Mo and W bis-MGD enzymes: nitrate reductases and formate dehydrogenases

Molybdenum and tungsten are second- and third-row transition elements, respectively, which are found in a mononuclear form in the active site of a diverse group of enzymes that generally catalyze oxygen atom transfer reactions. Mononuclear Mo-containing enzymes have been classified into three families: xanthine oxidase, DMSO reductase, and sulfite oxidase. The proteins of the DMSO reductase family present the widest diversity of properties among its members and our knowledge about this family was greatly broadened by the study of the enzymes nitrate reductase and formate dehydrogenase, obtained from different sources. We discuss in this review the information of the better characterized examples of these two types of Mo enzymes and W enzymes closely related to the members of the DMSO reductase family. We briefly summarize, also, the few cases reported so far for enzymes that can function either with Mo or W at their active site.     

Kinetics of substrate inhibition of periplasmic nitrate reductase

Periplasmic nitrate reductase catalyzes the reduction of nitrate into nitrite using a mononuclear molybdenum cofactor that has nearly the same structure in all enzymes of the DMSO reductase family. In previous electrochemical investigations, we found that the enzyme exists in several inactive states, some of which may have been previously isolated and mistaken for catalytic intermediates. In particular, the enzyme slowly and reversibly inactivates when exposed to high concentrations of nitrate. Here, we study the kinetics of substrate inhibition and its dependence on electrode potential and substrate concentration to learn about the properties of the active and inactive forms of the enzyme. We conclude that the substrate-inhibited enzyme never significantly accumulates in the EPR-active Mo(+ V) state. This conclusion is relevant to spectroscopic investigations where attempts are made to trap a Mo(+ V) catalytic intermediate using high concentrations of nitrate.     

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD⁺ to NADP⁺. In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.     

Pyranopterin Coordination Controls Molybdenum Electrochemistry in Escherichia coli Nitrate Reductase

We test the hypothesis that pyranopterin (PPT) coordination plays a critical role in defining molybdenum active site redox chemistry and reactivity in the mononuclear molybdoenzymes. The molybdenum atom of Escherichia coli nitrate reductase A (NarGHI) is coordinated by two PPT-dithiolene chelates that are defined as proximal and distal based on their proximity to a [4Fe-4S] cluster known as FS0. We examined variants of two sets of residues involved in PPT coordination: (i) those interacting directly or indirectly with the pyran oxygen of the bicyclic distal PPT (NarG-Ser⁷¹⁹, NarG-His¹¹⁶³, and NarG-His¹¹⁸⁴); and (ii) those involved in bridging the two PPTs and stabilizing the oxidation state of the proximal PPT (NarG-His¹⁰⁹² and NarG-His¹⁰⁹⁸). A S719A variant has essentially no effect on the overall Mo(VI/IV) reduction potential, whereas the H1163A and H1184A variants elicit large effects (ΔE_m values of −88 and −36 mV, respectively). Ala variants of His¹⁰⁹² and His¹⁰⁹⁸ also elicit large ΔE_m values of −143 and −101 mV, respectively. An Arg variant of His¹⁰⁹² elicits a small ΔE_m of +18 mV on the Mo(VI/IV) reduction potential. There is a linear correlation between the molybdenum E_m value and both enzyme activity and the ability to support anaerobic respiratory growth on nitrate. These data support a non-innocent role for the PPT moieties in controlling active site metal redox chemistry and catalysis.     

EPR and redox properties of periplasmic nitrate reductase from Desulfovibrio desulfuricans ATCC 27774

Nitrate reductases are enzymes that catalyze the conversion of nitrate to nitrite. We report here electron paramagnetic resonance (EPR) studies in the periplasmic nitrate reductase isolated from the sulfate-reducing bacteria Desulfovibrio desulfuricans ATCC 27774. This protein, belonging to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes, comprises a single 80-kDa subunit and contains a Mo bis(molybdopterin guanosine dinucleotide) cofactor and a [4Fe–4S] cluster. EPR-monitored redox titrations, carried out with and without nitrate in the potential range from 200 to −500 mV, and EPR studies of the enzyme, in both catalytic and inhibited conditions, reveal distinct types of Mo(V) EPR-active species, which indicates that the Mo site presents high coordination flexibility. These studies show that nitrate modulates the redox properties of the Mo active site, but not those of the [4Fe–4S] center. The possible structures and the role in catalysis of the distinct Mo(V) species detected by EPR are discussed.Electronic Supplementary Material Supplementary material is available for this article at and is accessible for authorized users.     

