Oxalate decarboxylase uses electron hole hopping for catalysis

The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.     

Suicide inactivation of cytochrome c oxidase: catalytic turnover in the absence of subunit III alters the active site

The catalytic core of cytochrome c oxidase is composed of three subunits: I, II, and III. Subunit III is a highly hydrophobic membrane protein that contains no redox centers; its role in cytochrome oxidase function is not obvious. Here, subunit III has been removed from the three-subunit mitochondrial-like oxidase of Rhodobacter sphaeroides by detergent washing. The resulting two-subunit oxidase, subunit III (-), is highly active. Ligand-binding analyses and resonance Raman spectroscopy show that its heme a(3)-Cu(B) active site is normal. However, subunit III (-) spontaneously and irreversibly inactivates during O(2) reduction. At pH 7.5, its catalytic lifetime is only 2% that of the normal oxidase. This suicide inactivation event primarily alters the active site. Its ability to form specific O(2) reduction intermediates is lost, and CO binding experiments suggest that the access of O(2) to reduced heme a(3) is inhibited. Reduced heme a accumulates in response to a decrease in the redox potential of heme a(3); electron transfer between the hemes is inhibited. Ligand-binding experiments and resonance Raman analysis show that increased flexibility in the structure of the active site accompanies inactivation. Cu(B) is partially lost. It is proposed that suicide inactivation results from the dissociation of a ligand of Cu(B) and that subunit III functions to prevent suicide inactivation by maintaining the structural integrity of the Cu(B) center via long-range interactions.     

Versatile peroxidase oxidation of high redox potential aromatic compounds: site-directed mutagenesis, spectroscopic and crystallographic investigation of three long-range electron transfer pathways

Versatile peroxidases (VP), a recently described family of ligninolytic peroxidases, show a hybrid molecular architecture combining different oxidation sites connected to the heme cofactor. High-resolution crystal structures as well as homology models of VP isoenzymes from the fungus Pleurotus eryngii revealed three possibilities for long-range electron transfer for the oxidation of high redox potential aromatic compounds. The possible pathways would start either at Trp164 or His232 of isoenzyme VPL, and at His82 or Trp170 of isoenzyme VPS1. These residues are exposed, and less than 11 A apart from the heme. With the purpose of investigating their functionality, two single mutations (W164S and H232F) and one double mutation (W164S/P76H) were introduced in VPL that: (i) removed the two pathways in this isoenzyme; and (ii) incorporated the absent putative pathway. Analysis of the variants showed that Trp164 is required for oxidation of two high redox potential model substrates (veratryl alcohol and Reactive Black 5), whereas the two other pathways (starting at His232 and His82) are not involved in long-range electron transfer (LRET). None of the mutations affected Mn2+ oxidation, which would take place at the opposite side of the enzyme. Substitution of Trp164 by His also resulted in an inactive variant, indicating that an indole side-chain is required for activity. It is proposed that substrate oxidation occurs via a protein-based radical. For the first time in a ligninolytic peroxidase such an intermediate species could be detected by low-temperature electron paramagnetic resonance of H2O2-activated VP, and was found to exist at Trp164 as a neutral radical. The H2O2-activated VP was self-reduced in the absence of reducing substrates. Trp164 is also involved in this reaction, which in the W164S variant was blocked at the level of compound II. When analyzing VP crystal structures close to atomic resolution, no hydroxylation of the Trp164 Cbeta atom was observed (even after addition of several equivalents of H2O2). This is in contrast to lignin peroxidase Trp171. Analysis of the crystal structures of both peroxidases showed differences in the environment of the protein radical-forming residue that could affect its reactivity. These variations would also explain differences found for the oxidation of some high redox potential aromatic substrates.     

Tuning the redox properties of a [4Fe-4S] center to modulate the activity of Mo-bisPGD periplasmic nitrate reductase

