Physicochemical Characterization of a Thermostable Alcohol Dehydrogenase from Pyrobaculum aerophilum

In this work we characterize an alcohol dehydrogenase (ADH) from the hyperthermophilic archaeon Pyrobaculum aerophilum (PyAeADHII). We have previously found that PyAeADHII has no activity when standard ADH substrates are used but is active when α-tetralone is used as substrate. Here, to gain insights into enzyme function, we screened several chemical libraries for enzymatic modulators using an assay employing α-tetralone. The results indicate that PyAeADHII activity in the presence of α-tetralone was inhibited by compounds such as flunarizine. We also examined metal coordination of the enzyme in solution by performing metal substitution of the enzyme-bound zinc (Zn²⁺) with cobalt. The solution-based absorption spectra for cobalt substituted PyAeADHII supports substitution at the structural Zn²⁺ site. To gain structural insight, we obtained the crystal structure of both wild-type and cobalt-substituted PyAeADHII at 1.75 Å and 2.20 Å resolution, respectively. The X-ray data confirmed one metal ion per monomer present only at the structural site with otherwise close conservation to other ADH enzymes. We next determined the co-crystal structure of the NADPH-bound form of the enzyme at 2.35 Å resolution to help define the active site region of the enzyme and this data shows close structural conservation with horse ADH, despite the lack of a catalytic Zn²⁺ ion in PyAeADHII. Modeling of α-tetralone into the NADPH bound structure suggests an arginine as a possible catalytic residue. The data presented here can yield a better understanding of alcohol dehydrogenases lacking the catalytic zinc as well as the structural features inherent to thermostable enzymes.     

Molecular Evolution Within the L-Malate and L-Lactate Dehydrogenase Super-Family

The NAD(P)-dependent malate (L-MalDH) and NAD-dependent lactate (L-LDH) form a large super-family that has been characterized in organisms belonging to the three domains of life. In the first part of this study, the group of [LDH-like] L-MalDH, which are malate dehydrogenases resembling lactate dehydrogenase, were analyzed and clearly defined with respect to the other enzymes. In the second part, the phylogenetic relationships of the whole super-family were presented by taking into account the [LDH-like] L-MalDH. The inferred tree unambiguously shows that two ancestral genes duplications, and not one as generally thought, are needed to explain both the distribution into two enzymatic functions and the observation of three main groups within the super-family: L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. In addition, various cases of functional changes within each group were observed and analyzed. The direction of evolution was found to always be polarized: from enzymes with a high stringency of substrate recognition to enzymes with a broad substrate specificity. A specific phyletic distribution of the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH over the Archaeal, Bacterial, and Eukaryal domains was observed. This was analyzed in the light of biochemical, structural, and genomic data available for the L-LDH, [LDH-like] L-MalDH, and dimeric L-MalDH. This analysis led to the elaboration of a refined evolutionary scenario of the super-family, in which the selection of L-LDH and the fate of L-MalDH during mitochrondrial genesis are presented.     

Nucleotide Sequence and Chromosomal Location of L-Lactate Dehydrogenase Gene from Streptococcus pneumoniae

We report here the 1244-bp sequence of a Streptococcus pneumoniae chromosomal fragment that contains the putative promoter and protein-coding region of the lactate dehydrogenase gene (ldh). The nucleotide sequence predicts a protein of 327 aa with a molecular weight of 35,202 daltons, after removal of the N-terminal methionine residue. The ldh gene is located on the ApaI fragment 1 and SmaI fragment 2 of the previously reported physical map of S. pneumoniae chromosome. Received: 3 December 1997 / Accepted: 20 January 1998     

Partial Purification and Characterization of the Maize Mitochondrial Pyruvate Dehydrogenase Complex

The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1β-, E1α-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg²⁺. Michaelis constants for the substrates were 47, 3, 77, and 1 μm for pyruvate, coenzyme A (CoA), NAD⁺, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD⁺ and CoA, and the inhibition constants were 15 and 47 μm, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg²⁺.     

