Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

Purification and characterization of NAD-dependent isocitrate dehydrogenase from Dictyostelium discoideum

Isocitrate dehydrogenase (threo-d_s-isocitrate: NAD oxidoreductase (decar☐ylating) EC 1.1.1.41) from Dictyostelium dicoideum was purified 161-fold. The purified enzyme was NAD specific and required Mn²⁺ for activity. Isocitrate consumption and 2-oxoglutarate and NADH production were stoichiometric; no NADH oxidase or glutamate dehydrogenase activities were detected. The pH optimum range for activity was pH 7.5–8.5. Reductive car☐ylation of 2-oxoglutarate with NADH could not be demonstrated. Lineweaver - Burk plots of data from initial velocity studies were linear. There was no evidence of allosteric control by reported effectors (AMP, ADP, citrate) of isocitrate dehydrogenase activity. The reaction was inhibited by NADH. The inhibition by NADH was competitive when either isocitrate or NAD was the variable substrate. 2-Oxoglutarate was not inhibitory at concentrations below 4 mm. The Michaelis constant (K_m) and dissociation constant (K_ib) for isocitrate were 0.16 mm; and K_m and dissociation constant (K_ia) for NAD were 0.34 mm. The inhibition constant for NADH was 0.02 mm. The data are consistent with a rapid equilibrium random bi-bi reaction mechanism (Cleland nomenclature). The NAD-linked isocitrate dehydrogenase activity was also demonstrated in crude extracts of isolated mitochondria.     

Identification of Janthinobacterium lividum from the soils of the islands of Scotia Ridge and from Antarctic peninsula

Summary Eight isolates of bacteria from the soils of maritime Antarctica and Antarctic peninsula have been identified as members of the genus Janthinobacterium. Based on their morphology, physiological characteristics, biochemical characteristics and mole percent G+C content of their DNA six of them have been identified as J. lividum and the remaining two as atypical J. lividum. The Antarctic J. lividum unlike the mesophilic type strains were unique in that they could grow at pH 4, could produce acid from trehalose and none of them could tolerate more than 2.9% NaCl.     

Proline dehydrogenase from Pseudomonas fluorescens: Gene cloning, purification, characterization and homology modeling

The gene encoding proline dehydrogenase (ProDH) from Pseudomonas fluorescens was isolated using PCR amplification and cloned into pET23a expression vector. The expression of the recombinant target enzyme was induced by addition of IPTG. The produced His-fusion enzyme was purified and its kinetic properties were studied. The 3D structure modeling was also performed to identify key amino acids involved in FAD-binding and catalysis. The PCR product contained a 1033 bp open reading frame encoding 345 amino acid residue polypeptide chain. SDS-PAGE analysis revealed a MW of 40 kDa, whereas the native enzyme exhibited a MW of 40 kDa suggesting a monomeric protein. The K _m and V _max values of the P. fluorescens ProDH were estimated to be 35 mM and 116 μmol/min, respectively. ProDH activity was stable at alkaline pH and the highest activity was observed at 30°C and pH 8.5. The modeling analysis of the three dimensional structure elucidated that Lys-173 and Asp-202, which were oriented near the hydroxyl group of the substrate, were essential residues for the ProDH activity. This study, to our knowledge, is the first data on the cloning and biochemical and structural properties of P. fluorescens ProDH.     

Purification and characterization of an oxygen-labile, NAD-dependent alcohol dehydrogenase from Desulfovibrio gigas.

A NAD-dependent, oxygen-labile alcohol dehydrogenase was purified from Desulfovibrio gigas. It was decameric, with subunits of M(r) 43,000. The best substrates were ethanol (Km, 0.15 mM) and 1-propanol (Km, 0.28 mM). N-terminal amino acid sequence analysis showed that the enzyme belongs to the same family of alcohol dehydrogenases as Zymomonas mobilis ADH2 and Bacillus methanolicus MDH.     

NAD-dependent formate dehydrogenase from methylotrophic bacterium, strain 1. Purification and characterization.

