Structure and mechanism of soluble quinoprotein glucose dehydrogenase.

Soluble glucose dehydrogenase (s-GDH; EC 1.1.99.17) is a classical quinoprotein which requires the cofactor pyrroloquinoline quinone (PQQ) to oxidize glucose to gluconolactone. The reaction mechanism of PQQ-dependent enzymes has remained controversial due to the absence of comprehensive structural data. We have determined the X-ray structure of s-GDH with the cofactor at 2.2 A resolution, and of a complex with reduced PQQ and glucose at 1.9 A resolution. These structures reveal the active site of s-GDH, and show for the first time how a functionally bound substrate interacts with the cofactor in a PQQ-dependent enzyme. Twenty years after the discovery of PQQ, our results finally provide conclusive evidence for a reaction mechanism comprising general base-catalyzed hydride transfer, rather than the generally accepted covalent addition-elimination mechanism. Thus, PQQ-dependent enzymes use a mechanism similar to that of nicotinamide- and flavin-dependent oxidoreductases.     

Determination of enzyme mechanisms by molecular dynamics: studies on quinoproteins, methanol dehydrogenase, and soluble glucose dehydrogenase

Molecular dynamics (MD) simulations have been carried out to study the enzymatic mechanisms of quinoproteins, methanol dehydrogenase (MDH), and soluble glucose dehydrogenase (sGDH). The mechanisms of reduction of the orthoquinone cofactor (PQQ) of MDH and sGDH involve concerted base-catalyzed proton abstraction from the hydroxyl moiety of methanol or from the 1-hydroxyl of glucose, and hydride equivalent transfer from the substrate to the quinone carbonyl carbon C5 of PQQ. The products of methanol and glucose oxidation are formaldehyde and glucolactone, respectively. The immediate product of PQQ reduction, PQQH- [-HC5(O-)-C4(=O)-] and PQQH [-HC5(OH)-C4(=O)-] converts to the hydroquinone PQQH2 [-C5(OH)=C4(OH)-]. The main focus is on MD structures of MDH * PQQ * methanol, MDH * PQQH-, MDH * PQQH, sGDH * PQQ * glucose, sGDH * PQQH- (glucolactone, and sGDH * PQQH. The reaction PQQ-->PQQH- occurs with Glu 171-CO2- and His 144-Im as the base species in MDH and sGDH, respectively. The general-base-catalyzed hydroxyl proton abstraction from substrate concerted with hydride transfer to the C5 of PQQ is assisted by hydrogen-bonding to the C5=O by Wat1 and Arg 324 in MDH and by Wat89 and Arg 228 in sGDH. Asp 297-COOH would act as a proton donor for the reaction PQQH(-)-->PQQH, if formed by transfer of the proton from Glu 171-COOH to Asp 297-CO2- in MDH. For PQQH-->PQQH2, migration of H5 to the C4 oxygen may be assisted by a weak base like water (either by crystal water Wat97 or bulk solvent, hydrogen-bonded to Glu 171-CO2- in MDH and by Wat89 in sGDH).     

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     

Physiological properties of a pyrroloquinoline quinone mutant of Methylobacterium organophilum

Abstract The grwoth of MTMl, a mutant of methylobacterium organophilum) blocked in the use of methanol as a carbon and energy source, was restored by addition of pyrroloquinoline quinone (PQQ) in the culture medium. No PQQ could be detected in crude medium. No PQQ could be of MTMl. Therefore, MTMl can be regarded as a mutant blocked in the biosynthesis of PQQ. Under the conditions of growth employed, growth rates of MTMl on methanol, comparable to those of the wild type, occured at a PQQ concentration of 1 μM. Since lower amounts of methanol dehydrogenase (MDH) wer found in cell-free extracts of PQQ-supplemented MTMl, the wild type strain synthesizes a surplus of MDH under these conditions. Growth of M. organophilum on ethanol proceeds via MDH as a catalyst for the first step, since (NAD(P) -dependent etanol. dehydrogenase was absent in cell-free extracts and growth of MTMl on ethanol only took place in the presence of PQQ. On the hand, growth of MTMl on mthylamine was unimpaired. This is in accordance with the fact that methylamine dehydrogenase was absent and N -methylamine mate dehydrogenase was present in cell-free extracts     

Engineering the quaternary structure of an enzyme: construction and analysis of a monomeric form of malate dehydrogenase from Escherichia coli.