Cellobiose Dehydrogenase from the Ligninolytic Basidiomycete Ceriporiopsis subvermispora

Cellobiose dehydrogenase (CDH), an extracellular flavocytochrome produced by several wood-degrading fungi, was detected in cultures of the selective delignifier Ceriporiopsis subvermispora when grown on a cellulose- and yeast extract-based liquid medium. CDH amounted to up to 2.5% of total extracellular protein during latter phases of the cultivation and thus suggested an important function for the fungus under the given conditions. The enzyme was purified 44-fold to apparent homogeneity. It was found to be present in two glycoforms of 98 kDa and 87 kDa with carbohydrate contents of 16 and 4%, respectively. The isoelectric point of both glycoforms is around 3.0, differing by 0.1 units, which is the most acidic value so far reported for a CDH. By using degenerated primers of known CDH sequences, one cdh gene was found in the genomic DNA, cloned, and sequenced. Alignment of the 774-amino-acid protein sequence revealed a high similarity to CDH from other white rot fungi. One notable difference was found in the longer interdomain peptide linker, which might affect the interdomain electron transfer at higher temperatures. The preferred substrate of C. subvermispora CDH is cellobiose, while glucose conversion is strongly discriminated by a 155,000-fold-lower catalytic efficiency. This is a typical feature of a basidiomycete CDH, as are the acidic pH optima for all tested electron acceptors in the range from 2.5 to 4.5.White rot fungi are the most efficient lignocellulose degraders in our ecosystem, and several species, e.g., Phanerochaete chrysosporium, Trametes versicolor, and Ceriporiopsis subvermispora, have been studied in great detail as model organisms for this complex process. The ability to degrade phenolic and nonphenolic lignin structures in wood has made these strains attractive for biotechnological applications mainly in the pulp and paper industry, where C. subvermispora exhibits a substantial advantage over P. chrysosporium and T. versicolor through its ability for selective removal of a large fraction of lignin without attacking the valuable cellulose (16, 38). The lignin-degrading system of these fungi is composed of extracellular enzymes together with low-molecular-mass cofactors (21, 46). Typically found ligninolytic enzymes are lignin peroxidase, manganese peroxidase (MnP), and laccase. The secretion pattern of these enzymes varies greatly in white rot fungi (22) and is influenced by culture conditions and medium composition. Whereas P. chrysoporium secretes high lignin and manganese peroxidase activities but no laccase activity (32, 33), C. subvermispora produces several MnP and laccase isoforms but no lignin peroxidase. T. versicolor is the only one of these model organisms known so far to express all three of these ligninolytic enzymes efficiently (5). Together with the cellulolytic enzyme system, these patterns of enzyme activities cause varied degrees of lignin and cellulose breakdown at different cultivation stages. The simultaneous attack of cellulose and lignin is the preferred strategy of T. versicolor, whereas C. subvermispora is a selective delignifier in the first stages of biotreatment, secreting only low activities of cellulolytic enzymes at a late culture stage (12, 23), and apparently lacks cellobiohydrolase activity (23).Cellobiose dehydrogenase (CDH; EC1.1.99.18; cellobiose (acceptor) 1-oxidoreductase) is an extracellular flavocytochrome secreted by some white rot and brown rot plant pathogenic and saprotrophic fungi from the dicaryotic phyla Basidiomycota and Ascomycota (50). It shows a strong preference for cellobiose and cello-oligosaccharides, which are oxidized to the corresponding lactones during the reductive half-reaction of the FAD cofactor, and further hydrolyze to aldonic acids in the bulk water. In the oxidative half-reaction FAD transfers two reduction equivalents to either one two-electron acceptor, e.g., various quinones, or to two one-electron acceptors, like complexed Fe(III) or Mn(II) ions. At low pH values (usually below 5.5), the heme cofactor can be involved in the electron transfer to one-electron acceptors. Even though CDH has been studied for a considerable time, the exact role and function of the two prosthetic groups are not fully understood. The pH optima with most electron acceptors are rather acidic, but oxygen, although a poor electron acceptor, is also reduced to H₂O₂ under neutral and alkaline conditions (30).In recent years CDH was shown to participate in the ligninolytic or cellulolytic metabolism of white rot fungi (3, 10, 24, 26, 50). The currently favored mechanism is the production of hydroxyl radicals through Fenton reaction chemistry by the ability of CDH to reduce Fe³⁺ to Fe²⁺ and to produce H₂O₂ (28, 31, 36, 37). CDH is believed to be involved in early stages of cellulose breakdown: knocking out the cdh gene in T. versicolor did not considerably affect its ability to grow on amorphous cellulosic substrates, while it could not grow on crystalline cellulose or recalcitrant substrates such as birch wood (13).Interestingly, no CDH activity has been reported so far from cultures of C. subvermispora, even though it is closely related to other white rotters producing this enzyme, e.g., Trametes spp. (35, 41) or Pycnoporus cinnabarinus (45). It has been speculated that the lack of CDH might contribute to the selectivity of C. subvermispora in degrading lignin while growing on wood. It was therefore the aim of our study to show unequivocally whether C. subvermispora carries a cdh gene and can produce the enzyme under certain growth conditions.     