Molybdoenzymes are ubiquitous in living organisms and catalyze, for most of them, oxidation-reduction reactions using a large range of substrates. Periplasmic nitrate reductase (NapAB) from Rhodobacter sphaeroides catalyzes the 2-electron reduction of nitrate into nitrite. Its active site is a Mo bis-(pyranopterin guanine dinucleotide), or Mo-bisPGD, found in most prokaryotic molybdoenzymes. A [4Fe-4S] cluster and two c-type hemes form an intramolecular electron transfer chain that deliver electrons to the active site. Lysine 56 is a highly conserved amino acid which connects, through hydrogen-bonds, the [4Fe-4S] center to one of the pyranopterin ligands of the Mo-cofactor. This residue was proposed to be involved in the intramolecular electron transfer, either defining an electron transfer pathway between the two redox cofactors, and/or modulating their redox properties.In this work, we investigated the role of this lysine by combining site-directed mutagenesis, activity assays, redox titrations, EPR and HYSCORE spectroscopies. Removal of a positively-charged residue at position 56 strongly decreased the redox potential of the [4Fe-4S] cluster at pH?8 by 230?mV to 400?mV in the K56H and K56M mutants, respectively, thus affecting the kinetics of electron transfer from the hemes to the [4Fe-4S] center up to 5 orders of magnitude. This effect was partly reversed at acidic pH in the K56H mutant likely due to protonation of the imidazole ring of the histidine. Overall, our study demonstrates the critical role of a charged residue from the second coordination sphere in tuning the reduction potential of the [4Fe-4S] cluster in RsNapAB and related molybdoenzymes.     

Branched activation- and catalysis-specific pathways for electron relay to the manganese/iron cofactor in ribonucleotide reductase from Chlamydia trachomatis

A conventional class I (subclass a or b) ribonucleotide reductase (RNR) employs a tyrosyl radical (Y (*)) in its R2 subunit for reversible generation of a 3'-hydrogen-abstracting cysteine radical in its R1 subunit by proton-coupled electron transfer (PCET) through a network of aromatic amino acids spanning the two subunits. The class Ic RNR from the human pathogen Chlamydia trachomatis ( Ct) uses a Mn (IV)/Fe (III) cofactor (specifically, the Mn (IV) ion) in place of the Y (*) for radical initiation. Ct R2 is activated when its Mn (II)/Fe (II) form reacts with O 2 to generate a Mn (IV)/Fe (IV) intermediate, which decays by reduction of the Fe (IV) site to the active Mn (IV)/Fe (III) state. Here we show that the reduction step in this sequence is mediated by residue Y222. Substitution of Y222 with F retards the intrinsic decay of the Mn (IV)/Fe (IV) intermediate by approximately 10-fold and diminishes the ability of ascorbate to accelerate the decay by approximately 65-fold but has no detectable effect on the catalytic activity of the Mn (IV)/Fe (III)-R2 product. By contrast, substitution of Y338, the cognate of the subunit interfacial R2 residue in the R1 <--> R2 PCET pathway of the conventional class I RNRs [Y356 in Escherichia coli ( Ec) R2], has almost no effect on decay of the Mn (IV)/Fe (IV) intermediate but abolishes catalytic activity. Substitution of W51, the Ct R2 cognate of the cofactor-proximal R1 <--> R2 PCET pathway residue in the conventional class I RNRs (W48 in Ec R2), both retards reduction of the Mn (IV)/Fe (IV) intermediate and abolishes catalytic activity. These observations imply that Ct R2 has evolved branched pathways for electron relay to the cofactor during activation and catalysis. Other R2s predicted also to employ the Mn/Fe cofactor have Y or W (also competent for electron relay) aligning with Y222 of Ct R2. By contrast, many R2s known or expected to use the conventional Y (*)-based system have redox-inactive L or F residues at this position. Thus, the presence of branched activation- and catalysis-specific electron relay pathways may be functionally important uniquely in the Mn/Fe-dependent class Ic R2s.     

Introduction of unnatural amino acids into chalcone isomerase.

The active site cysteine residue of chalcone isomerase was rapidly and selectively modified under denaturing conditions with a variety of electrophilic reagents. These denatured and modified enzyme were renatured to produce enzyme derivatives containing a series of unnatural amino acids in the active site. Addition of methyl, ethyl, butyl, heptyl, and benzyl groups to the cysteine sulfur does not abolish catalytic activity, although the activity decreases as the steric bulk of the amino acid side-chain increases. Modification of the cysteine to introduce a charged homoglutamate or a neutral homoglutamine analogue results in retention of 22% of the catalytic activity. Addition of a methylthio group (SMe) to the cysteine residue of native chalcone isomerase preserves 85% of the catalytic activity measured with 2',4',4-trihydroxychalcone, 2',4',6',4-tetrahydroxychalcone, or 2'-hydroxy-4-methoxychalcone as substrates. The competitive inhibition constant for 4',4-dihydroxychalcone, the substrate inhibition constant for 2',4',4-trihydroxychalcone, and other steady-state kinetic parameters for the methanethiolated enzyme are very similar to those of the native enzyme. The strong binding of 4',4-dihydroxychalcone to the methanethiolated enzyme shows that there is no steric repulsion between this modified amino acid residue and the substrate analogue. This structure-activity study clearly demonstrates that the active site cysteine residue does not function as an acid-base or nucleophilic group in producing the catalysis or substrate inhibition observed with chalcone isomerase. The method presented in this paper allows for the rapid introduction of a series of unnatural amino acids into the active site as a means of probing the structure-function relationship.     

