Environmental regulation of alcohol metabolism in thermotolerant methylotrophic Bacillus strains

The thermotolerant methylotroph Bacillus sp. C1 possesses a novel NAD-dependent methanol dehydrogenase (MDH), with distinct structural and mechanistic properties. During growth on methanol and ethanol, MDH was responsible for the oxidation of both these substrates. MDH activity in cells grown on methanol or glucose was inversely related to the growth rate. Highest activity levels were observed in cells grown on the C₁-substrates methanol and formaldehyde. The affinity of MDH for alcohol substrates and NAD, as well as V _max, are strongly increased in the presence of a M _r 50,000 activator protein plus Mg²⁺-ions [Arfman et al. (1991) J Biol Chem 266: 3955–3960]. Under all growth conditions tested the cells contained an approximately 18-fold molar excess of (decameric) MDH over (dimeric) activator protein. Expression of hexulose-6-phosphate synthase (HPS), the key enzyme of the RuMP cycle, was probably induced by the substrate formaldehyde. Cells with high MDH and low HPS activity levels immediately accumulated (toxic) formaldehyde when exposed to a transient increase in methanol concentration. Similarly, cells with high MDH and low CoA-linked NAD-dependent acetaldehyde dehydrogenase activity levels produced acetaldehyde when subjected to a rise in ethanol concentration. Problems frequently observed in establishing cultures of methylotrophic bacilli on methanol- or ethanol-containing media are (in part) assigned to these phenomena.Abbreviations MDH NAD-dependent methanol dehydrogenase - ADH NAD-dependent alcohol dehydrogenase - A1DH CoA-linked NAD-dependent aldehyde dehydrogenase - HPS hexulose-6-phosphate synthase - G6Pdh glucose-6-phosphate dehydrogenase     

Purification and characterization of methanol dehydrogenase of Methylobacterium nodulans rhizosphere phytosymbionts

Methanol dehydrogenase (MDH) of the facultative methylotrophic phytosymbiont Methylobacterium nodulans has been purified for the first time to an electrophoretically homogeneous state and characterized. The native protein with a molecular mass of 70 kDa consists of large (60 kDa) and small (6.5 kDa) subunits. The purified protein displayed a spectrum identical to that of pyrroloquinoline quinone (PQQ)-containing MDH, pI 8.7, pH optimum in the range 9–10. The enzyme was inactive in the absence of ammonium or methylamine and exhibited a wide substrate specificity with regard to C₁–C₅ alcohols with the high-est affinity to methanol (K _M = 70 μM), but it did not oxidize benzyl and secondary alcohols. The apparent K _M values to primary alcohols increased with the length of the carbon chain. The enzyme was characterized by a high stability level even in the absence of a substrate. An immobilized enzyme was used for amperometric methanol detection.     

人骨骼肌M蛋白的提取与纯化

依据Ｔｒｉｎｉｃｋ－Ｅｐｐｅｎｂｅｒｇｅｒ对鸡骨骼肌Ｍ蛋白的提取方法,由人骨骼肌中得到的Ｍ蛋白粗提物除含分子量为１６５０００的Ｍ蛋白外,还含有分子量为１８５０００和１４００００（Ｃ成分）的两组分。由于在粗提物中未发现分子量为９００００的磷酸化酶,我们将最终纯化步骤中的亲和层析改为制备电泳,同样获得了纯化的Ｍ蛋白。     

Cloning and primary structure of putative cytosolic and mitochondrial malate dehydrogenase from the mollusc Nucella lapillus (L.)

The evolutionary history of the malate dehydrogenase (MDH) gene family [NAD-dependent MDH; EC 1.1.1.37 and NAD(P)-dependent MDH; EC 1.1.1.82] has received much attention. MDHs have also featured extensively as electrophoretic markers in population genetics and evolutionary ecology, and in many cases, intraspecific variation in MDH has been correlated with environmental variables. However, while the amino acid residues essential for MDH function are known, no studies have examined intraspecific nucleotide variation despite evidence indicating that natural selection may be operating on this locus. This study presents two sets of degenerate oligonucleotide PCR primers to facilitate the cloning of cytosolic MDH (cMDH) and mitochondrial MDH (mMDH) from a broad range of animals (cMDH) and animals and plants (mMDH). These primers were used to obtain putative cMDH and mMDH cDNAs from the mollusc Nucella lapillus. The N. lapillus cMDH cDNA was found to encode a putative cMDH protein of 334aa and 36kDa, while the mMDH cDNA encoded a putative mature mMDH protein of 315aa and 33kDa. The putative amino acid sequences of the two compartmentalised N. lapillus MDHs are presented and compared to other known MDH sequences.     

