可用于二氧化碳捕获过程的微生物碳酸酐酶的挖掘与改造

二氧化碳排放量的急剧上升引起全球温室效应加剧。碳酸酐酶是地球上反应速率最快的几种酶之一,可以大幅提高CO_2捕获和生物矿化的效率,从而降低大气中CO_2的排放量。但捕获过程在高温条件,而CO_2生物矿化形成CaCO_3的过程则需要碱性条件。因此,迫切需要筛选出既嗜热又耐碱的碳酸酐酶以用于CO_2捕获,极端微生物是这类酶的重要来源之一。文中系统、深入地介绍了目前从极端微生物或利用蛋白质工程技术获取嗜热、耐碱的碳酸酐酶的最新研究进展,同时简要介绍了一些新型固定化碳酸酐酶的方法。最后指出当前研究的重点应致力于拓宽寻找碳酸酐酶的范围,改良蛋白质工程改造技术,研发高效廉价、易于放大的固定化方法,为减轻温室效应、延缓全球变暖这一迫切需要解决的问题提供新思路。     

Biocatalytic carbon capture via reversible reaction cycle catalyzed by isocitrate dehydrogenase

The practice of carbon capture and storage (CCS) requires efficient capture and separation of carbon dioxide from its gaseous mixtures such as flue gas, followed by releasing it as a pure gas which can be subsequently compressed and injected into underground storage sites. This has been mostly achieved via reversible thermochemical reactions which are generally energy-intensive. The current work examines a biocatalytic approach for carbon capture using an NADP(H)-dependent isocitrate dehydrogenase (ICDH) which catalyzes reversibly carboxylation and decarboxylation reactions. Different from chemical carbon capture processes that rely on thermal energy to realize purification of carbon dioxide, the biocatalytic strategy utilizes pH to leverage the reaction equilibrium, thereby realizing energy-efficient carbon capture under ambient conditions. Results showed that over 25 mol of carbon dioxide could be captured and purified from its gas mixture for each gram of ICDH applied for each carboxylation/decarboxylation reaction cycle by varying pH between 6 and 9. This work demonstrates the promising potentials of pH-sensitive biocatalysis as a green-chemistry route for carbon capture.     

The mechanisms of reductive carboxylation reactions. Carbon dioxide or bicarbonate as substrate of nicotinamide-adenine dinucleotide phosphate-linked isocitrate dehydrogenase and `malic'' enzyme

1. A simple kinetic method was devised to show whether dissolved CO(2) or HCO(3)- ion is the substrate in enzyme-catalysed carboxylation reactions. 2. The time-course of the reductive carboxylation of 2-oxoglutarate by NADPH, catalysed by isocitrate dehydrogenase, was studied by a sensitive fluorimetric method at pH7.3 and pH6.4, with large concentrations of substrate and coenzyme and small carbon dioxide concentrations. 3. Reaction was initiated by the addition of carbon dioxide in one of three forms: (i) as the dissolved gas in equilibrium with bicarbonate; (ii) as unbuffered bicarbonate solution; (iii) as the gas or as an unbuffered solution of the gas in water. Different progress curves were obtained in the three cases. 4. The results show that dissolved CO(2) is the primary substrate of the enzyme, and that HCO(3)- ion is at best a very poor substrate. The progress curves are in quantitative agreement with this conclusion and with the known rates of the reversible hydration of CO(2) under the conditions of the experiments. The effects of carbonic anhydrase confirm the conclusions. 5. Similar experiments on the reductive carboxylation of pyruvate catalysed by the ;malic' enzyme show that dissolved CO(2) is the primary substrate of this enzyme also. 6. The results are discussed in relation to the mechanisms of these enzymes, and the effects of pH on the reactions. 7. The advantages of the method and its possible applications to other enzymes involved in carbon dioxide metabolism are discussed.     