The oxidation of veratryl alcohol, dimeric lignin models and lignin by lignin peroxidase: The redox cycle revisited

Abstract: The mechanism of oxidation of veratryl alcohol and β-0–4 dimeric lignin models is reviewed. Veratryl alcohol radicals are intermediates in both oxidation pathways. The possible role of the veratryl alcohol radical cation as a mediator is discussed. The lignin peroxidase (LIP) redox cycle is analyzed in terms of the Marcus theory of electron transfer. Reduction of both LiP-Compound I (LiP-I) and LiP-Compound II (LiP-II) by veratryl alcohol occurs in the endergonic region of the driving force. The reduction of LiP-II has a higher reorganization energy due to the change in spin state and the accompanying conformational change in the protein. It is suggested that a reversible nucleophilic addition of a carbohydrate residue located at the entrance of the active site channel plays a key role in the LiP redox cycle. Moreover. (polymeric) hydroxysubstituted benzyl radicals may reduce LiP-II via long-range electron transfer.     

The role of the conserved tryptophan272 of the Paracoccus denitrificans cytochrome c oxidase in proton pumping

The catalytic mechanism of heme-copper oxidases - electron transfer coupled to proton pumping - is not yet fully understood. Single turnover experiments in which fully reduced cytochrome aa(3) from Paracoccus denitrificans reacts with O(2) using the microsecond freeze-hyperquenching sampling technique enabled trapping of transient catalytic intermediates and analysis by low temperature UV-Visible, X-band and Q-band EPR spectroscopy. Our recent findings (Wiertz et al. (2007) J. Biol. Chem. 282, 31580-31591), which show that the strictly conserved W272 is a redox active residue are reviewed here. The W272 forms a tryptophan neutral radical in the transition F-->F(W)-->O(H) in which the novel intermediate F(W) harbors the tryptophan radical. The potential role of W272 in proton pumping is highlighted.     

Structure,functionality and tuning up of laccases for lignocellulose and other industrial applications

Laccases (EC 1.10.3.2) are copper-containing oxidoreductases that have a relatively high redox potential which enables them to catalyze oxidation of phenolic compounds, including lignin-derived phenolics. The laccase-catalyzed oxidation of phenolics is accompanied by concomitant reduction of dioxygen to water via copper catalysis and involves a series of electron transfer reactions balanced by a stepwise re-oxidation of copper ions in the active site of the enzyme. The reaction details of the catalytic four-copper mechanism of laccase-mediated catalysis are carefully re-examined and clarified. The substrate range for laccase catalysis can be expanded by means of supplementary mediators that essentially function as vehicles for electron transfer. Comparisons of amino acid sequences and structural traits of selected laccases reveal conservation of the active site trinuclear center geometry but differences in loop conformations. We also evaluate the features and regions of laccases in relation to modification and evolution of laccases for various industrial applications including lignocellulosic biomass processing.     

The cloning of a new peroxidase found in lignocellulose cultures of Pleurotus eryngii and sequence comparison with other fungal peroxidases

We report cloning and sequencing of gene ps1 encoding a versatile peroxidase combining catalytic properties of lignin peroxidase (LiP) and manganese peroxidase (MnP) isolated from lignocellulose cultures of the white-rot fungus Pleurotus eryngii. The gene contains 15 putative introns, and the deduced amino acid sequence consists of a 339-residue mature protein with a 31-residue signal peptide. Several putative response elements were identified in the promoter region. Amino acid residues involved in oxidation of Mn(2+) and aromatic substrates by direct electron transfer to heme and long-range electron transfer from superficial residues as predicted by analogy with Phanerochaete chrysosporium MnP and LiP, respectively. A dendrogram is presented illustrating sequence relationships between 29 fungal peroxidases.     