Purification and characterization of a methanol dehydrogenase derived fromMethylomicrobium sp. HG-1 cultivated using a compulsory circulation diffusion system

Methanotrophs are microorganisms that possess the unique ability to utilize methane as their sole source of carbon and energy. A novel culture system, known as the compulsory circulation diffusion system, was developed for rapid growth of methanotrophic bacteria. Methanol dehydrogenase (MDH, EC 1.1.99.8) fromMethylomicrobium sp. HG-1, which belongs to the type 1 group of methanotrophic bacteria, can catalyze the oxidation of methanol directly into formaldehyde. This enzyme was purified 8-fold to electrophoretic homogeneity by means of a 4 step procedure and was found in the soluble fraction. The relative molecular weight of the native enzyme was estimated by gel filtration to be 120 kDa. The enzyme consisted of two identical dimers which, in turn, consisted of large and small subunits in anα ₂ β ₂ conformation. The isoelectric point was 5.4. The enzymatic activity of purified MDH was optimum at pH 9.0 and 60°C, and remained stable at that temperature for 20 min. MDH was able to oxidize primary alcohols from methanol to octanol and formaldehyde.     

Interaction of the N-terminal domain of Escherichia coli heat-shock protein ClpB and protein aggregates during chaperone activity

The Escherichia coli heat-shock protein ClpB reactivates protein aggregates in cooperation with the DnaK chaperone system. The ClpB N-terminal domain plays an important role in the chaperone activity, but its mechanism remains unknown. In this study, we investigated the effect of the ClpB N-terminal domain on malate dehydrogenase (MDH) refolding. ClpB reduced the yield of MDH refolding by a strong interaction with the intermediate. However, the refolding kinetics was not affected by deletion of the ClpB N-terminal domain (ClpBDeltaN), indicating that MDH refolding was affected by interaction with the N-terminal domain. In addition, the MDH refolding yield increased 50% in the presence of the ClpB N-terminal fragment (ClpBN). Fluorescence polarization analysis showed that this chaperone-like activity is explained best by a weak interaction between ClpBN and the reversible aggregate of MDH. The dissociation constant of ClpBN and the reversible aggregate was estimated as 45 muM from the calculation of the refolding kinetics. Amino acid substitutions at Leu 97 and Leu 110 on the ClpBN surface reduced the chaperone-like activity and the affinity to the substrate. In addition, these residues are involved in stimulation of ATPase activity in ClpB. Thus, Leu 97 and Leu 110 are responsible for the substrate recognition and the regulation of ATP-induced ClpB conformational change.     

依赖PQQ甲醇脱氢酶对底物催化专一性差的原因分析

采用甲基营养杆菌NO .2为实验菌株 ,经超声波破细胞 ,酸处理 ,DEAE 纤维素和CM 纤维素柱层析等改进的纯化程序 ,可得到比活力为 12 .5u/mg的甲醇脱氢酶 (MDH)样品。该酶在测活系统中除能氧化甲醇等醇类化合物外 ,还能以较大速率氧化氯化铵、甲胺、脲等物质 ,MDH对不同底物亲和力的差异性主要取决于其辅基吡咯喹啉醌 (PQQ)与底物的结合力。甲醇脱氢酶与底物结合前后在特定区域的光谱有一定的差异性     

一碳代谢关键酶——甲醇脱氢酶的研究进展与展望

甲醇和甲烷等一碳原料来源广泛,价格低廉,是生物制造的理想原料。甲醇脱氢酶(Methanol dehydrogenase,MDH)催化甲醇生成甲醛是一碳代谢的关键反应。目前已从天然甲基营养菌中发现了多种利用不同辅因子,具有不同酶学性质的MDH。其中,烟酰胺腺嘌呤双核苷酸(NAD)依赖型MDH被广泛应用于构建人工甲基营养菌。但是,NAD依赖型MDH的甲醇氧化活性较低,对甲醇的亲和力较差,导致甲醇氧化成为人工甲基营养菌代谢甲醇的限速步骤。为了提高甲醇氧化速率,进而提高人工甲基营养菌的甲醇利用效率,近年来大量研究集中于MDH的挖掘与改造研究。文中系统综述了不同类型MDH的发现、表征、改造以及在人工甲基营养菌中的应用进展,详细阐述了MDH的定向进化和多酶复合体的构建,并展望了通过细胞生长偶联的蛋白质进化和蛋白质理性设计获得高活性MDH的潜在策略。     

Complete primary structure and tissue expression of chicken pectoralis M-protein.