Cathepsin L Plays an Important Role in the Lysosomal Degradation of L-Lactate Dehydrogenase

A cystatin α-sensitive cysteine proteinase that plays an important role in the lysosomal inactivation and degradation of L-lactate dehydrogenase (LDH) was purified by column chromatography from an ammonium sulfate precipitate of lysosome extract prepared from rat livers. It was eluted with marked delay from cathepsins B and H in a Sephacryl S-200 column by its specific interaction with the gel, and then effectively separated from cathepsins B and H and other proteins. It was eluted with 0.5 M NaCl after washing with 0.2 M NaCl in a CM-Sephadex column, indicating that it showed the same elution behavior as cathepsin L from the CM-Sephadex column. It had activity to hydrolyze z-Phe-Arg-NH-Mec, a synthetic substrate for cysteine proteinases, including cathepsins B and L. The N-terminal sequences of the final preparation of LDH-inactivating enzyme were identical with those of rat cathepsin L. Inactivation and degradation of LDH by the final preparation were observed and effectively inhibited by a low level of cystatin α as well as a general cysteine proteinase inhibitor, leupeptin or (L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine (3-methylbutyl)amide (E-64-c). From these results, it is concluded that cathepsin L plays a critical role in the lysosomal degradation of native LDH.     

Characterization of Flavin-Containing Opine Dehydrogenase from Bacteria

Opines, in particular nopaline and octopine, are specific compounds found in crown gall tumor tissues induced by infections with Agrobacterium species, and are synthesized by well-studied NAD(P)H-dependent dehydrogenases (synthases), which catalyze the reductive condensation of α-ketoglutarate or pyruvate with L-arginine. The corresponding genes are transferred into plant cells via a tumor-inducing (Ti) plasmid. In addition to the reverse oxidative reaction(s), the genes noxB-noxA and ooxB-ooxA are considered to be involved in opine catabolism as (membrane-associated) oxidases; however, their properties have not yet been elucidated in detail due to the difficulties associated with purification (and preservation). We herein successfully expressed Nox/Oox-like genes from Pseudomonas putida in P. putida cells. The purified protein consisted of different α-, β-, and γ-subunits encoded by the OdhA, OdhB, and OdhC genes, which were arranged in tandem on the chromosome (OdhB-C-A), and exhibited dehydrogenase (but not oxidase) activity toward nopaline in the presence of artificial electron acceptors such as 2,6-dichloroindophenol. The enzyme contained FAD, FMN, and [2Fe-2S]-iron sulfur as prosthetic groups. On the other hand, the gene cluster from Bradyrhizobium japonicum consisted of OdhB₁-C-A-B₂, from which two proteins, OdhAB₁C and OdhAB₂C, appeared through the assembly of each β-subunit together with common α- and γ-subunits. A poor phylogenetic relationship was detected between OdhB₁ and OdhB₂ in spite of them both functioning as octopine dehydrogenases, which provided clear evidence for the acquisition of novel functions by “subunit-exchange”. To the best of our knowledge, this is the first study to have examined flavin-containing opine dehydrogenase.     