Trapping of peptide-based surrogates in an artificially created channel of cytochrome c peroxidase

As recently described, the deliberate removal of the proposed electron transfer pathway from cytochrome c peroxidase resulted in the formation of an extended ligand-binding channel. The engineered channel formed a template for the removed peptide segment, suggesting that synthetic surrogates might be introduced to replace the native electron transfer pathway. This approach could be united with the recent development of sensitizer-linked substrates to initiate and study electron transfer, allowing access to unresolved issues about redox mechanism of the enzyme. Here, we present the design, synthesis, and screening of a peptide library containing natural and unnatural amino acids to identify the structural determinants for binding this channel mutant. Only one peptide, (benzimidazole-propionic acid)-Gly-Ala-Ala, appeared to interact, and gave evidence for both reversible and kinetically trapped binding, suggesting multiple conformations for the channel protein. Notably, this peptide was the most analogous to the removed electron transfer sequence, supporting the use of a cavity-template strategy for design of specific sensitizer-linked substrates as replacements for the native electron transfer pathway.     

Biochemical Evaluation of the Decarboxylation and Decarboxylation-Deamination Activities of Plant Aromatic Amino Acid Decarboxylases

Plant aromatic amino acid decarboxylase (AAAD) enzymes are capable of catalyzing either decarboxylation or decarboxylation-deamination on various combinations of aromatic amino acid substrates. These two different activities result in the production of arylalkylamines and the formation of aromatic acetaldehydes, respectively. Variations in product formation enable individual enzymes to play different physiological functions. Despite these catalytic variations, arylalkylamine and aldehyde synthesizing AAADs are indistinguishable without protein expression and characterization. In this study, extensive biochemical characterization of plant AAADs was performed to identify residues responsible for differentiating decarboxylation AAADs from aldehyde synthase AAADs. Results demonstrated that a tyrosine residue located on a catalytic loop proximal to the active site of plant AAADs is primarily responsible for dictating typical decarboxylase activity, whereas a phenylalanine at the same position is primarily liable for aldehyde synthase activity. Mutagenesis of the active site phenylalanine to tyrosine in Arabidopsis thaliana and Petroselinum crispum aromatic acetaldehyde synthases primarily converts the enzymes activity from decarboxylation-deamination to decarboxylation. The mutation of the active site tyrosine to phenylalanine in the Catharanthus roseus and Papaver somniferum aromatic amino acid decarboxylases changes the enzymes decarboxylation activity to a primarily decarboxylation-deamination activity. Generation of these mutant enzymes enables the production of unusual AAAD enzyme products including indole-3-acetaldehyde, 4-hydroxyphenylacetaldehyde, and phenylethylamine. Our data indicates that the tyrosine and phenylalanine in the catalytic loop region could serve as a signature residue to reliably distinguish plant arylalkylamine and aldehyde synthesizing AAADs. Additionally, the resulting data enables further insights into the mechanistic roles of active site residues.     

VARIATIONS IN AROMATIC AMINO ACID DECARBOXYLASE ACTIVITY TOWARDS DOPA AND 5-HYDROXYTRYPTOPHAN CAUSED BY pH CHANGES AND DENATURATION

—DOPA and 5-hydroxytryptophan (5-HTP) are generally supposed to be decarboxylated in mammalian tissues by a single enzyme, the two activities being present in constant ratio through a variety of purification procedures. It has now been shown that the ratio of activity of the liver enzyme towards the two substrates can be altered by mild treatments, such as might be used in solubilization of brain preparations. DOPA decarboxylase activity was preferentially inactivated by sodium dodecyl sulphate treatment, and 5-HTP decarboxylation by urea. Previous reports that the two substrates show different pH optima but are mutually competitive, have been confirmed. The K_m of the enzyme towards 5-HTP was lowest at pH 7.8 (the optimum pH for decarboxylation of this amino acid), but the variation with pH of the K_m towards DOPA was unrelated to the pH optimum for decarboxylation. There appeared to be no relation between the probable ionization state of the substrates and the pH dependence of the enzyme. Studies on the binding characteristics of the enzyme for the two products, dopamine and serotonin, did not show any specific saturable binding. It is proposed that the enzyme has a complex active site, with separate affinity sites for the two substrates, adjacent to a single catalytic site.     