The citric acid cycle enzyme, malate dehydrogenase (MDH), is a dimer of identical subunits. In the crystal structures of 2 prokaryotic and 2 eukaryotic forms, the subunit interface is conformationally homologous. To determine whether or not the quaternary structure of MDH is linked to the catalytic activity, mutant forms of the enzyme from Escherichia coli have been constructed. Utilizing the high-resolution structure of E. coli MDH, the dimer interface was analyzed critically for side chains that were spatially constricted and needed for electrostatic interactions. Two such residues were found, D45 and S226. At their nearest point in the homodimer, they are in different subunits, hydrogen bond across the interface, and do not interact with any catalytic residues. Each residue was mutated to a tyrosine, which should disrupt the interface because of its large size. All mutants were cloned and purified to homogeneity from an mdh- E. coli strain (BHB111). Gel filtration of the mutants show that D45Y and D45Y/S226Y are both monomers, whereas the S226Y mutant remains a dimer. The monomeric D45Y and D45Y/S226Y mutants have 14,000- and 17,500-fold less specific activity, respectively, than the native enzyme. The dimeric S226Y has only 1.4-fold less specific activity. All forms crystallized, indicating they were not random coils. Data have been collected to 2.8 A resolution for the D45Y mutant. The mutant is not isomorphous with the native protein and work is underway to solve the structure by molecular replacement.     

Wiring of PQQ-dehydrogenases

The performance of pyrroloquinoline quinone (PQQ) dependent alcohol dehydrogenase (ADH) and two types of PQQ-glucose dehydrogenases in solution and when immobilized on the carbon paste electrodes modified with ferrocene derivatives is investigated. The immobilization of ADH consisting of PQQ and four hemes improves its stability up to 10 times. Both PQQ and heme moieties are involved in the electron transport from substrate to electrode. The ferrocene derivatives improve the electron transport 10-fold. Membrane-bound alcohol dehydrogenase from Gluconobacter sp. 33, intracellular soluble glucose dehydrogenase from Acinetobacter calcoaceticus L.M.D. 79.41 (s-GDH), and the membrane-bound enzyme (m-GDH) from Erwinia sp. 34-1 were purified and investigated. Soluble and membrane-bound PQQ-glucose dehydrogenases display different behavior during the immobilization on the modified carbon electrodes. The immobilization of s-GDH leads to a decrease in both stability and substrate specificity of the enzyme. This suggests that PQQ dissociates from the enzyme active center and operates as a free-diffusing mediator. The rate-limiting step of the process is likely the loading of PQQ onto the apo-enzyme. The immobilization of m-GDH leads to its substantial stabilization and improves the substrate specificity. The nature of m-GDH binding to the electrode surface is presumably similar to the binding to the cell membrane through its anchor-subunit. The enzyme operates as an enzyme and mediator complex.     

甲基营养菌甲醇脱氢酶基因的克隆及表达

目的:从甲基营养菌MP681中扩增甲醇脱氢酶(MDH)基因,在大肠杆菌中表达并检测其活性,同时在MP681中考察该基因对吡咯喹啉醌(PQQ)产生的影响。方法:根据MP681基因组序列设计引物,PCR扩增靶基因mdh,构建表达载体,考察活性,利用接合转移转化至MP681,考察PQQ的合成。结果:扩增得到甲基营养菌MP681甲醇脱氢酶基因,在大肠杆菌中的表达产物能够催化甲醇脱氢;将携带mdh基因的质粒转入MP681后,PQQ产量略有提高。结论:获得编码MDH的基因,该基因能够在大肠杆菌中表达,且表达产物具有生物活性;甲醇脱氢酶基因表达对宿主菌的PQQ合成可能有一定影响。     