M-Protein (165 kDa) is a structural constituent of myofibrillar M-band in striated muscle. We generated a monoclonal antibody which recognized a 165-kDa protein from chicken pectoralis muscle in immunoblot analysis and stained the M-band under immunofluorescence microscopy. By screening a lambda gt11 cDNA library from chicken embryonic pectoralis muscle with this antibody, we isolated a cDNA clone encoding the M-protein. Northern blot analysis showed that M-protein mRNA is expressed in pectoralis and cardiac muscle but not in gizzard smooth muscle or non-muscle tissues. Moreover, the anterior latissimus dorsi muscle, which consists almost exclusively of slow fiber types, contains no detectable levels of the mRNA. The full-length cDNA sequence predicted a 1,450-amino acid polypeptide with a calculated molecular weight of 163 x 10(3). The encoded protein contains several copies of two different repetitive motifs: five copies of fibronectin type III repeats are in the middle part of the predicted molecule, and two and four copies of the immunoglobulin C2-type repeats are located toward the NH2-terminal and COOH-terminal regions, respectively. This indicates that M-protein, along with other thick filament-associated proteins such as C-protein, twichin, and titin, belongs to the superfamily of cytoskeletal proteins with immunoglobulin/fibronectin repeats.     

Purification and characterization of an activator protein for methanol dehydrogenase from thermotolerant Bacillus spp

All thermotolerant methanol-utilizing Bacillus spp. investigated by us possess a NAD-dependent methanol dehydrogenase (MDH) activity which is stimulated by a protein present in the soluble fraction of Bacillus sp. C1 cells. This activator protein was purified to homogeneity from Bacillus sp. C1 cells grown at a low dilution rate in a methanol-limited chemostat culture. The native activator protein (Mr = 50,000) is a dimer of Mr = 27,000 subunits. The N-terminal amino acid sequence revealed no significant similarity with any published sequences. Stimulation of MDH activity by the activator protein required the presence of Mg2+ ions. Plots of specific MDH activity versus activator protein concentration revealed Michaelis-Menten type kinetics. In the presence of activator protein, MDH displayed biphasic kinetics (v versus substrate concentration) toward C1-C4 primary alcohols and NAD. The data suggest that in the presence of activator protein plus Mg2+ ions, MDH possesses a high affinity active site for alcohols and NAD, in addition to an activator- and Mg2(+)-independent low affinity active site. The activation mechanism remains to be elucidated.     

The Agrobacterium tumefaciens DnaK: ATPase cycle, oligomeric state and chaperone properties

DnaK is a molecular chaperone that promotes cell survival during stress by preventing protein misfolding. The chaperone activity is regulated by nucleotide binding and hydrolysis events in the N-terminal ATPase domain, which in turn mediate substrate binding and release in the C-terminal substrate binding domain. In this study we determined that ATP hydrolysis was the rate limiting step in the ATPase cycle of Agrobacterium tumefaciens DnaK (Agt DnaK); however the data suggested that Agt DnaK had a significantly lower affinity for ATP than Escherichia coli DnaK. We show for the first time that Agt DnaK was very effective at preventing thermal aggregation of malate dehydrogenase (MDH) in a concentration dependent manner. This is in contrast to E. coli DnaK which was ineffective at preventing thermal aggregation of MDH. A mutant Agt DnaK-V431F, with a blocked hydrophobic pocket in the substrate binding domain, was unable to suppress the thermosensitivty of an E. coli dnaK103 deletion strain. However the mutation did not inhibit Agt DnaK-V431F from preventing the thermal aggregation of MDH. The oligomeric state of Agt DnaK was studied using size exclusion chromatography. We demonstrated that dilution of the Agt DnaK protein, the addition of ATP and the removal of the 10kDa C-terminal alpha-helical subdomain reduced higher order associations but did not abrogate dimerisation. Our research implies that the C-terminal alpha-helical subdomain is involved in higher order associations, while the substrate binding domain is possibly involved in dimerisation.     

