrogenphosphate solution (pH 4.5) containing 15% (w/v) polyethylene glycol 8,000 and 1% (v/v) n-propanol. The crystals belong to the monoclinic space group C2 with unit cell dimensions of a = 92.21 Å, b = 62.78 Angst; c = 50.84 Å, and β = 122.4°, and contain two dehalogenase dimers in the unit cell. They are of good quality and diffract up to 1.5 Å resolution.     

一种来源于运动替斯崔纳菌KA081020-065的新型卤醇脱卤酶的表达纯化及性质分析

卤醇脱卤酶(Halohydrin dehalogenase)不仅对于含氯污染物的生物降解、净化环境具有重要意义,而且在手性医药中间体合成中也是一种重要的生物催化剂,但其数量较少。采用基因挖掘在基因组数据库中获得一种来自运动替斯崔纳菌Tistrella mobilis KA081020-065的新型卤醇脱卤酶基因Hhe TM,将该基因克隆、表达在大肠杆菌BL21(DE3)中,利用镍柱亲和层析将该酶进行纯化并研究了其酶学性质。Hhe TM是一种同源四聚体;最适温度为50℃;不同的p H缓冲液对其活性有较大影响;在碱性、30℃以下的条件下稳定性高。在氰根离子存在的条件下,Hhe TM能够催化(S)-4-氯-3-羟基丁酸乙酯合成(R)-4-氰基-3-羟基丁酸乙酯,为酶法合成阿托伐他汀关键中间体提供了一种新的卤醇脱卤酶。     

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

The gene encoding thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL was isolated, and its over-expression system was constructed. Gene library was prepared fromSau3AI fragments of total DNA fromPs. sp. YL, pUC118 as a vector andEscherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.Abbreviations IPTG isopropyl -D-thiogalactopyranoside - KPB potassium phosphate buffer - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside     

R-2-卤代酸脱卤酶的同源模建及底物对映体选择性研究

对来自假单胞菌ZJU26中的R-2-卤代酸脱卤酶(DehI-R)进行同源模建,分析其与底物的相互作用,为解析酶的底物对映体选择性提供理论依据.采用Sybyl中的APM模块首次构建并优化了R-2-卤代酸脱卤酶的三维结构,并用Procheck 验证结构模型的合理性.使用Suflex-Docking模块将结构模型与底物分别进行对接,分析相互之间的作用.序列比对结果显示,R-2-卤代酸脱卤酶与恶臭假单胞菌PP3中DehI的相似性达23.71％.Deh-R模建后的结构与模板很好的吻合.模型比对分析DehI-R中参与催化的残基,除Asn2.03外大部分都比较保守.分子对接结果表明,R-2-氯丙酸和S-2-氯丙酸都可以结合到活性位点上,决定其选择性的是值点Asn203,在RS-2-卤代酸脱卤酶所对应位点的残基为Ala,相比之下,Aan具备较大的空间位阻,从而阻止了S-2-氯丙酸的反应.利用Sybyl中的Biopolymer模块对R-2-卤代酸脱卤酶中的Asn203突变成具有不同空间位阻的Ala、Gly和Gln.突变酶与底物对接结果进一步证实了Asn203位点对R-2-卤代酸脱卤酶的底物对映体选择性作用.     

Purification and properties of a new enzyme, DL-2-haloacid dehalogenase, from Pseudomonas sp.

A new enzyme, DL-2-haloacid dehalogenase, was isolated and purified to homogeneity from the cells of Pseudomonas sp. strain 113. This enzyme catalyzed non-stereospecific dehalogenation of both of the optical isomers of 2-chloropropionate through an SN2 type of reaction; L- and D-lactates were formed from D- and L-2-chloropropionates, respectively. The enzyme acted on 2-halogenated aliphatic carboxylic acids whose carbon chain lengths were less than five. It also dehalogenated trichloroacetate to form oxalate and showed maximum activity at pH 9.5. The Michaelis constants for substrates were as follows: 5.0 mM for monochloroacetate, 1.1 mM for L-2-chloropropionate, and 4.8 mM for D-2-chloropropionate. DL-2-Haloacid dehalogenase was inhibited by HgCl2, ZnSO4, and MnSO4, but was not affected by thiol reagents, such as p-chloromercuribenzoate and iodoacetamide. This enzyme had a molecular weight of about 68,000 and appeared to be composed of two subunits identical in molecular weight.     