The Incorporation of 14CO2 into Green Peas in the Dark

Shelled green peas (ripening seeds of Pisum sativum var. Onward)were kept in the dark for 2 or 3 days in an atmosphere, eitherof air or 10 per cent carbon dioxide in air, containing ¹⁴CO₂.Samples were removed at intervals, the acids of the T.C.A.C.extracted, estimated, and their content of ¹⁴C measured. Incorporationof ¹⁴C was mainly into the carboxyl carbon atoms of the acidswhich, with the exception of citrate, rose in specific activityrapidly for the first 4–6 h and then levelled off androse slowly for the rest of the experiment. The final valuesattained, again with the exception of C-6 of citrate, were muchbelow that of the tissue carbon dioxide. The cause of this failureto equilibrate with tissue carbon dioxide is discussed. Twomain carboxylation reactions are proposed, one from pyruvate(or other three carbon acid) to malate and the other from -oxoglutarateto oxalsuccinate and so to C-6 of citrate. The value of thevelocity constant of the first of these reactions was shownto be markedly decreased by increase in tissue carbon dioxidewhile that for the second carboxylation was unaffected. ¹⁴Cmoved into soluble amino-acids rapidly at first and then moreslowly; protein received ¹⁴C at a high rate throughout the experimentsmainly by isotopic exchange reactions. Calculations were madeof the rate of movement of carbon in the T.C.A.C. which wouldhave been required to give the observed changes in specificactivity of the metabolites but no scheme tested fitted allthe results satisfactorily.     

Lysine carboxylation in proteins: OXA-10 beta-lactamase

An increasing number of proteins are being shown to have an N(zeta)-carboxylated lysine in their structures, a posttranslational modification of proteins that proceeds without the intervention of a specific enzyme. The role of the carboxylated lysine in these proteins is typically structural (hydrogen bonding or metal coordination). However, carboxylated lysines in the active sites of OXA-10 and OXA-1 beta-lactamases and the sensor domain of BlaR signal-transducer protein serve in proton transfer events required for the functions of these proteins. These examples demonstrate the utility of this unusual amino acid in acid-base chemistry, in expansion of function beyond those of the 20 standard amino acids. In this study, the ONIOM quantum-mechanical/molecular-mechanical (QM/MM) method is used to study the carboxylation of lysine in the OXA-10 beta-lactamase. Lys-70 and the active site of the OXA-10 beta-lactamase were treated with B3LYP/6-31G(d,p) density functional calculations and the remainder of the enzyme with the AMBER molecular mechanics force field. The barriers for unassisted carboxylation of neutral lysine by carbon dioxide or bicarbonate are high. However, when the reaction with CO2 is catalyzed by a molecule of water in the active site, it is exothermic by about 13 kcal/mol, with a barrier of approximately 14 kcal/mol. The calculations show that the carboxylation and decarboxylation of Lys-70 are likely to be accompanied by deprotonation and protonation of the carbamate, respectively. The analysis may also be relevant for other proteins with carboxylated lysines, a feature that may be more common in nature than previously appreciated.     

Immobilized carbonic anhydrase: preparation,characteristics and biotechnological applications

Carbonic anhydrase (CA) is an essential metalloenzyme in living systems for accelerating the hydration and dehydration of carbon dioxide. CA-catalyzed reactions can be applied in vitro for capturing industrially emitted gaseous carbon dioxide in aqueous solutions. To facilitate this type of practical application, the immobilization of CA on or inside solid or soft support materials is of great importance because the immobilization of enzymes in general offers the opportunity for enzyme recycling or long-term use in bioreactors. Moreover, the thermal/storage stability and reactivity of immobilized CA can be modulated through the physicochemical nature and structural characteristics of the support material used. This review focuses on (i) immobilization methods which have been applied so far, (ii) some of the characteristic features of immobilized forms of CA, and (iii) biotechnological applications of immobilized CA. The applications described not only include the CA-assisted capturing and sequestration of carbon dioxide, but also the CA-supported bioelectrochemical conversion of CO₂ into organic molecules, and the detection of clinically important CA inhibitors. Furthermore, immobilized CA can be used in biomimetic materials synthesis involving cascade reactions, e.g. for bone regeneration based on calcium carbonate formation from urea with two consecutive reactions catalyzed by urease and CA.     