PQQ-dependent methanol dehydrogenases: rare-earth elements make a difference

Methanol dehydrogenase (MDH) catalyzes the first step in methanol use by methylotrophic bacteria and the second step in methane conversion by methanotrophs. Gram-negative bacteria possess an MDH with pyrroloquinoline quinone (PQQ) as its catalytic center. This MDH belongs to the broad class of eight-bladed β propeller quinoproteins, which comprise a range of other alcohol and aldehyde dehydrogenases. A well-investigated MDH is the heterotetrameric MxaFI-MDH, which is composed of two large catalytic subunits (MxaF) and two small subunits (MxaI). MxaFI-MDHs bind calcium as a cofactor that assists PQQ in catalysis. Genomic analyses indicated the existence of another MDH distantly related to the MxaFI-MDHs. Recently, several of these so-called XoxF-MDHs have been isolated. XoxF-MDHs described thus far are homodimeric proteins lacking the small subunit and possess a rare-earth element (REE) instead of calcium. The presence of such REE may confer XoxF-MDHs a superior catalytic efficiency. Moreover, XoxF-MDHs are able to oxidize methanol to formate, rather than to formaldehyde as MxaFI-MDHs do. While structures of MxaFI- and XoxF-MDH are conserved, also regarding the binding of PQQ, the accommodation of a REE requires the presence of a specific aspartate residue near the catalytic site. XoxF-MDHs containing such REE-binding motif are abundantly present in genomes of methylotrophic and methanotrophic microorganisms and also in organisms that hitherto are not known for such lifestyle. Moreover, sequence analyses suggest that XoxF-MDHs represent only a small part of putative REE-containing quinoproteins, together covering an unexploited potential of metabolic functions.     

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured byk _cat/K _m) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.Abbreviations used BSLDH Bacillus stearothermophilus lactate dehydrogenase - FBP fructose-1,6-bisphosphate - HP hydroxypyruvate - KB ketobutyrate - KC ketocaproate - KV ketovalerate - MDH malate dehydrogenase - PP phenylpyruvate - PYR pyruvate - RBE relative binding energy     

The quinoprotein dehydrogenases for methanol and glucose

This review summarises our current understanding of two of the main types of quinoprotein dehydrogenase in which pyrroloquinoline quinone (PQQ) is the only prosthetic group. These are the soluble methanol dehydrogenase and the membrane glucose dehydrogenase (mGDH). The membrane GDH has an additional N-terminal domain by which it is tightly anchored to the membrane, and a periplasmic domain whose structure has been modelled on the X-ray structure of the alpha-subunit of MDH which contains PQQ in the active site. This review discusses their structures and mechanisms, concentrating particularly on the pathways for electron transfer from the reduced PQQ, through the protein, to their electron acceptors. In MDH, this is the specific cytochrome c(L), the electron transfer pathway probably involving the unique disulphide ring in the active site. By contrast, mGDH contains a permanently bound ubiquinone, which acts as a single electron carrier, mediating electron transfer through the protein to the membrane ubiquinone.     