Bacterial assimilation of d- and l-2-chloropropionates and occurrence of a new dehalogenase

The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing dl-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both d- and l-isomers of 2-chloropropionate and (2) strains utilizing only the l-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of d- and l-2-chloropropionates to yield l- and d-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the d- and l-isomers. Apparent K _m values for d- and l-2-chloropropionates were 4.5 and 1.0 mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed witg the crude enzyme preparation showed that the dehalogenation of d- and l-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and dl-2-chlorobutyrate is due to a single protein.Abbreviations MCA monochloroacetic acid - DCA dichloroacetic acid - TCA trichloroacetic acid - 2 MCPA 2-monochloropropionic acid - 22 DCPA 2,2-dichloropropionic acid - 3 MCPA 3-monochloropropionic acid - 2 MCBA 2-monochlorobutyric acid - 3 MCBA 3-monochlorobutyric acid - 4 MCBA 4-monochlorobutyric acid     

Structure and activity of DmmA, a marine haloalkane dehalogenase

DmmA is a haloalkane dehalogenase (HLD) identified and characterized from the metagenomic DNA of a marine microbial consortium. Dehalogenase activity was detected with 1,3-dibromopropane as substrate, with steady-state kinetic parameters typical of HLDs (K_m = 0.24 ± 0.05 mM, k_cat = 2.4 ± 0.1 s⁻¹). The 2.2-Å crystal structure of DmmA revealed a fold and active site similar to other HLDs, but with a substantially larger active site binding pocket, suggestive of an ability to act on bulky substrates. This enhanced cavity was shown to accept a range of linear and cyclic substrates, suggesting that DmmA will contribute to the expanding industrial applications of HLDs.     

A transmembrane helix-bundle from G-protein coupled receptor CB2: biosynthesis, purification, and NMR characterization

The cannabinoid receptor subtype 2 (CB2) is a member of the G-protein coupled receptor (GPCR) superfamily. As the relationship between structure and function for this receptor remains poorly understood, the present study was undertaken to characterize the structure of a segment including the first and second transmembrane helix (TM1 and TM2) domains of CB2. To accomplish this, a transmembrane double-helix bundle from this region was expressed, purified, and characterized by NMR. Milligrams of this hydrophobic fragment of the receptor were biosynthesized using a fusion protein overexpression strategy and purified by affinity chromatography combined with reverse phase HPLC. Chemical and enzymatic cleavage methods were implemented to remove the fusion tag. The resultant recombinant protein samples were analyzed and confirmed by HPLC, mass spectrometry, and circular dichroism (CD). The CD analyses of HPLC-purified protein in solution and in DPC micelle preparations suggested predominant alpha-helical structures under both conditions. The 13C/15N double-labeled protein CB2(27-101) was further verified and analyzed by NMR spectroscopy. Sequential assignment was accomplished for more than 80% of residues. The 15N HSQC NMR results show a clear chemical shift dispersion of the amide nitrogen-proton correlation indicative of a pure double-labeled polypeptide molecule. The results suggest that this method is capable of generating transmembrane helical bundles from GPCRs in quantity and purity sufficient for NMR and other biophysical studies. Therefore, the biosynthesis of GPCR transmembrane helix bundles represents a satisfactory alternative strategy to obtain and assemble NMR structures from recombinant "building blocks."     

Catalytic action of L-2-halo acid dehalogenase on long-chain L-2-haloalkanoic acids in organic solvents

A Lyophilized preparation of L-2-halo acid dehalogenase was not only stable but also catalytically active in anhydrous dimethyl sulfoxide, toluene, and other organic solvents. 2-Halo acids with long alkyl (C(5)-C(16)) or aromatic (phenyl and benzyl) side chains were inert in water but dehalogenated effectively in anhydrous dimethyl sulfoxide by the lyophilized enzyme. Long chain 2-haloalkanoic acids such as 2-bromohexadecanoic acids were better as substrate than short-chain halo acids (e.g., 2-chloropropanoic acid). The dehalogenation proceed with inversion of C(2) configuration to produce the corresponding (2R)-2-hydroxy acids in anhydrous dimethyl sulfoxide in the same way as found in water.     