Identification of naphthalene carboxylase as a prototype for the anaerobic activation of non-substituted aromatic hydrocarbons

Polycyclic aromatic hydrocarbons such as naphthalene are recalcitrant environmental pollutants that are only slowly metabolized by bacteria under anoxic conditions. Based on metabolite analyses of culture supernatants, carboxylation or methylation of naphthalene have been proposed as initial enzymatic activation reactions in the pathway. However, the extremely slow growth of anaerobic naphthalene degraders with doubling times of weeks and the little biomass obtained from such cultures hindered the biochemical elucidation of the initial activation reaction, so far. Here, we provide biochemical evidence that anaerobic naphthalene degradation is initiated via carboxylation. Crude cell extracts of the sulfate-reducing enrichment culture N47 converted naphthalene and (13) C-labelled bicarbonate to 2-[carboxyl-(13) C]naphthoic acid at a rate of 0.12?nmol min(-1) mg protein(-1) . The enzyme, namely naphthalene carboxylase, catalysed a much faster exchange of (13) C-labelled bicarbonate with the carboxyl group of 2-[carboxyl-(12) C]naphthoic acid at a rate of 3.2?nmol min(-1) mg protein(-1) , indicating that the rate limiting step of the carboxylation reaction is the activation of the naphthalene molecule rather than the carboxylation itself. Neither the carboxylation nor the exchange reaction activities necessitate the presence of ATP or divalent metal ions and they were not inhibited by avidin or EDTA. The new carboxylation reaction is unprecedented in biochemistry and opens the door to understand the anaerobic degradation of polycyclic aromatic hydrocarbons which are among the most hazardous environmental contaminants.     

Carboxylation reaction catalyzed by 2-oxoglutarate:ferredoxin oxidoreductases from Hydrogenobacter thermophilus

Hydrogenobacter thermophilus TK-6 is a thermophilic, chemolithoautotrophic, hydrogen-oxidizing bacterium that fixes carbon dioxide via the reductive tricarboxylic acid (rTCA) cycle. 2-Oxoglutarate:ferredoxin oxidoreductase (OGOR) is the key enzyme in this cycle that fixes carbon dioxide. The genome of strain TK-6 encodes at least two distinct OGOR enzymes, termed For and Kor. We report here a method for measuring the carboxylation of succinyl-CoA catalyzed by OGORs. The method involves the in vitro coupling of OGOR with ferredoxin and pyruvate:ferredoxin oxidoreductase from strain TK-6, and glutamate dehydrogenase from Sulfolobus tokodaii. Using this method, we determined both the apparent maximum velocities and the K _m values of For and Kor for the carboxylation of succinyl-CoA. This is the first reported kinetic analysis of carbon fixation catalyzed by OGOR enzymes from the rTCA cycle.     

Thermodynamic constraints shape the structure of carbon fixation pathways

Thermodynamics impose a major constraint on the structure of metabolic pathways. Here, we use carbon fixation pathways to demonstrate how thermodynamics shape the structure of pathways and determine the cellular resources they consume. We analyze the energetic profile of prototypical reactions and show that each reaction type displays a characteristic change in Gibbs energy. Specifically, although carbon fixation pathways display a considerable structural variability, they are all energetically constrained by two types of reactions: carboxylation and carboxyl reduction. In fact, all adenosine triphosphate (ATP) molecules consumed by carbon fixation pathways - with a single exception - are used, directly or indirectly, to power one of these unfavorable reactions. When an indirect coupling is employed, the energy released by ATP hydrolysis is used to establish another chemical bond with high energy of hydrolysis, e.g. a thioester. This bond is cleaved by a downstream enzyme to energize an unfavorable reaction. Notably, many pathways exhibit reduced ATP requirement as they couple unfavorable carboxylation or carboxyl reduction reactions to exergonic reactions other than ATP hydrolysis. In the most extreme example, the reductive acetyl coenzyme A (acetyl-CoA) pathway bypasses almost all ATP-consuming reactions. On the other hand, the reductive pentose phosphate pathway appears to be the least ATP-efficient because it is the only carbon fixation pathway that invests ATP in metabolic aims other than carboxylation and carboxyl reduction. Altogether, our analysis indicates that basic thermodynamic considerations accurately predict the resource investment required to support a metabolic pathway and further identifies biochemical mechanisms that can decrease this requirement.     