1. NAD-dependent formate dehydrogenase was isolated from gram-negative methylotrophic bacteria, strain 1, grown on methanol. The purification procedure involved ammonium sulfate fractionation, ion-exchange chromatography and preparative isotachophoresis or gel filtration; it resulted in a yield of 40%. 2. The final enzyme preparations were homogeneous as judged by sedimentation in an ultracentrifuge. Formate dehydrogenase purified in the presence of EDTA reveals two bands on electrophoresis in polyacrylamide gel both after protein and activity staining. Two components are transformed into a single one after prolonged storage in the presence of 2-mercaptoethanol. 3. Formate dehydrogenase is a dimer composed of identical or very similar subunits. The molecular weight of the enzyme is about 80 000. 4. Amino acid composition and some other physico-chemical properties of the enzyme were studied. 5. Formate dehydrogenase is specific for formate and NAD as electron acceptor. The Michaelis constant was 0.11 mM for NAD and 15 mM for formate (pH 7.0, 37 degrees C). 6. Formate dehydrogenase was rapidly inactivated in the absence of -SH compounds. The enzyme retained full activity upon storage at ambient temperature in solution for half a year in the presence of 2-mercaptoethanol or EDTA.     

Cloning and sequencing of a gene encoding the 69-kDa extracellular chitinase of Janthinobacterium lividum

Abstract Janthinobacterium lividum secretes a major 56-kDa chitinase and a minor 69-kDa chitinase. A chitinase gene was defined on a 3-kb fragment of clone pRKT10, by virtue of fluorescent colonies in the presence of 4-methylumbelliferyl-β-d-N,N',N"-chitotrioside. Nucleotide sequencing revealed an 1998-bp open reading frame with the potential to encode a 69 716-Da protein with amino acid sequences similar to those in other chitinases, suggesting it encodes the minor chitinase (Chi69). Chitinase activity of Escherichia coli (pRKTIO) lysates was detected mainly in the periplasmic fraction and immunoblotting detected a 70-kDa protein in this fraction. Chi69 has an N-terminal secretory leader peptide preceding two probable chitin-binding domains and a catalytic domain. These functional domains are separated by linker regions of proline-threonine repeats. Amino acid sequencing of cyanogen bromide cleavage-derived peptides from the major 56-kDa chitinase suggested that Chi69 may be a precursor of Chi56. In addition, an N-terminally truncated version of Chi69 retained chitinase activity as expected if in vivo processing of Chi69 generates Chi56.     

Gene cloning,purification and characterization of thermostable alanine dehydrogenase of Bacillus stearothermophilus

The alanine dehydrogenase (l-alanine: NAD⁺ oxidoreductase, deaminating, EC 1.4.1.1) gene of Bacillus stearothermophilus IFO12550 was cloned and expressed in Escherichia coli C600 with a recombinant plasmid, pICD301, which was constructed from pBR322 and the alanine dehydrogenase gene derived from B. stearothermophilus. The enzyme overproduced in the clone was purified about 30 fold to homogeneity by heat treatment and two subsequent steps with a yield of 46%. The enzyme of E. coli-pICD301 was immunochemically identical with that of B. stearothermophilus. The enzyme has a molecular weight of about 240,000 and consists of six subunits identical in molecular weight (40,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75°C for 30 min; at 55°C and various pHs between 6.0 and 11.5 for 10 min. The enzymological properties are very similar to those of the mesophilic B. sphaericus enzyme (Ohshima, T. and Soda, K., Eur. J. Biochem., 100, 29–39, 1979) except for thermostability.     

Partial purification and characterization of NAD-dependent 7alpha-hydroxysteroid dehydrogenase from Bacteroides thetaiotaomicron.