Probing the reactivity of Ni in the active site of methyl-coenzyme M reductase with substrate analogues

Methyl-coenzyme M reductase (MCR) catalyses the reduction of methyl-coenzyme M (CH₃-S-CoM) with coenzyme B (HS-CoB) to methane and CoM-S-S-CoB. It contains the nickel porphyrinoid F₄₃₀ as prosthetic group which has to be in the Ni(I) oxidation state for the enzyme to be active. The active enzyme exhibits an axial Ni(I)-derived EPR signal MCR-red1. We report here on experiments with methyl-coenzyme M analogues showing how they affect the activity and the MCR-red1 signal of MCR from Methanothermobacter marburgensis. Ethyl-coenzyme M was the only methyl-coenzyme M analogue tested that was used by MCR as a substrate. Ethyl-coenzyme M was reduced to ethane (apparent K _M=20 mM; apparent V _max=0.1 U/mg) with a catalytic efficiency of less than 1% of that of methyl-coenzyme M reduction to methane (apparent K _M=5 mM; apparent V _max=30 U/mg). Propyl-coenzyme M (apparent K _i=2 mM) and allyl-coenzyme M (apparent K _i=0.1 mM) were reversible inhibitors. 2-Bromoethanesulfonate ([I]_{0.5 V}=2 µM), cyano-coenzyme M ([I]_{0.5 V}=0.2 mM), 3-bromopropionate ([I]_{0.5 V}=3 mM), seleno-coenzyme M ([I]_{0.5 V}=6 mM) and trifluoromethyl-coenzyme M ([I]_{0.5 V}=6 mM) irreversibly inhibited the enzyme. In their presence the MRC-red1 signal was quenched, indicating the oxidation of Ni(I) to Ni(II). The rate of oxidation increased over 10-fold in the presence of coenzyme B, indicating that the Ni(I) reactivity was increased in the presence of coenzyme B. Enzyme inactivated in the presence of coenzyme B showed an isotropic signal characteristic of a radical that is spin coupled with one hydrogen nucleus. The coupling was also observed in D₂O. The signal was abolished upon exposure of the enzyme to O₂. 3-Bromopropanesulfonate ([I]_{0.5 V}=0.1 µM), 3-iodopropanesulfonate ([I]_{0.5 V}=1 µM), and 4-bromobutyrate also inactivated MCR. In their presence the EPR signal of MCR-red1 was converted into a Ni-based EPR signal MCR-BPS that resembles in line shape the MCR-ox1 signal. The signal was quenched by O₂. 2-Bromoethanesulfonate and 3-bromopropanesulfonate, which both rapidly reacted with Ni(I) of MRC-red1, did not react with the Ni of MCR-ox1 and MCR-BPS. The Ni-based EPR spectra of both inactive forms were not affected in the presence of high concentrations of these two potent inhibitors.     

Characterization of the first eukaryotic cold-adapted patatin-like phospholipase from the psychrophilic Euplotes focardii: Identification of putative determinants of thermal-adaptation by comparison with the homologous protein from the mesophilic Euplotes crassus

The ciliated protozoon Euplotes focardii, originally isolated from the coastal seawaters of Terra Nova Bay in Antarctica, shows a strictly psychrophilic phenotype, including optimal survival and multiplication rates at 4–5 °C. This characteristic makes E. focardii an ideal model species for identifying the molecular bases of cold adaptation in psychrophilic organisms, as well as a suitable source of novel cold-active enzymes for industrial applications. In the current study, we characterized the patatin-like phospholipase from E. focardii (EfPLP), and its enzymatic activity was compared to that of the homologous protein from the mesophilic congeneric species Euplotes crassus (EcPLP). Both EfPLP and EcPLP have consensus motifs conserved in other patatin-like phospholipases.     