Partial purification and characterization of a copper-induced anionic peroxidase of sunflower roots

Treatment of 14-day-old sunflower seedlings with a toxic amount of copper (50 μM of CuSO₄) during 5 days caused significant increase in peroxidase activity in roots. Qualitative analysis of soluble proteins using native anionic PAGE followed by detection of peroxidase activity with guaïacol as electron donor in the presence of H₂O₂ revealed five stimulated peroxidases, named A1, A2, A3, A4, and A5. These peroxidases had differential behavior during the period of treatment. A1, A2, A3 and A4 were stimulated in the first period of stress, but rapidly suppressed at 72 h. A5 showed a progressive stimulation which was even increased at 120 h. A1 was partially purified, identified using liquid chromatography coupled to mass spectrometry (LC-MS/MS), and characterized. Effects of pH and temperature on its activity were determined with guaïacol as electron donor. Optima were obtained at pH 8 and at 40 °C. Analysis of substrate specificity showed that A1 was active on coniferyl alcohol but not on IAA. Enzymatic activity was inhibited by a high concentration of H₂O₂.     

Endo-beta-mannosidase, a plant enzyme acting on N-glycan: purification, molecular cloning, and characterization

Endo-beta-mannosidase is a novel endoglycosidase that hydrolyzes the Manbeta1-4GlcNAc linkage in the trimannosyl core structure of N-glycans. This enzyme was partially purified and characterized in a previous report (Sasaki, A., Yamagishi, M., Mega, T., Norioka, S., Natsuka, S., and Hase, S. (1999) J. Biochem. 125, 363-367). Here we report the purification and molecular cloning of endo-beta-mannosidase. The enzyme purified from lily flowers gave a single band on native-PAGE and three bands on SDS-PAGE with molecular masses of 42, 31, and 28 kDa. Amino acid sequence information from these three polypeptides allowed the cloning of a homologous gene, AtEBM, from Arabidopsis thaliana. AtEBM was engineered for expression in Escherichia coli, and the recombinant protein comprised a single polypeptide chain with a molecular mass of 112 kDa corresponding to the sum of molecular masses of three polypeptides of the lily enzyme. The recombinant protein hydrolyzed pyridylamino derivatives (PA) of Manalpha1-6Manbeta1-4Glc-NAcbeta1-4GlcNAc into Manalpha1-6Man and GlcNAcbeta1-4Glc-NAc-PA, showing that AtEBM is an endo-beta-mannosidase. AtEBM hydrolyzed Man(n)Manalpha1-6Manbeta1-4GlcNAcbeta1-4GlcNAc-PA (n = 0-2) but not PA-sugar chains containing Manalpha1-3Manbeta or Xylosebeta1-2Manbeta as for the lily endo-beta-mannosidase. AtEBM belonged to the clan GH-A of glycosyl hydrolases. Site-directed mutagenesis experiments revealed that two glutamic acid residues (Glu-464 and Glu-549) conserved in this clan were critical for enzyme activity. The amino acid sequence of AtEBM has distinct differences from those of the bacterial, fungal, and animal exo-type beta-mannosidases. Indeed, AtEBM-like genes are only found in plants, indicating that endo-beta-mannosidase is a plant-specific enzyme. The role of this enzyme in the processing and/or degradation of N-glycan will be discussed.     

Demonstration of a new amidase acting on glycopeptides

An enzyme preparation from almond emulsin cleaved the peptide-carbohydrate linkage of stem bromelain glycopeptide, Asn-Asn (oligosaccharide)-Glu-Ser-Ser. The resulting products were determined to be an intact peptide, Asn-$\text{Asp}$-Glu-Ser-Ser, and an intact oligosaccharide unit with two moles of N-acetylglucosamine. So far as tested the enzyme hydrolyzed glycopeptides with 3 to 10 amino acids, while both asparagine-oligosaccharide from ovalbumin and Asn-GlcNAc were not. Thus, the enzyme is a new amidase capable of hydrolyzing aspartylglycosylamine linkage in glycopeptides with multiple amino acid residues.     

From genome to proteome: back to the future. Report on the 7th Siena meeting, September 3-7, 2006

This report reviews the 7th Siena Meeting 'From Genome to Proteome: Back to the Future' which took place in Italy from 3-7 September, 2006. There was a significant rise in the number of delegates attending compared with previous Siena meetings. A diversity of speakers and presentations addressed the theme of the meeting in moving proteomics forward to integrate with biology as a whole entity rather than in isolated fractions. In addition, technological advancements in sample preparation and separation as well as identification were discussed.     