How does an enzyme recognize CO2?

Phosphoenolpyruvate carboxykinase (PCK) reversibly catalyzes the carboxylation of phosphoenolpyruvate to oxaloacetate. Carbon dioxide, and not bicarbonate ion, is the substrate utilized. Assays of the carboxylation reaction show that initial velocities are 7.6-fold higher when CO(2) is used instead of HCO(3)(-). Two Escherichia coli PCK-CO(2) crystal structures are presented here. The location of CO(2) is the same for both structures; however the orientation of CO(2) is significantly different, likely from the presence of a manganese ion in one of the structures. PCK and the other three known protein-CO(2) crystal structure complexes have been compared; all have CO(2) hydrogen bonding with a basic amino acid side chain (Arg65 or Lys213 in PCK), likely to polarize CO(2) to make the central carbon atom more electrophilic and thus more reactive. Kinetic studies found that the PCK mutant Arg65Gln increased the K(M) for substrates PEP and oxaloacetate but not for CO(2). The unchanged K(M) for CO(2) can be explained since the Arg65Gln mutant likely maintains a hydrogen bond to one of the oxygen atoms of carbon dioxide.     

Phylogenetic and evolutionary relationships of RubisCO and the RubisCO-like proteins and the functional lessons provided by diverse molecular forms

Ribulose 1,5-bisphosphate (RuBP) carboxylase/oxygenase (RubisCO) catalyses the key reaction by which inorganic carbon may be assimilated into organic carbon. Phylogenetic analyses indicate that there are three classes of bona fide RubisCO proteins, forms I, II and III, which all catalyse the same reactions. In addition, there exists another form of RubisCO, form IV, which does not catalyse RuBP carboxylation or oxygenation. Form IV is actually a homologue of RubisCO and is called the RubisCO-like protein (RLP). Both RubisCO and RLP appear to have evolved from an ancestor protein in a methanogenic archaeon, and comprehensive analyses indicate that the different forms (I, II, III and IV) contain various subgroups, with individual sequences derived from representatives of all three kingdoms of life. The diversity of RubisCO molecules, many of which function in distinct milieus, has provided convenient model systems to study the ways in which the active site of this protein has evolved to accommodate necessary molecular adaptations. Such studies have proven useful to help provide a framework for understanding the molecular basis for many important aspects of RubisCO catalysis, including the elucidation of factors or functional groups that impinge on RubisCO carbon dioxide/oxygen substrate discrimination.     

In vivo quantification of parallel and bidirectional fluxes in the anaplerosis of Corynebacterium glutamicum

The C(3)-C(4) metabolite interconversion at the anaplerotic node in many microorganisms involves a complex set of reactions. C(3) carboxylation to oxaloacetate can originate from phosphoenolpyruvate and pyruvate, and at the same time multiple C(4)-decarboxylating enzymes may be present. The functions of such parallel reactions are not yet fully understood. Using a (13)C NMR-based strategy, we here quantify the individual fluxes at the anaplerotic node of Corynebacterium glutamicum, which is an example of a bacterium possessing multiple carboxylation and decarboxylation reactions. C. glutamicum was grown with a (13)C-labeled glucose isotopomer mixture as the main carbon source and (13)C-labeled lactate as a cosubstrate. 58 isotopomers as well as 15 positional labels of biomass compounds were quantified. Applying a generally applicable mathematical model to include metabolite mass and carbon labeling balances, it is shown that pyruvate carboxylase contributed 91 +/- 7% to C(3) carboxylation. The total in vivo carboxylation rate of 1.28 +/- 0.14 mmol/g dry weight/h exceeds the demand of carboxylated metabolites for biosyntheses 3-fold. Excess oxaloacetate was recycled to phosphoenolpyruvate by phosphoenolpyruvate carboxykinase. This shows that the reactions at the anaplerotic node might serve additional purposes other than only providing C(4) metabolites for biosynthesis.     