Mutation of arginine 357 of the CP43 protein of photosystem II severely impairs the catalytic S-state cycle of the H2O oxidation complex

Basic amino acid side chains situated in active sites may mediate critical proton transfers during an enzymatic catalytic cycle. In the case of photosynthetic water oxidation, a strong base is postulated to facilitate the deprotonation of the active site Mn4-Ca cluster, thereby allowing the otherwise thermodynamically constrained transfer of an electron away from the Mn4-Ca cluster to the oxidized redox active tyrosine radical, YZ*, generated by photosynthetic charge separation. Arginine 357 of the CP43 polypeptide may be located in the second coordination shell of the O2-evolving Mn4-Ca cluster of photosystem II (PSII) according to current structural models. An ostensibly conservative substitution mutation, CP43-357K, was investigated using polarographic and fluorescence techniques in evaluating its potential impact on S-state cycling. Cells containing the CP43-357K mutation lost their capacity for autotrophic growth and exhibited a drastic reduction in O2 evolving activity ( approximately 15% of that of the wild type) despite the fact that mutant cells contained more than 80% of the concentration of charge-separating PSII reaction centers and more than half of these contained photooxidizable Mn. Fluorescence kinetics indicated that acceptor side electron transfer, dominated by the transfer of electrons from QA- to QB, was unaffected, but the fraction of centers containing Mn clusters capable of forming the S2 state was reduced to approximately 40% of that of the wild type. Analysis of O2 yields using a bare platinum electrode indicated a severe defect in the S-state cycling properties of the mutant H2O oxidation complexes. Although O2 evolution was delayed to the third flash during a train of single-turnover saturating flashes, the pattern of O2 emission did not exhibit a discernible periodicity indicating a very high miss factor, which was estimated to be approximately 45% compared to the wild-type value of approximately 10%. On the other hand, the multiflash fluorescence measurements indicate that the yield of formation of the S2 state from S1 is diminished by approximately 20%, although this latter estimate is complicated by the presence of damaged PSII centers. Taken together, the experiments indicate that the high miss factor observed during S-state cycling is likely due to a defect in the higher S-state transitions. These results are discussed in relation to the idea that CP43-R357 may serve as a ligand to bicarbonate or as the catalytic base proposed to mediate proton-coupled electron transfer (PCET) in the higher S states of the catalytic cycle of H2O oxidation.     

Tuning of functional heme reduction potentials in Shewanella fumarate reductases

The fumarate reductases from S. frigidimarina NCIMB400 and S. oneidensis MR-1 are soluble and monomeric enzymes located in the periplasm of these bacteria. These proteins display two redox active domains, one containing four c-type hemes and another containing FAD at the catalytic site. This arrangement of single-electron redox co-factors leading to multiple-electron active sites is widespread in respiratory enzymes. To investigate the properties that allow a chain of single-electron co-factors to sustain the activity of a multi-electron catalytic site, redox titrations followed by NMR and visible spectroscopies were applied to determine the microscopic thermodynamic parameters of the hemes. The results show that the redox behaviour of these fumarate reductases is similar and dominated by a strong interaction between hemes II and III. This interaction facilitates a sequential transfer of two electrons from the heme domain to FAD via heme IV.     

Electrochemical studies of a truncated laccase produced in Pichia pastoris

The cDNA that encodes an isoform of laccase from Trametes versicolor (LCCI), as well as a truncated version (LCCIa), was subcloned and expressed by using the yeast Pichia pastoris as the heterologous host. The amino acid sequence of LCCIa is identical to that of LCCI except that the final 11 amino acids at the C terminus of LCCI are replaced with a single cysteine residue. This modification was introduced for the purpose of improving the kinetics of electron transfer between an electrode and the copper-containing active site of laccase. The two laccases (LCCI and LCCIa) are compared in terms of their relative activity with two substrates that have different redox potentials. Results from electrochemical studies on solutions containing LCCI and LCCIa indicate that the redox potential of the active site of LCCIa is shifted to more negative values (411 mV versus normal hydrogen electrode voltage) than that found in other fungal laccases. In addition, replacing the 11 codons at the C terminus of the laccase gene with a single cysteine codon (i.e., LCCI-->LCCIa) influences the rate of heterogeneous electron transfer between an electrode and the copper-containing active site (k(het) for LCCIa = 1.3 x 10(-4) cm s(-1)). These results demonstrate for the first time that the rate of electron transfer between an oxidoreductase and an electrode can be enhanced by changes to the primary structure of a protein via site-directed mutagenesis.     

Crystal structure of quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni: structural basis for substrate oxidation and electron transfer.