Electron transfer in quinoproteins

Soluble quinoprotein dehydrogenases oxidize a wide range of sugar, alcohol, amine, and aldehyde substrates. The physiological electron acceptors for these enzymes are not pyridine nucleotides but are other soluble redox proteins. This makes these enzymes and their electron acceptors excellent systems with which to study mechanisms of long-range interprotein electron transfer reactions. The tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH) transfers electrons to a blue copper protein, amicyanin. It has been possible to alter the rate of electron transfer by using different redox forms of MADH, varying reaction conditions, and performing site-directed mutagenesis on these proteins. From kinetic and thermodynamic analyses of the reaction rates, it was possible to determine whether a change in rate is due a change in Delta G(0), electronic coupling, reorganization energy or kinetic mechanism. Examples of each of these cases are discussed in the context of the known crystal structures of the electron transfer protein complexes. The pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase transfers electrons to a c-type cytochrome. Kinetic and thermodynamic analyses of this reaction indicated that this electron transfer reaction was conformationally coupled. Quinohemoproteins possess a quinone cofactor as well as one or more c-type hemes within the same protein. The structures of a PQQ-dependent quinohemoprotein alcohol dehydrogenase and a TTQ-dependent quinohemoprotein amine dehydrogenase are described with respect to their roles in intramolecular and intermolecular protein electron transfer reactions.     

Crystal structure of quinone‐dependent alcohol dehydrogenase from Pseudogluconobacter saccharoketogenes. A versatile dehydrogenase oxidizing alcohols and carbohydrates

The quinone‐dependent alcohol dehydrogenase (PQQ‐ADH, E.C. 1.1.5.2) from the Gram‐negative bacterium Pseudogluconobacter saccharoketogenes IFO 14464 oxidizes primary alcohols (e.g. ethanol, butanol), secondary alcohols (monosaccharides), as well as aldehydes, polysaccharides, and cyclodextrins. The recombinant protein, expressed in Pichia pastoris, was crystallized, and three‐dimensional (3D) structures of the native form, with PQQ and a Ca²⁺ ion, and of the enzyme in complex with a Zn²⁺ ion and a bound substrate mimic were determined at 1.72 Å and 1.84 Å resolution, respectively. PQQ‐ADH displays an eight‐bladed β‐propeller fold, characteristic of Type I quinone‐dependent methanol dehydrogenases. However, three of the four ligands of the Ca²⁺ ion differ from those of related dehydrogenases and they come from different parts of the polypeptide chain. These differences result in a more open, easily accessible active site, which explains why PQQ‐ADH can oxidize a broad range of substrates. The bound substrate mimic suggests Asp333 as the catalytic base. Remarkably, no vicinal disulfide bridge is present near the PQQ, which in other PQQ‐dependent alcohol dehydrogenases has been proposed to be necessary for electron transfer. Instead an associated cytochrome c can approach the PQQ for direct electron transfer.     

Cooning of the genes encoding the two different glucose dehydrogenases fromAcinetobacter calcoaceticus

Glucose dehydrogenase (GDH) is a PQQ dependent bacterial enzyme which converts aldoses to their corresponding acids.A. calcoaceticus contains two different PQQ dependent glucose dehydrogenases designated GDH-A which is activein vivo and GDH-B of which onlyin vitro activity can be shown. We cloned the genes coding for the two GDH enzymes. The DNA sequences of bothgdh genes were determined. There is no obvious homology betweengdhA andgdhB. Both GDH enzymes oxidize D-glucosein vitro but disaccharides are specific GDH-B substrates and 2-deoxyglucose is specifically oxidized by GDH-A.     

甲基营养菌MP688甲醇脱氢酶基因mpq1818的敲除及功能研究

目的:研究甲醇脱氢酶基因mpq1818在甲基营养菌MP688生长代谢中的作用。方法:利用同源重组原理构建中间为庆大霉素抗性基因Gmr、两侧mpq1818基因上下游序列同源的敲除载体pAK0-up-Gmr-down,接合转移导入MP688,通过庆大霉素抗性和组合PCR方法筛选基因敲除菌,并检测其生长、甲醇脱氢酶活性、甲醇利用及吡咯喹啉醌(PQQ)生物合成能力等方面的差异。结果:抗性和PCR验证显示mpq1818缺失株构建成功;与野生菌相比,缺失株的甲醇脱氢酶活力及利用甲醇的能力降低,而且菌株的生长和PQQ产量也有显著下降。结论:基因mpq1818的缺失影响菌株前期生长与PQQ合成。     

The 1.7 A crystal structure of the apo form of the soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus reveals a novel internal conserved sequence repeat.