A NAD-dependent 7alpha-hydroxysteroid dehydrogenase was purified 18-fold over the activity in crude cell extracts prepared from Bacteroides thetaiotaomicron NCTC 10852 using Bio-Gel A 1.5-M column chromatography. A molecular weight of 320 000 was estimated for the partially purified intact enzyme. Substrate saturation kinetics were performed using the 18-fold purified enzyme and the lowest Km values were obtained for 3alpha,7alpha-dihydroxy bile acid and bile salt substrates including chenodeoxycholic acid (Km 0.048 mM), glycochenodeoxycholic acid (Km 0.083 mM) and taurochenodeoxycholic acid (Km 0.059 mM). In contrast, 3alpha,7alpha,12alpha-trihydroxy bile acid and bile salts had higher Km values, i.e. cholic acid (Km 0.22 mM), glycoholic acid Km 0.32 mM) and taurocholic acid Km 0.26 mM). NAD had a Km value of 0.20 mM. The possible physiological significance of 7alpha-hydroxy bile acid oxidation to intestinal bacteroides strains was accessed by determining the rate of conversion of [14C]-cholic acid to 7-ketodeoxy[14C]cholic acid by whole cell suspensions under different incubation conditions. The rate of biotransformation of bile acid to keto-bile acid incubated anaerobically under N2 gas increased markedly when potential electron acceptors such as fumarate (10 mM) or menadione (4 mM) was added exogenously. These results suggest that bile acid oxidation reactions may be linked to energy-generating systems in this bacterium.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).     

Gene cloning,purification, and characterization of the highly thermostable leucine dehydrogenase of Bacillus sp.

The leucine dehydrogenase (l-leucine: NAD⁻ oxidoreductase, deaminating, EC 1.4.1.9) gene from Bacillus sp. DSM730 was cloned into Escherichia coli C600 with a vector plasmid, pBR322. The E. coli cells carrying a recombinant plasmid, pKULD1 (9.5 kb), produced a highly thermostable leucine dehydrogenase. The enzyme from E. coli cells carrying pKULD103, a deletion plasmid (6.5 kb) of pKULD1, was purified to homogeneity from the crude extract of clone cells by only one ion-change chromatography application with a yield of 73%. The leucine dehydrogenase of Bacillus sp. DSM730 is very similar in enzymological properties to those of other bacteria, except for molecular weight and stability. It has a molecular weight of about 280,000 and consists of six subunits identical in molecular weight (47,000). The enzyme is not inactivated by heat treatment at 80°C for 10 min, and incubation in the pII range of 5.4 to 10.3 at 55°C for 10 min. The Bacillus sp. DSM730 leucine dehydrogenase is the most thermostable of the leucine dehydrogenases so far purified, and is very useful for structure and stability studies, as well as being applicable to l-leucine production.     

芽胞杆菌(Bacillus sp.)YX-1耐有机溶剂葡萄糖脱氢酶的基因克隆与酶学性质

[目的] 从芽胞杆菌(Bacillus sp.)YX-1基因组中克隆出一种有机溶剂耐受型的葡萄糖脱氢酶基因,实现了该基因在大肠杆菌中的高效表达,研究了重组蛋白的酶学性质.[方法] 依据芽胞杆菌属中葡萄糖脱氢酶氨基酸序列的保守性,设计合理引物,钓取来源于Bacillus sp.YX-1的葡萄糖脱氢酶基因,构建诱导型表达载体pET28a-gdh,于大肠杆菌中进行表达.镍柱亲和层析法纯化重组蛋白,考察了重组蛋白的酶学性质.[结果] 葡萄糖脱氢酶基因全长为786 bp,编码261个氨基酸.酶学研究结果表明:该酶最适反应温度为45℃,最适pH值为8.0;具有良好的有机溶剂耐受性,于50％的辛烷、环己烷、癸烷中室温放置1h后,酶活仍能保持90％以上;具有较宽的底物谱,对多种糖均具有一定的催化活性,其中催化D-葡萄糖的活力最高,产生还原型辅酶因子;对辅酶NADH和NADPH具有相似的依赖性,对NAD+和NADP+的催化比活分别为8.37 U/mg和8.62 U/mg.[结论]利用生物信息学成功地挖掘出Bacillus sp.YX-1一种耐有机溶剂的葡萄糖脱氢酶,为氧化还原酶在有机相反应中的的辅酶再生循环提供了新型的生物催化剂.     