Photoreductive path of carbon fixation in green plant photosynthesis. Reaction pathway of six-carbon ribulose 1,5-bisphosphate carboxylation adduct intermediate

In this paper we examine the six-carbon intermediate pathway of ribulose 1,5-bisphosphate (RuBP) carboxylation reaction in photosynthesis. Based on the observed reactions of purified RuBP carboxylase, mechanisms are described for carbon dioxide assimilation leading to the hydrolytic splitting of the six-carbon intermediate to two enzyme-bound glycerate-3-P (3-PGA) molecules. It is concluded that, under photosynthetic conditions, the reduction of enzyme-bound NADP+ by the chlorophyll is responsible for the rapid carboxylase turnover rate given by the lifetime, tau L = 0.4 s, which is nearly two orders of magnitude shorter than the corresponding value, tau D = 11 +/- 3 s, for the dark decay of enzyme-bound RuBP. The nocturnal inhibition and photoactivation of RuBP carboxylation are described in terms of the reversible light-dark cycles of the NADP+/NADPH redox couple and endogenous changes that accompany the 2-carboxy-D-arabinitol-1-phosphate binding to the enzyme active site.     

Genes and enzymes involved in the biodegradation of the quaternary carbon compound pivalate in the denitrifying Thauera humireducens strain PIV-1

Quaternary carbon-containing compounds exist in natural and fossil oil-derived products and are used in chemical and pharmaceutical applications up to industrial scale. Due to the inaccessibility of the quaternary carbon atom for a direct oxidative or reductive attack, they are considered as persistent in the environment. Here, we investigated the unknown degradation of the quaternary carbon-containing model compound pivalate (2,2-dimethyl-propionate) in the denitrifying bacterium Thauera humireducens strain PIV-1 (formerly Thauera pivalivorans). We provide multiple evidence for a pathway comprising the activation to pivalyl-CoA and the carbon skeleton rearrangement to isovaleryl-CoA. Subsequent reactions proceed similar to the catabolic leucine degradation pathway such as the carboxylation to 3-methylglutaconyl-CoA and the cleavage of 3-methyl-3-hydroxyglutaryl-CoA to acetyl-CoA and acetoacetate. The completed genome of Thauera humireducens strain PIV-1 together with proteomic data was used to identify pivalate-upregulated gene clusters including genes putatively encoding pivalate CoA ligase and adenosylcobalamin-dependent pivalyl-CoA mutase. A pivalate-induced gene encoding a putative carboxylic acid CoA ligase was heterologously expressed, and its highly enriched product exhibited pivalate CoA ligase activity. The results provide the first experimental insights into the biodegradation pathway of a quaternary carbon-containing model compound that serves as a blueprint for the degradation of related quaternary carbon-containing compounds.     

Comparative mechanisms of vitamin B6-catalyzed beta-decarboxylation and beta-dephosphonylation in model systems

Reaction pathways and mechanisms of vitamin B6-catalyzed beta-decarboxylation and beta-dephosphonylation of aminocarboxylic and aminophosphonic acids in model systems are compared. It was found that both reactions require prior transamination of an aldimine intermediate to a ketimine. For ketimines having carboxylate or phosphonate groups substituted on the beta-carbon atoms of the keto acid residue, there is a hydrogen ion or metal ion-activated covalent bond pathway which involves a shift of electron pairs toward the coordinated ketimine nitrogen, leading to beta-gamma, C-C or C-P bond fission and release of carbon dioxide or metaphosphate, respectively. Comparison of these reactions indicates that beta-decarboxylation is 10(6) faster than the corresponding dephosphonylation reaction. Since only a few studies of vitamin B6-catalyzed dephosphonylation have been carried out, suggestions are made for further studies with substrates designed to elucidate the reaction mechanisms involved.     