The crystal structure of a dimeric apo form of the soluble quinoprotein glucose dehydrogenase (s-GDH) from Acinetobacter calcoaceticus has been solved by multiple isomorphous replacement followed by density modification, and was subsequently refined at 1. 72 A resolution to a final crystallographic R-factor of 16.5% and free R-factor of 20.8% [corrected]. The s-GDH monomer has a beta-propeller fold consisting of six four-stranded anti-parallel beta-sheets aligned around a pseudo 6-fold symmetry axis. The enzyme binds three calcium ions per monomer, two of which are located in the dimer interface. The third is bound in the putative active site, where it may bind and functionalize the pyrroloquinoline quinone (PQQ) cofactor. A data base search unexpectedly showed that four uncharacterized protein sequences are homologous to s-GDH with many residues in the putative active site absolutely conserved. This indicates that these homologs may have a similar structure and that they may catalyze similar PQQ-dependent reactions.A structure-based sequence alignment of the six four-stranded beta-sheets in s-GDH's beta-propeller fold shows an internally conserved sequence repeat that gives rise to two distinct conserved structural motifs. The first structural motif is found at the corner of the short beta-turn between the inner two beta-strands of the beta-sheets, where an Asp side-chain points back into the beta-sheet to form a hydrogen-bond with the OH/NH of a Tyr/Trp side-chain in the same beta-sheet. The second motif involves an Arg/Lys side-chain in the C beta-strand of one beta-sheet, which forms a bidentate salt-bridge with an Asp/Glu in the CD loop of the next beta-sheet. These intra and inter-beta-sheet hydrogen-bonds are likely to contribute to the stability of the s-GDH beta-propeller fold.     

Physical and genetic interactions of cytosolic malate dehydrogenase with other gluconeogenic enzymes

A truncated form (deltanMDH2) of yeast cytosolic malate dehydrogenase (MDH2) lacking 12 residues on the amino terminus was found to be inadequate for gluconeogenic function in vivo because the mutant enzyme fails to restore growth of a Deltamdh2 strain on minimal medium with ethanol or acetate as the carbon source. The DeltanMDH2 enzyme was also previously found to be refractory to the rapid glucose-induced inactivation and degradation observed for authentic MDH2. In contrast, kinetic properties measured for purified forms of MDH2 and deltanMDH2 enzymes are very similar. Yeast two-hybrid assays indicate weak interactions between MDH2 and yeast phosphoenolpyruvate carboxykinase (PCK1) and between MDH2 and fructose-1,6-bisphosphatase (FBP1). These interactions are not observed for deltanMDH2, suggesting that differences in cellular function between authentic and truncated forms of MDH2 may be related to their ability to interact with other gluconeogenic enzymes. Additional evidence was obtained for interaction of MDH2 with PCK1 using Hummel-Dreyer gel filtration chromatography, and for interactions of MDH2 with PCK1 and with FBP1 using surface plasmon resonance. Experiments with the latter technique demonstrated a much lower affinity for interaction of deltanMDH2 with PCK1 and no interaction between deltanMDH2 and FBP1. These results suggest that the interactions of MDH2 with other gluconeogenic enzymes are dependent on the amino terminus of the enzyme, and that these interactions are important for gluconeogenic function in vivo.     