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4‐amino‐4,6‐dideoxy‐d‐glucose, also known as d‐viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide‐linked sugar, which in the Mimivirus is thought to be UDP‐d‐glucose. The enzyme required for the installment of the amino group at the C‐4′ position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5′‐phosphate‐dependent enzyme, referred to as L136. For this analysis, three high‐resolution X‐ray structures were determined: the wildtype enzyme/pyridoxamine 5′‐phosphate/dTDP complex and the site‐directed mutant variant K185A in the presence of either UDP‐4‐amino‐4,6‐dideoxy‐d‐glucose or dTDP‐4‐amino‐4,6‐dideoxy‐d‐glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP‐d‐glucose or dTDP‐d‐glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three‐dimensional architecture was previously reported by this laboratory. As determined in this investigation,DesI shows a profound preference in its catalytic efficiency for the dTDP‐linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three‐dimensional model for a virally encoded PLP‐dependent enzyme and thus provides new information on sugar aminotransferases in general.     

Native and Acid-denatured L-Lactate Dehydrogenase Are Different in the Lysosomal Proteinases Involving in Their Overall Degradation

When native and acid-denatured lactate dehydrogenase (LDH) were incubated with total lysosomal enzymes in vitro, amino acids from their degradation were produced at various acidic pH. The pH profile in the overall degradation of native LDH was markedly different from that of acid-denatured LDH. Disappearance of the 35-kDa subunit of native LDH was markedly suppressed by a low level of cystatin α as well as by a general cysteine proteinase inhibitor, N-(L-3-trans-carboxyoxirane-2-carbonyl)-L-leucine-3-methylbutylamide (E-64-c). On the other hand, the degradation of acid-denatured LDH was only slightly suppressed by these inhibitors. It was concluded that at least a part of the proteinases involved in the overall degradation of native LDH is different from the proteinases involved in the degradation of acid-denatured form and a role of a cystatin α-sensitive cysteine proteinase is critical in the lysosomal degradation of native LDH, but not in that of acid-denatured form.     

Enhanced Enzymatic Activity of Glycerol-3-Phosphate Dehydrogenase from the Cryophilic Saccharomyces kudriavzevii

During the evolution of the different species classified within the Saccharomyces genus, each one has adapted to live in different environments. One of the most important parameters that have influenced the evolution of Saccharomyces species is the temperature. Here we have focused on the study of the ability of certain species as Saccharomyces kudriavzevii to grow at low temperatures, in contrast to Saccharomyces cerevisiae. We observed that S. kudriavzevii strains isolated from several regions are able to synthesize higher amounts of glycerol, a molecule that has been shown to accumulate in response to freeze and cold stress. To explain this observation at the molecular level we studied the expression of glycerol biosynthetic pathway genes and we observed a higher expression of GPD1 gene in S. kudriavzevii compared to S. cerevisiae in micro-vinification conditions. We observed higher enzymatic activity of Gpd1p in S. kudriavzevii in response to osmotic and cold stress. Also, we determined that S. kudriavzevii Gpd1p enzyme presents increased catalytic properties that will contribute to increase glycerol production. Finally, we evaluated the glycerol production with S. cerevisiae, S. kudriavzevii or a recombinant Gpd1p variant in the same background and observed that the S. kudriavzevii enzyme produced increased glycerol levels at 12 or 28°C. This suggests that glycerol is increased in S. kudriavzevii mainly due to increased V_max of the Gpd1p enzyme. All these differences indicate that S. kudriavzevii has changed the metabolism to promote the branch of the glycolytic pathway involved in glycerol production to adapt to low temperature environments and maintain the NAD⁺/NADH ratio in alcoholic fermentations. This knowledge is industrially relevant due to the potential use, for example, of S. cerevisiae-S. kudriavzevii hybrids in the wine industry where glycerol content is an important quality parameter.     