Partial purification and characterization of an NAD-dependent 3 beta-hydroxysteroid dehydrogenase from Clostridium innocuum.

In nine strains of Clostridium innocuum, 3 beta-hydroxysteroid-dehydrogenating activities were detected. 3 beta, 7 alpha, 12 alpha-Trihydroxy- and 3 beta-hydroxy-12-keto-5 beta-cholanoic acids were identified as reduction products of the respective 3-keto bile acids by gas-liquid chromatography and gas-liquid chromatography-mass spectrometry. One strain was shown to contain a NAD-dependent 3 beta-hydroxysteroid dehydrogenase. Enzyme production was constitutive in the absence of added bile acids. The specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids, with trisubstituted acids being more effective than disubstituted ones. A pH optimum of 10.0 to 10.2 was found after partial purification by DEAE-cellulose chromatography. A molecular weight of about 56,000 was established. 3 beta-hydroxysteroid dehydrogenase activity was also found in the membrane fraction after solubilization with Triton X-100, suggesting that the enzyme was originally membrane bound. The enzyme reduced a 3-keto group in unconjugated and conjugated bile acids, lower Km values being demonstrated with disubstituted than with trisubstituted bile acids. Keto functions at C-7 and C-12 further reduced the Km value. The enzyme was found to be partially heat labile (86% inactivation at 50 degrees C for 10 min).     

Molecular cloning, characterization, and engineering of xylitol dehydrogenase from Debaryomyces hansenii

Because of its natural ability to utilize both xylose and arabinose, the halotolerant and osmotolerant yeast Debaryomyces hansenii is considered as a potential microbial platform for exploiting lignocellulosic biomass. To gain better understanding of the xylose metabolism in D. hansenii, we have cloned and characterized a xylitol dehydrogenase gene (DhXDH). The cloned gene appeared to be essential for xylose metabolism in D. hansenii as the deletion of this gene abolished the growth of the cells on xylose. The expression of DhXDH was strongly upregulated in the presence of xylose. Recombinant DhXdhp was expressed and purified from Escherichia coli. DhXdhp was highly active against xylitol and sorbitol as substrate. Our results showed that DhXdhp was thermo-sensitive, and except this, its biochemical properties were quite comparable with XDH from other yeast species. Furthermore, to make this enzyme suitable for metabolic engineering of D. hansenii, we have improved its thermotolerance and modified cofactor requirement through modelling and mutagenesis approach.     

Gene cloning and biochemical characterization of 4-N-trimethylaminobutyraldehyde dehydrogenase II from Pseudomonas sp. 13CM

The gene encoding 4-N-trimethylaminobutyraldehyde dehydrogenase (TMABaldehyde-DH) from Pseudomonas sp. 13CM, responsible for the conversion of 4-N-trimethylaminobutyraldehyde (TMABaldehyde) to γ-butyrobetaine in the carnitine biosynthesis pathway, isolated by shotgun cloning and expressed in Escherichia coli DH5α. The recombinant TMABaldehyde-DH was purified 19.5 fold to apparent homogeneity by hydrophobic and affinity chromatography and biochemically characterized. The enzyme was found to be a trimer with identical 52 kDa subunits. The isoelectric point was found to be 4.5. Optimum pH and temperature were found respectively as pH 9.5 and 40 °C. The K_m values for TMABaldehyde, 4-dimethylaminobutyraldehyde, and NAD+ were respectively, 0.31, 0.62, and 1.16 mM. The molecular and catalytic properties differed from those of TMABaldehyde-DH I, which was discovered initially in Pseudomonas sp. 13CM. The new enzyme, designated TMABaldehyde-DH II, structural gene was inserted into an expression vector pET24b (+) and over-expressed in E. coli BL21 (DE3) under the control of a T7 promoter. The recombinant TMABaldehyde-DH from Pseudomonas sp. 13CM can now be obtained in large quantity necessary for further biochemical characterization and applications.     