斯氏并殖吸虫乳酸脱氢酶,苹果酸脱氢酶及酯酶同工酶的研究

本文采用Disc-PAGE电泳,首次对我国独有的斯氏并殖吸虫(Paragonimus skrjabini Chen,1959)成虫、童虫、囊蚴的乳酸脱氢酶(以下简称LDH)、苹果酸脱氢酶(以下简称MDH)和酯酶(以下简称EST)同工酶进行了研究。 在成虫、童虫、囊蚴间,LDH、MDH、EST同工酶在酶带数、排列型式、Rf值、相对活性和优势酶带的位置都存在差异。 根据虫体和宿主组织同工酶谱的不同,可以认为是本虫本身所具有。 同工酶作为其分类指标时,不仅要比较不同虫种成虫稳定的同工酶谱,也要比较同工酶在个体发育型式间的差异。     

Malate dehydrogenases--structure and function

Malate dehydrogenases (MDH, L-malate:NAD oxidoreductase, EC 1.1.1.37), catalyze the NAD/NADH-dependent interconversion of the substrates malate and oxaloacetate. This reaction plays a key part in the malate/aspartate shuttle across the mitochondrial membrane, and in the tricarboxylic acid cycle within the mitochondrial matrix. They are homodimeric molecules in most organisms, including all eukaryots and the most bacterial species. The enzymes share a common catalytic mechanism and their kinetic properties are similar, which demonstrates a high degree of structural similarity. The three-dimensional structures and elements essential for catalysis are conserved between mitochondrial and cytoplasmic forms of MDH in eukaryotic cells even though these isoenzymes are only marginally related at the level of primary structure.     

Soluble aldose sugar dehydrogenase from Escherichia coli: a highly exposed active site conferring broad substrate specificity

A water-soluble aldose sugar dehydrogenase (Asd) has been purified for the first time from Escherichia coli. The enzyme is able to act upon a broad range of aldose sugars, encompassing hexoses, pentoses, disaccharides, and trisaccharides, and is able to oxidize glucose to gluconolactone with subsequent hydrolysis to gluconic acid. The enzyme shows the ability to bind pyrroloquinoline quinone (PQQ) in the presence of Ca2+ in a manner that is proportional to its catalytic activity. The x-ray structure has been determined in the apo-form and as the PQQ-bound active holoenzyme. The beta-propeller fold of this protein is conserved between E. coli Asd and Acinetobacter calcoaceticus soluble glucose dehydrogenase (sGdh), with major structural differences lying in loop and surface-exposed regions. Many of the residues involved in binding the cofactor are conserved between the two enzymes, but significant differences exist in residues likely to contact substrates. PQQ is bound in a large cleft in the protein surface and is uniquely solvent-accessible compared with other PQQ enzymes. The exposed and charged nature of the active site and the activity profile of this enzyme indicate possible factors that underlie a low affinity for glucose but generic broad substrate specificity for aldose sugars. These structural and catalytic properties of the enzymes have led us to propose that E. coli Asd provides a prototype structure for a new subgroup of PQQ-dependent soluble dehydrogenases that is distinct from the A. calcoaceticus sGdh subgroup.     

Stabilization of a tetrameric malate dehydrogenase by introduction of a disulfide bridge at the dimer-dimer interface

Malate dehydrogenase (MDH) from the moderately thermophilic bacterium Chloroflexus aurantiacus (CaMDH) is a tetrameric enzyme, while MDHs from mesophilic organisms usually are dimers. To investigate the potential contribution of the extra dimer-dimer interface in CaMDH with respect to thermal stability, we have engineered an intersubunit disulfide bridge designed to strengthen dimer-dimer interactions. The resulting mutant (T187C, containing two 187-187 disulfide bridges in the tetramer) showed a 200-fold increase in half-life at 75 degrees C and an increase of 15 deg. C in apparent melting temperature compared to the wild-type. The crystal structure of the mutant (solved at 1.75 A resolution) was essentially identical with that of the wild-type, with the exception of the added inter-dimer disulfide bridge and the loss of an aromatic intra-dimer contact. Remarkably, the mutant and the wild-type had similar temperature optima and activities at their temperature optima, thus providing a clear case of uncoupling of thermal stability and thermoactivity. The results show that tetramerization may contribute to MDH stability to an extent that depends strongly on the number of stabilizing interactions in the dimer-dimer interface.