Gluconobacter oxydans NH-10中短链D-阿拉伯糖醇脱氢酶的研究

以氧化葡萄糖酸杆菌(Gluconobacter oxydans)NH-10基因组DNA为模板,扩增得到D-阿拉伯糖醇脱氢酶基因arDH,将其克隆到大肠杆菌表达载体JM109(DE3)中进行诱导表达。SDS-PAGE电泳分析ArDH的分子量约为30 kDa,是一个短链脱氢酶,既能催化D-阿拉伯糖醇氧化为D-木酮糖,又能催化D-木酮糖还原为D-阿拉伯糖醇。催化氧化反应时,对D-阿拉伯糖醇的K_m为60.67 mmol/L,V_max为0.803 U/mg;它能同时依赖于NAD⁺和NADP⁺,但是更加偏好辅酶NAD⁺;最适pH为12.0。还原反应对D-木酮糖的 K_m为36.39 mmol/L,V_max为1.71 U/mg;最优pH为7.0,最适温度均为30℃。     

Purification and Characterization of Sorbitol Dehydrogenase from Bovine Brain

Sorbitol dehydrogenase (EC 1.1.1.14) was isolated from bovine brain and purified 3,000-fold to apparent homogeneity, as judged by polyacrylamide gel electrophoresis. The purified enzyme had a specific activity of 36 units/mg of protein; a molecular weight of 39,000 for each of the four identical subunits and 155,000 for the intact enzyme were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel exclusion chromatography, respectively. The presence of one Zn2+ per subunit was confirmed by atom absorption spectroscopy; inactivation of the enzyme by metal-chelating agents points to the essential role that Zn2+ plays in the catalytically competent enzyme. The enzyme is also inactivated by thiol-blocking reagents; with respect to inactivation by sodium pyrophosphate, sorbitol dehydrogenase is different from closely related alcohol dehydrogenase.     

Structural Characterization of Bacterioferritin from Blastochloris viridis

Iron storage and elimination of toxic ferrous iron are the responsibility of bacterioferritins in bacterial species. Bacterioferritins are capable of oxidizing iron using molecular oxygen and import iron ions into the large central cavity of the protein, where they are stored in a mineralized form. We isolated, crystallized bacterioferritin from the microaerophilic/anaerobic, purple non-sulfur bacterium Blastochloris viridis and determined its amino acid sequence and X-ray structure. The structure and sequence revealed similarity to other purple bacterial species with substantial differences in the pore regions. Static 3- and 4-fold pores do not allow the passage of iron ions even though structural dynamics may assist the iron gating. On the other hand the B-pore is open to water and larger ions in its native state. In order to study the mechanism of iron import, multiple soaking experiments were performed. Upon Fe(II) and urea treatment the ferroxidase site undergoes reorganization as seen in bacterioferritin from Escherichia coli and Pseudomonas aeruginosa. When soaking with Fe(II) only, a closely bound small molecular ligand is observed close to Fe₁ and the coordination of Glu94 to Fe₂ changes from bidentate to monodentate. DFT calculations indicate that the bound ligand is most likely a water or a hydroxide molecule representing a product complex. On the other hand the different soaking treatments did not modify the conformation of other pore regions.     

保加利亚乳杆菌ATCC11842 L-乳酸脱氢酶同工酶基因的克隆与表达分析

通过对保加利亚乳杆菌(Lactobacillus delbrueckii subsp.bulgaricus)L-乳酸脱氢酶(L-lactate dehydrogenase, L-LDH)同工酶基因的异源表达、酶活测定和摇瓶发酵研究L-LDH在乳酸合成中的作用。将保加利亚乳杆菌ATCC11842中L-乳酸脱氢酶基因ldb0120和ldb0094分别克隆至载体pET28a(+)中,构建重组表达载体pET28aldb0120和pET28aldb0094,并转化到大肠埃希菌(Escherichia coli) BL21(DE3)中进行表达。进一步对重组蛋白进行Ni-NTA柱亲和层析和酶学活性测定,结果显示,LDB0120和LDB0094的比活力分别为0和25 U/mg,表明LDB0094是具有低活性的L-乳酸脱氢酶,而LDB0120不具有活性。对两株重组菌分别进行好氧和微好氧发酵,重组菌E.coli BL21/pET28aldb0094在好氧和微好氧条件可以合成L-乳酸,浓度分别为41.9和227.9 mg/L,而菌株E.coli BL21/pET28aldb0120在两种培养条件下均基本不合...