Reversal of coenzyme specificity and improvement of catalytic efficiency of <Emphasis Type="Italic">Pichia</Emphasis><Emphasis Type="Italic">stipitis</Emphasis> xylose reductase by rational site-directed mutagenesis

A major problem when xylose is used for ethanol production is the intercellular redox imbalance arising from different coenzyme specificities of xylose reductase (XR) and xylitol dehydrogenase. The residue Lys21 in XR from Pichia stipitis was subjected to site-directed mutagenesis to alter its coenzyme specificity. The N272D mutant exhibited improved catalytic efficiency when NADH was the coenzyme. Both K21A and K21A/N272D preferred NADH to NADPH, their catalytic efficiencies for NADPH were almost zero. The catalytic efficiency of K21A/N272D for NADH was almost 9-fold and 2-fold that of K21A and the wild-type enzyme, respectively. Complete reversal of coenzyme specificity toward NADH and improved catalytic efficiency were achieved. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. Qi-Kai Zeng, Hong-Li Du, Jing-Fang Wang have contributed equally to this work.     

Structural characterization of quadruplex DNA with in-cell EPR approaches

Guanosine-rich DNA sequences have the potential to adopt four-stranded conformations termed quadruplexes. The chromosomes of higher organisms are capped by so-called telomeres that are composed of repeats of the sequence TTAGGG. Up to 200 nucleotides of the G-rich strand form an overhang that is suspected to fold into intramolecular G-quadruplexes. Since induction of quadruplexes at the telomeres results in anti-proliferative effects, the intracellular structure of G-quadruplexes is of high interest as an anti-cancer drug target. Here we give a perspective on the elucidation of DNA sequence folds by electron paramagnetic resonance (EPR) distance measurements. The technique complements X-ray crystallography and NMR spectroscopy, as it can be applied in noncrystalline states, is not intrinsically limited by the size of the bio-macromolecular complex, and is able to analyze flexible structures or coexisting DNA conformation.     

定点突变技术在DNA缺失改造上的应用

本实验采用寡聚核苷酸指导的定点突变法,缺失了分别存在于YFD42和YFD58中的a-因子信号肽序列与a-hANP基因和a-因子信号肽序列与a-1FN基因间接头区域的27和18个核苷酸。由于被缺失部分恰好含有一个酶切位点,利用这一特点,酶切检查初步筛选出缺失了一个HindⅢ酶切位点的突变子。经DNA序列分析,证实缺失的核苷酸序列和设计完全一致。     

An innovative spectroelectrochemical reflection cell for rapid protein electrochemistry and ultraviolet/visible/infrared spectroscopy

A novel electrochemical reflection cell combining electrochemical techniques and spectroscopy which uses a solid gold working electrode as an optical mirror is described. This cell can be used at path lengths as low as a few micrometers and thus is suitable for ultraviolet/visible (UV/Vis) and infrared spectroscopy even for aqueous solutions and suspensions. The cell was designed for small sample volumes of only a few microliters, thus reducing the effort for sample preparation. Due to the short path length of some micrometers, the entire volume is within the Nernst diffusion layer, hence resulting in fast equilibration. Evaluation of the technique is described with direct electrochemistry of horse heart cytochrome c at the gold electrode modified with 4,4'-dithiodipyridine. Cyclic voltammograms indicate rapid and reversible electrochemistry with the correct midpoint potential (52 mV vs Ag/AgCl/3 M KCl). Chronoamperometry and coulometry confirm rapid and complete oxidation and reduction; the cell volume can be entirely fully reduced within less than 10-20 s. Spectroscopy in the UV/Vis region, with potentials at the working electrode stepped between -390 and 390 mV, show perfect titration of the cytochrome c heme bands. A Nernst fit of the alpha band absorption, with redox potential Em and number of electrons n left as parameters, yields a midpoint potential of 49 mV and n=0.9. The potential of this cell in the investigation of biological electron transfer reactions and in the study of bioenergetic systems is discussed.     

Insult

INSULT, a novel method for the creation of insertions, deletions, and point mutations without subcloning, requires only one new primer per mutant, and produces circular plasmids, obviating the need for special “ultracompetent” cells. The method includes cycles of linear amplification with a thermophilic polymerase, and nick repair after each cycle with a thermophilic ligase. After production of multiple single-stranded copies of circular mutation-bearing plasmid DNA, addition of a “generic” primer followed by one or more polymerase reaction cycles generates double-stranded circular DNA bearing the desired mutation.     