Structural basis for cold adaptation. Sequence, biochemical properties, and crystal structure of malate dehydrogenase from a psychrophile Aquaspirillium arcticum

Aquaspillium arcticum is a psychrophilic bacterium that was isolated from arctic sediment and grows optimally at 4 degrees C. We have cloned, purified, and characterized malate dehydrogenase from A. arcticum (Aa MDH). We also have determined the crystal structures of apo-Aa MDH, Aa MDH.NADH binary complex, and Aa MDH.NAD.oxaloacetate ternary complex at 1.9-, 2.1-, and 2.5-A resolutions, respectively. The Aa MDH sequence is most closely related to the sequence of a thermophilic MDH from Thermus flavus (Tf MDH), showing 61% sequence identity and over 90% sequence similarity. Stability studies show that Aa MDH has a half-life of 10 min at 55 degrees C, whereas Tf MDH is fully active at 90 degrees C for 1 h. Aa MDH shows 2-3-fold higher catalytic efficiency compared with a mesophilic or a thermophilic MDH at the temperature range 4-10 degrees C. Structural comparison of Aa MDH and Tf MDH suggests that the increased relative flexibility of active site residues, favorable surface charge distribution for substrate and cofactor, and the reduced intersubunit ion pair interactions may be the major factors for the efficient catalytic activity of Aa MDH at low temperatures.     

Gene cloning,purification, and characterization of thermostable and halophilic leucine dehydrogenase from a halophilic thermophile,Bacillus licheniformis TSN9

A halophilic and thermophilic isolate from the sand of Tottori Dune was found to produce a thermostable and halophilic leucine dehydrogenase (EC 1.4.1.9). It was identified to be a new strain of Bacillus licheniformis. The enzyme gene was cloned into Escherichia coli JM109 with a vector plasmid pUC18. The enzyme was purified to homogeneity from the clone cell extract by ion-exchange column chromatography with a yield of 31%. The enzyme was found to be composed of eight subunits identical in relative molecular mass (43 000). The amino acid sequence of the enzyme, deduced from the nucleotide sequence of the gene, showed an identity of 84.6% with that of the B. stearothermphilus enzyme [Nagata S, Tanizawa K, Esaki N, Sakamoto Y, Ohshima T, Tanaka H, Soda K (1988) Biochemistry 27:9056–9062], although both enzymes were similar to each other in various enzymological properties such as thermostability, substrate and coenzyme specificities, and stereospecificity for hydrogen transfer from the C-4 of NADH. However, they were markedly distinct from each other in halophilicity: the B. licheniformis enzyme was much more stable than the other in the presence of high concentrations of salts.     

Purification, characterization and cloning of isovaleryl-CoA dehydrogenase from higher plant mitochondria.

Between the different types of Acyl-CoA dehydrogenases (ACADs), those specific for branched chain acyl-CoA derivatives are involved in the catabolism of amino acids. In mammals, isovaleryl-CoA dehydrogenase (IVD), an enzyme of the leucine catabolic pathway, is a mitochondrial protein, as other acyl-CoA dehydrogenases involved in fatty acid beta-oxidation. In plants, fatty acid beta-oxidation takes place mainly in peroxisomes, and the cellular location of the enzymes involved in the catabolism of branched-chain amino acids had not been definitely assigned. Here, we describe that highly purified potato mitochondria have important IVD activity. The enzyme was partially purified and cDNAs from two different genes were obtained. The partially purified enzyme has enzymatic constant values with respect to isovaleryl-CoA comparable to those of the mammalian enzyme. It is not active towards straight-chain acyl-CoA substrates tested, but significant activity was also found with isobutyryl-CoA, implying an additional role of the enzyme in the catabolism of valine. The present study confirms recent reports that in plants IVD activity resides in mitochondria and opens the way to a more detailed study of amino-acid catabolism in plant development.