Enantioselectivity of haloalkane dehalogenase LinB on the degradation of 1,2-dichloropropane: A QM/MM study

The hydrolysis dechlorination mechanism of a chiral organochlorine pollutant, 1,2-dichloropropane (DCP), catalyzed by haloalkane dehalogenase LinB has been investigated by using a combined quantum mechanics/molecular mechanics method. LinB was confirmed to be enantioselective towards the catabolism of the racemic mixture. Based on the S_N2 nucleophilic substitution mechanism, the dechlorination process was identified as the rate-determining step in LinB-catalyzed degradation of 1,2-dichloropropane, the Boltzmann-weighted average potential barrier of which is 18.8 kcal/mol for the (R)-isomer and 24.0 kcal/mol for the (S)-isomer. A particular water molecule near (S)-DCP in the reaction system can strongly disturb the dechlorination process, which can account for the enantioselectivity of LinB. Further electrostatic influence analysis indicates that proper mutation of Gly37 may improve the catalytic efficiency of LinB towards DCP.     

The purification and characterisation of 4-chlorobenzoate:CoA ligase and 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain TM-1

4-Chlorobenzoate:CoA ligase, the first enzyme in the pathway for 4-chlorobenzoate dissimilation, has been partially purified from Arthrobacter sp. strain TM-1, by sequential ammonium sulphate precipitation and chromatography on DEAE-Sepharose and Sephacryl S-200. The enzyme, a homodimer of subunit molecular mass approximately 56 kD, is dependent on Mg2+-ATP and coenzyme A, and produces 4-chlorobenzoyl CoA and AMP. Besides Mg2+, Mn2+, Co2+, Fe2+ and Zn2+ are also stimulatory, but not Ca2+. Maximal activity is exhibited at pH 7.0 and 25 degrees C. The ligase demonstrates broad specificity towards other halobenzoates, with 4-chlorobenzoate as best substrate. The apparent Michaelis constants (Km) of the enzyme for 4-chlorobenzoate, CoA and ATP were determined as 3.5, 30 and 238 microM respectively. 4-Chlorobenzoyl CoA dehalogenase, the second enzyme, has been purified to homogeneity by sequential column chromatography on hydroxyapatite, DEAE-Sepharose and Sephacryl S-200. It is a homotetramer of 33 kD subunits with an isoelectric point of 6.4. At pH 7.5 and 30 degrees C, Km and kcat for 4-CBCoA are 9 microM and 1 s(-1) respectively. The optimum pH is 7.5, and maximal enzymic activity occurs at 45 degrees C. The properties of this enzyme are compared with those of the 4-chlorobenzoyl CoA dehalogenases from Arthrobacter sp. strain 4-CB1 and Pseudomonas sp. strain CBS-3, which differ variously in their N-terminal amino acid sequences, optimal pH values, pI values and/or temperatures of maximal activity.     

嗜热菌Bacillus fordii 3-2海因酶的纯化及性质

对自行筛选的海因酶法L-苯丙氨酸生产菌株Bacillus fordii 3-2中的海因酶进行了分离纯化及相关性质研究.该海因酶协同L-氨甲酰水解酶是海因酶法生产L-氨基酸的关键酶.B.fordii 3-2菌悬液经压力破碎离心后取上清液为粗酶液,粗酶液通过硫酸铵分级沉淀、Phenyl FF(high sub) 疏水层析以及Source 15Q离子交换层析,经SDS-PAGE分析达到电泳纯,亚基相对分子质量为55×103,海因酶的纯化回收率为20.5%,纯化倍数为149.23.该海因酶在pH 8.0～10.0的范围内具有较高的活性,在45～70℃具有很高的酶活力, 最适反应pH和温度分别为10.0 ℃和65 ℃.纯酶易氧化失活,DTT对该酶有一定的保护作用.二价金属离子,Mn2 、Co2 及Fe2 对酶活性有显著的促进作用,而Ca2 、Cu2 等对酶活性有抑制作用.相关研究可为该菌株及海因酶的进一步开发与应用提供理论指导.