Isolation of a type IV collagen binding protein from human platelets.

In order to study the molecular basis of platelet interaction with collagen IV of the basement membrane separating the arterial endothelium from the underlying subendothelial connective tissue, the possibility of presence of platelet membrane protein with affinity to type IV collagen was examined by subjecting the platelet membrane extract to affinity chromatography on collagen IV-sepharose. Urea (4 M) eluate was found to contain a protein with an apparent mol. wt of 68 kDa. The radioiodinated protein was isolated and used to test its specificity. By dot blot assay on nitrocellulose disks and solid-phase assays, the 68 kDa protein was found to bind with high affinity to collagen IV. Lack of significant binding to fibronectin and laminin when compared to albumin control indicated its high specificity for collagen. The radioiodinated protein was inserted into egg yolk lecithin liposomes. While these liposomes attached to microtitre plates coated with collagen IV, there was no significant binding to fibronectin or laminin coated wells, suggesting the membrane associated character of the protein as well as its specificity for collagen. These results indicate that presence of a 68 kDa protein in platelet membrane which interacts with very high specificity to collagen IV.     

From malate dehydrogenase to phenyllactate dehydrogenase. Incorporation of unnatural amino acids to generate an improved enzyme-catalyzed activity

Malate dehydrogenase (MDH) from Escherichia coli is highly specific for its keto acid substrate. The placement of the active site-binding groups in MDH effectively discriminates against both the shorter and the longer keto dicarboxylic acids that could potentially serve as alternative substrates. A notable exception to this specificity is the alternative substrate phenylpyruvate. This aromatic keto acid can be reduced by MDH, albeit at a somewhat slower rate and with greatly diminished affinity, despite the presence of several substrate-binding arginyl residues and the absence of a hydrophobic pocket in the active site. The specificity of MDH for phenylpyruvate has now been enhanced, and that for the physiological substrate oxaloacetate has been diminished, through the replacement of one of the binding arginyl residues with several unnatural alkyl and aryl amino acid analogs. This approach, called site-specific modulation, incorporates systematic structural variations at a site of interest. Molecular modeling studies have suggested a structural basis for the affinity of native MDH for phenylpyruvate and a rationale for the improved catalytic activity that is observed with these new, modified phenyllactate dehydrogenases.     

Production and proteolytic assay of lethal factor from Bacillus anthracis

Bacillus anthracis is the causative agent of anthrax. The major virulence factors are a poly-D-glutamic acid capsule and three-protein component exotoxin, protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa), respectively. These three proteins individually have no known toxic activities, but in combination with PA form two toxins (lethal toxin or edema toxin), causing different pathogenic responses in animals and cultured cells. In this study, we constructed and produced rLF as a form of GST fusion protein in Escherichia coli. rLF was rapidly purified through a single affinity purification step to near homogeneity. Furthermore, we developed an in vitro immobilized proteolytic assay of LF under the condition containing full-length native substrate, MEK1, rather than short synthetic peptide. The availability of full-length substrate and of an immobilized LF assay could facilitate not only the in-depth investigation of structure-function relationship of the enzyme toward its substrate but also wide spectrum screening of inhibitor collections based on the 96-well plate system.     

Physiological regulation of Paracoccus denitrificans methanol dehydrogenase synthesis and activity.

An enzyme-linked immunosorbent assay and a whole-cell activity assay were developed which allowed detection of methanol dehydrogenase (MDH) of Paracoccus denitrificans with increased sensitivity. By these methods, it was shown that MDH was not induced by its natural substrate, methanol. Relief from a catabolite repression-like mechanism seemed responsible for low-level MDH synthesis, while product induction was the hypothesized mechanism for synthesis of high amounts of MDH. In the latter process, formaldehyde may play an important role as effector. For a variety of culture conditions, inconsistencies were observed in the relation between amounts of MDH protein synthesized and enzyme activities measured in vitro. Regulation of pyrrolo-quinoline-quinone biosynthesis or a modulation of its incorporation and stability in MDH may constitute an overriding mechanism to ensure a correct tuning between metabolic rates of methanol consumption and the required methanol oxidation rates.     