寡核苷酸探针(CAC)5/(GTG) 5指纹的非放射性标记和检测

在ＤＮＡ指纹分析中,通常采用的是同位素３２Ｐ标记ＤＮＡ分子探针,可检测到微微克（ｐｇ）靶序列。但３２Ｐ半衰期很短（仅有１４天）,使用和运输均不方便,３２Ｐ还放出硬射线,对实验者身体造成损害,需要一定的防护措施,并且容易污染环境。因此,科学家们研制出了...     

Describing the structure and assembly of protein filaments by EPR spectroscopy of spin-labeled side chains

In this review we summarize our approach to the study of Intermediate Filament (IF) structure and assembly by electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels. Using vimentin, a homopolymeric type III IF protein, we demonstrate that this approach serves as a general paradigm for studying protein filament structure and assembly. These strategies will be useful in exploring the structure and assembly properties of other filamentous or aggregation-prone systems.     

The effect of mutation of F87 on the properties of CYP102A1-CYP4C7 chimeras: altered regiospecificity and substrate selectivity

CYP102A1 is a highly active water-soluble bacterial monooxygenase that contains both substrate-binding heme and diflavin reductase subunits, all in a single polypeptide that has been called a "self-sufficient enzyme." Several years ago we developed a procedure called "scanning chimeragenesis," where we focused on residues 73-82 of CYP102A1, which contact approximately 40% of the substrates palmitoleic acid and N-palmitoylglycine [Murataliev et al. (2004) Biochemistry 43:1771-1780]. These residues were replaced with the homologous residues of CYP4C7. In the current work, that study has been expanded to include residue 87. Phenylalanine 87 of wild-type CYP102A1 was replaced with the homologous residue of CYP4C7, leucine, as well as with alanine. The full-sized chimeric proteins C(73-78, F87L), C(73-78, F87A), C(75-80, F87L), C(75-80, F87A), C(78-82, F87L) and C(78-82, F87A) have been purified and characterized. Wild-type CYP102A1 is most active toward fatty acids (both lauric and palmitic acids produce omega-1, omega-2, and omega-3 hydroxylated fatty acids), but it also catalyzes the oxidation of farnesol to three products (2, 3- and 10,11-epoxyfarnesols and 9-hydroxyfarnesol). All of the F87-mutant chimeric proteins show dramatic decreases in activities with the natural CYP102A1 substrates. In contrast, C(78-82, F87A) and C(78-82, F87L) have markedly increased activities with farnesol, with the latter showing a 5.7-fold increase in catalytic activity as compared to wild-type CYP102A1. C(78-82, F87L) produces 10,11-epoxyfarnesol as the single primary metabolite. The results show that chimeragenesis involving only the second half of SRS-1 plus F87 is sufficient to change the substrate selectivity of CYP102A1 from fatty acids to farnesol and to produce a single primary product.     

Crystal structure of a NO-forming nitrite reductase mutant: an analog of a transition state in enzymatic reaction

I257E was obtained by site directed mutagenesis of nitrite reductase from Achromobacter cycloclastes. The mutant has no enzyme activity. Its crystal structure determined at 1.65A resolution shows that the side-chain carboxyl group of the mutated residue, Glu257, coordinates with the type 2 copper in the mutant and blocks the contact between the type 2 copper and its solvent channel, indicating that the accessibility of the type 2 copper is essential for maintaining the activity of nitrite reductase. The carboxylate is an analog of the substrate, nitrite, but the distances between the type 2 copper and the two oxygen atoms of the side-chain carboxyl group are reversed in comparison to the binding of nitrite to the native enzyme. In the mutant, both the type 2 copper and the N epsilon atom on the imidazole ring of its coordinated residue His135 move in the substrate binding direction relative to the native enzyme. In addition, an EPR study showed that the type 2 copper in the mutant is in a reduced state. We propose that mutant I257E is in a state corresponding to a transition state in the enzymatic reaction.