Quinoprotein alcohol dehydrogenases are redox enzymes that participate in distinctive catabolic pathways that enable bacteria to grow on various alcohols as the sole source of carbon and energy. The x-ray structure of the quinohemoprotein alcohol dehydrogenase from Comamonas testosteroni has been determined at 1.44 A resolution. It comprises two domains. The N-terminal domain has a beta-propeller fold and binds one pyrroloquinoline quinone cofactor and one calcium ion in the active site. A tetrahydrofuran-2-carboxylic acid molecule is present in the substrate-binding cleft. The position of this oxidation product provides valuable information on the amino acid residues involved in the reaction mechanism and their function. The C-terminal domain is an alpha-helical type I cytochrome c with His(608) and Met(647) as heme-iron ligands. This is the first reported structure of an electron transfer system between a quinoprotein alcohol dehydrogenase and cytochrome c. The shortest distance between pyrroloquinoline quinone and heme c is 12.9 A, one of the longest physiological edge-to-edge distances yet determined between two redox centers. A highly unusual disulfide bond between two adjacent cysteines bridges the redox centers. It appears essential for electron transfer. A water channel delineates a possible pathway for proton transfer from the active site to the solvent.     

Paramagnetic relaxation enhancement of membrane proteins by incorporation of the metal-chelating unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA)

The use of paramagnetic constraints in protein NMR is an active area of research because of the benefits of long-range distance measurements (>10 Å). One of the main issues in successful execution is the incorporation of a paramagnetic metal ion into diamagnetic proteins. The most common metal ion tags are relatively long aliphatic chains attached to the side chain of a selected cysteine residue with a chelating group at the end where it can undergo substantial internal motions, decreasing the accuracy of the method. An attractive alternative approach is to incorporate an unnatural amino acid that binds metal ions at a specific site on the protein using the methods of molecular biology. Here we describe the successful incorporation of the unnatural amino acid 2-amino-3-(8-hydroxyquinolin-3-yl)propanoic acid (HQA) into two different membrane proteins by heterologous expression in E. coli. Fluorescence and NMR experiments demonstrate complete replacement of the natural amino acid with HQA and stable metal chelation by the mutated proteins. Evidence of site-specific intra- and inter-molecular PREs by NMR in micelle solutions sets the stage for the use of HQA incorporation in solid-state NMR structure determinations of membrane proteins in phospholipid bilayers.     

Pseudomonas cepacia 2,2-dialkylglycine decarboxylase. Sequence and expression in Escherichia coli of structural and repressor genes

A 3969-base pair PstI-PstI fragment of Pseudomonas cepacia DNA containing the gene for the pyridoxal 5'-phosphate dependent 2,2-dialkylglycine decarboxylase (pyruvate) (EC 4.1.1.64) was cloned in Escherichia coli. The insert was sequenced by the dideoxy method using nested deletions from both ends, revealing a central 1302-base pair region that codes for the decarboxylase subunit. The recombinant enzyme was expressed in E. coli, purified to homogeneity, and sequenced at the amino terminus. Also, a cofactor-labeled active site peptide was sequenced. The carboxyl terminus of the deduced amino acid sequence is homologous with the carboxyl terminus of mammalian ornithine aminotransferase; the active site sequence is similar to the active site sequences of several other aminotransferases. No homologies with known decarboxylase sequences could be found. Expression of the decarboxylase gene is negatively controlled by a 687-nucleotide sequence upstream of and diverging from the structural gene. Expression is induced by S-isovaline, 2-methylalanine, and D-2-aminobutanoic acid, but not by glycine, D- or L-alanine, L-2-aminobutanoic acid, R-isovaline, or other alkyl amino acids.     

A method for the direct detection of aromatic amino acid decarboxylase in electrophoretic media

Aromatic amino acid decarboxylase is an enzyme of broad specificity which decarboxylates a range of aromatic amino acids including 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, which yield dopamine and serotonin, respectively. The name aromatic amino acid decarboxylase was applied to the enzyme by workers (1–3) who found that, on purification, a single enzyme appeared to be responsible for activities previously ascribed to two separate enzymes. The separate enzymes had been named 5-hydroxy-l-tryptophan carboxylyase (EC 4.1.1.28) and 3,4-dihydroxy-l-phenylalanine carboxylyase (EC 4.1.1.26). More recent papers (4–7) still leave unresolved the question of whether one enzyme or two is responsible for the two activities.