Characterization of a novel 14 kDa bile acid-binding protein from rat ileal cytosol

A 14 kDa polypeptide in rat ileal cytosol has been identified as the major intestinal cytosolic bile acid-binding protein (I-BABP) by photoaffinity labeling with the radiolabeled 7,7-azo derivative of taurocholate (7,7-azo-TC). To further characterize I-BABP, the protein was purified by lysylglycocholate Sepharose 4B affinity and DE-52 anion-exchange chromatography. The purified I-BABP contained a single 14 kDa band on SDS-PAGE. The 14 kDa protein showed a 26-fold increase in binding affinity for [3H]7,7-azo-TC compared to cytosolic protein. Immunoblotting of protein fractions separated by affinity chromatography showed that neither liver fatty acid binding protein (L-FABP) nor intestinal fatty acid binding protein (I-FABP) bind to the affinity column and that the 14 kDa protein which bound to the column and was subsequently eluted with detergent did not cross-react with anti-L-FABP or anti-I-FABP. The 14 kDa protein labeled with [3H]7,7-azo-TC was radioimmunoprecipitated from cytosol by rabbit antiserum raised against purified I-BABP. I-BABP was shown to have a blocked N-terminus; however, its mixed internal sequence generated from cyanogen bromide-cleaved protein and amino acid composition indicated that it was related to (although clearly distinct from) both I-FABP and L-FABP. These studies have isolated a 14 kDa bile acid-binding protein from rat ileal cytosol which is immunologically and biochemically distinct from I-FABP and L-FABP.     

Purification and physical and kinetic characterization of an NAD+-dependent malate dehydrogenase from leaves of pineapple (Ananas comosus)

An NAD⁺-dependent malate dehydrogenase (MDH, EC 1.1.1.37) was purified and characterized from leaves of pineapple ( Ananas comosus ), a plant with Crassulacean acid metabolism (CAM). The purified enzyme had a subunit molecular mass of 39.5 kDa. Its activity showed a maximum at pH 6.8–7.0 and decreased sharply towards pH 8.0. This activity profile coincided with a change in the aggregation state, as determined by gel filtration on high-performance liquid chromatography from a dimer at pH 7.0 to a tetramer at pH 8.0. This isozyme is one of at least 5 MDH in pineapple leaves distinguishable by non-denaturing isoelectric focusing and displayed an isoelectric point of 5.8. The ratio of oxaloacetate reduction versus malate oxidation rates varied between 431 and 52 at pH 6.8 and 7.5, respectively. Antibodies raised against the purified pineapple leaf MDH immunodecorated a single 39.5-kDa polypeptide in denatured crude leaf extracts, but did not cross-react with extracts from purified pineapple mitochondria possessing high MDH activity. The purified MDH was recognized by monoclonal antibodies raised against the cytosolic MDH from Echinococcus granulosus . These and other distinctive traits, such as its isoelectric point and its subunit mass, suggest that the purified isozyme is the cytosolic MDH. Its properties are consistent with an implied function in the night acidification typical of CAM plants, although it is less clear if it also has a role in the daytime decarboxylation of malate.     

The interaction of methanol dehydrogenase and its electron acceptor, cytochrome cL in methylotrophic bacteria.

The interactions of methanol dehydrogenase (MDH, EC1.1.99.8) with its specific electron acceptor cytochrome cL has been investigated in Methylobacterium extorquens and Methylophilus methylotrophus. The MDHs of these two very different methylotrophs have the same alpha 2 beta 2 structure; the interaction of these MDHs with their specific electron acceptor, cytochrome cL, has been studied using a novel assay system. Electrostatic reactions are involved in 'docking' of the two proteins. EDTA inhibits the reaction by a process involving neither metal chelation nor the 'docking' process. Chemical modification studies showed that the two proteins interact by a 'docking' process involving interactions of lysyl residues on MDH and carboxyl residues on cytochrome cL. When 'zero length', two stage cross-linking was done (with proteins from both bacteria), the alpha-subunits of MDH cross-linked with cytochrome cL by way of lysyl groups on MDH and carboxyl groups on the cytochrome. Tuna mitochondrial cytochrome c provided a model for cytochrome cH which is the electron acceptor for cytochrome cL in the 'methanol oxidase' electron transport chain. Tuna cytochrome c was shown to form crosslinked products with carboxyl-modified cytochrome cL. MDH and tuna cytochrome c competed for the same domain on cytochrome cL. It was concluded that MDH reacts with cytochrome cL by an electrostatic reaction which involves carboxyl groups on cytochrome cL and amino groups on the alpha-subunit of MDH. The same domain on cytochrome cL is involved in subsequent 'docking' with its electron acceptor.