Comparison of Mechanisms of Alkane Metabolism under Sulfate-Reducing Conditions among Two Bacterial Isolates and a Bacterial Consortium

Recent studies have demonstrated that fumarate addition and carboxylation are two possible mechanisms of anaerobic alkane degradation. In the present study, we surveyed metabolites formed during growth on hexadecane by the sulfate-reducing isolates AK-01 and Hxd3 and by a mixed sulfate-reducing consortium. The cultures were incubated with either protonated or fully deuterated hexadecane; the sulfate-reducing consortium was also incubated with [1,2-¹³C₂]hexadecane. All cultures were extracted, silylated, and analyzed by gas chromatography-mass spectrometry. We detected a suite of metabolites that support a fumarate addition mechanism for hexadecane degradation by AK-01, including methylpentadecylsuccinic acid, 4-methyloctadecanoic acid, 4-methyloctadec-2,3-enoic acid, 2-methylhexadecanoic acid, and tetradecanoic acid. By using d₃₄-hexadecane, mass spectral evidence strongly supporting a carbon skeleton rearrangement of the first intermediate, methylpentadecylsuccinic acid, was demonstrated for AK-01. Evidence indicating hexadecane carboxylation was not found in AK-01 extracts but was observed in Hxd3 extracts. In the mixed sulfate-reducing culture, however, metabolites consistent with both fumarate addition and carboxylation mechanisms of hexadecane degradation were detected, which demonstrates that multiple alkane degradation pathways can occur simultaneously within distinct anaerobic communities. Collectively, these findings underscore that fumarate addition and carboxylation are important alkane degradation mechanisms that may be widespread among phylogenetically and/or physiologically distinct microorganisms.     

Carboxylases in natural and synthetic microbial pathways

Carboxylases are among the most important enzymes in the biosphere, because they catalyze a key reaction in the global carbon cycle: the fixation of inorganic carbon (CO₂). This minireview discusses the physiological roles of carboxylases in different microbial pathways that range from autotrophy, carbon assimilation, and anaplerosis to biosynthetic and redox-balancing functions. In addition, the current and possible future uses of carboxylation reactions in synthetic biology are discussed. Such uses include the possible transformation of the greenhouse gas carbon dioxide into value-added compounds and the production of novel antibiotics.     

Anaerobic degradation of homocyclic aromatic compounds via arylcarboxyl‐coenzyme A esters: organisms,strategies and key enzymes

Next to carbohydrates, aromatic compounds are the second most abundant class of natural organic molecules in living organic matter but also make up a significant proportion of fossil carbon sources. Only microorganisms are capable of fully mineralizing aromatic compounds. While aerobic microbes use well‐studied oxygenases for the activation and cleavage of aromatic rings, anaerobic bacteria follow completely different strategies to initiate catabolism. The key enzymes related to aromatic compound degradation in anaerobic bacteria are comprised of metal‐ and/or flavin‐containing cofactors, of which many use unprecedented radical mechanisms for C–H bond cleavage or dearomatization. Over the past decade, the increasing number of completed genomes has helped to reveal a large variety of anaerobic degradation pathways in Proteobacteria, Gram‐positive microbes and in one archaeon. This review aims to update our understanding of the occurrence of aromatic degradation capabilities in anaerobic microorganisms and serves to highlight characteristic enzymatic reactions involved in (i) the anoxic oxidation of alkyl side chains attached to aromatic rings, (ii) the carboxylation of aromatic rings and (iii) the reductive dearomatization of central arylcarboxyl‐coenzyme A intermediates. Depending on the redox potential of the electron acceptors used and the metabolic efficiency of the cell, different strategies may be employed for identical overall reactions.     

Carbon isotope effect on carboxylation of ribulose bisphosphate catalyzed by ribulosebisphosphate carboxylase from Rhodospirillum rubrum

The carbon isotope effect at CO2 has been measured in the carboxylation of ribulose 1,5-bisphosphate by the ribulosebisphosphate carboxylase from Rhodospirillum rubrum. The isotope effect is obtained by comparing the isotopic composition of carbon 1 of the 3-phosphoglyceric acid formed in the reaction with that of the carbon dioxide source. A correction is made for carbon 1 of 3-phosphoglyceric acid which arises from carbon 3 of the starting ribulose bisphosphate. The isotope effect is k12/k13 = 1.0178 +/- 0.0008 at 25 degrees C, pH 7.8. This value is smaller than the corresponding value for the spinach enzyme. It appears that substrate addition with the R. rubrum enzyme is principally ordered, with ribulose bisphosphate binding first, whereas substrate addition is random with the spinach enzyme. The carboxylation step is partially rate limiting with both enzymes.