Isolation and Characterization of L-Tryptophan Ammonia Lyase from Rubrivivax benzoatilyticus Strain JA2.

Ammonia lyase belongs to the family of enzymes that catalyzes the deamination of amino acids. Depending on the relative activity towards the substrates, L-tryptophan ammonia lyase converts L-tryptophan to indole 3-acrylic acid and ammonia. Here, we isolated, purified, and characterized an L-tryptophan ammonia lyase from phototrophic purple non-sulfur bacterium Rubrivivax benzoatilyticus JA2. The isolated L-tryptophan ammonia lyase found to catalyze the reaction of L-tryptophan to produce indole 3-acrylic acid and NH3. The enzyme is a heterotetramer and has the highest affinity to L-tryptophan. The optimum pH and temperature for the enzymatic action were 7.5 and 35°C, respectively and the Km and Vmax were 40.4 ± 23.1 nM and 0.964±0.2046 s(-1), respectively. These results suggest that the isolated enzyme is highly bioactive and could be a new class. Further molecular analyses are required to confirm the novelty of the enzyme.

4-methylideneimidazole-5-one-containing aminomutases in enediyne biosynthesis.

Many natural products contain unusual aromatic ß-amino acids or moieties derived therefrom. The biosynthesis of these ß-amino acids was first elucidated during a biosynthetic study of the enediyne antitumor antibiotic C-1027, when an enzyme, SgcC4, was discovered to convert L-tyrosine to (S)-ß-tyrosine. SgcC4 is similar in sequence and structure to 4-methylideneimidazole-5-one (MIO)-containing ammonia lyases. Whereas the ammonia lyases use the electrophilic power of the MIO group to catalyze the release of ammonia from aromatic amino acids to generate α,ß-unsaturated carboxylic acids as final products, SgcC4 retains the α,ß-unsaturated carboxylic acid and amine as intermediates and reappends the amino group to the ß-carbon, affording a ß-amino acid as the final product. The study of SgcC4 led to the subsequent discovery of other MIO-containing aminomutases with altered substrate specificity and product stereochemistry, including MdpC4 from the biosynthetic pathway of the enediyne antitumor antibiotic maduropeptin. This chapter describes protocols for the enzymatic and structural characterization of these MIO-containing aminomutases as exemplified by SgcC4 and MdpC4. These protocols are applicable to the study of other aminomutases.

Tailoring enzymes acting on carrier protein-tethered substrates in natural product biosynthesis.

Carrier proteins (CPs) are integral components of fatty acid synthases, polyketide synthases, and nonribosomal peptide synthetases and play critical roles in the biosynthesis of fatty acids, polyketides, and nonribosomal peptides. An emerging role CPs play in natural product biosynthesis involves tailoring enzymes that act on CP-tethered substrates. These enzymes provide a new opportunity to engineer natural product diversity by exploiting CPs to increase substrate promiscuity for the tailoring steps. This chapter describes protocols for in vitro biochemical characterization of SgcC3 and SgcC that catalyze chlorination and hydroxylation of SgcC2-tethered (S)-ß-tyrosine and analogues in the biosynthesis of the enediyne chromophore of the chromoprotein C-1027. These protocols are applicable to mechanistic characterization and engineered exploitation of other tailoring enzymes that act on CP-tethered substrates in natural product biosynthesis and structural diversification. The ultimate goal is to use the in vitro findings to guide in vivo engineering of designer natural products.

Expression and characterization of a second L-amino acid deaminase isolated from Proteus mirabilis in Escherichia coli.

L-amino acid deaminases catalyze the deamination of natural L-amino acids. Two types of L-amino acid deaminase have been identified in Proteus species. One exhibits high levels of activity toward a wide range of aliphatic and aromatic L-amino acids, typically L-phenylalanine, whereas the other acts on a relatively narrow range of basic L-amino acids, typically L-histidine. In this study, we cloned, expressed, and characterized a second amino acid deaminase, termed Pm1, from P. mirabilis KCTC 2566. Homology alignment of the deduced amino acid sequence of Pm1 demonstrated that the greatest similarity (96%) was with the L-amino acid deaminase (LAD) of P. vulgaris, and that homology with Pma was relatively low (72%). Also, similar to LAD, Pm1 was most active on L-histidine, indicating that Pm1 belongs to the second type of amino acid deaminase. In agreement with this conclusion, the V(max) and K(m) values of Pm1 were 119.7 (µg phenylpyruvic acid/mg/min) and 31.55 mM phenylalanine, respectively, values lower than those of Pma. The Pml deaminase will be very useful industrially in the preparation of commercially valuable materials including urocanic acid and α-oxoglutarate.

Purification and characterization of 3,4-dihydroxyphenylalanine oxidative deaminase from Rhodobacter sphaeroides OU5.

An enzyme involved in the catabolism of 3,4-dihydroxyphenylalanine (DOPA) was isolated from Rhodobacter sphaeroides OU5. The enzyme catalyzes the formation of 3,4-dihydroxyphenylpyruvic acid (DOPP) and ammonia from DOPA. Formation of ammonia by DOPA oxidative deaminase was O2 dependent and the enzyme isolated to its homogeneity has 100% affinity for DOPA. DOPA oxidative deaminase is functional at low concentrations of the substrate (< 100 micromol.L(-1)) and is independent of NADH. The molecular mass of the purified enzyme is approximately 190 kDa and the enzyme could be a pentamer of 54, 42, 34, 25, and 23 kDa subunits as determined by SDS-PAGE.

Identification, characterization and functional expression of a tyrosine ammonia-lyase and its mutants from the photosynthetic bacterium Rhodobacter sphaeroides.

A tyrosine ammonia-lyase (TAL) enzyme from the photosynthetic bacterium Rhodobacter sphaeroides (RsTAL) was identified, cloned and functionally expressed in Escherichia coli, where conversion of tyrosine to p-hydroxycinnamic acid (pHCA) was demonstrated. The RsTAL enzyme is implicated in production of pHCA, which serves as the cofactor for synthesis of the photoactive yellow protein (PYP) in photosynthetic bacteria. The wild type RsTAL enzyme, while accepting both tyrosine and phenylalanine as substrate, prefers tyrosine, but a serendipitous RsTAL mutant identified during PCR amplification of the RsTAL gene, demonstrates much higher preference for phenylalanine as substrate and deaminates it to produces cinnamic acid. Sequence analysis showed the presence of three mutations: Met4 --> Ile, Ile325 --> Val and Val409 --> Met in this mutant. Sequence comparison with Rhodobacter capsulatus TAL (RcTAL) shows that Val409 is conserved between RcTAL and RsTAL. Two single mutants of RsTAL, Val409 --> Met and Val 409 --> Ile, generated by site-directed mutagenesis, demonstrate greater preference for phenylalanine compared to the wild type enzyme. Our studies illustrate that relatively minor changes in the primary structure of an ammonia-lyase enzyme can significantly affect its substrate specificity.

Biosynthesis of pipecolic acid by RapL, a lysine cyclodeaminase encoded in the rapamycin gene cluster.

Rapamycin, FK506, and FK520 are immunosuppressant macrolactone natural products comprised of predominantly polyketide-based core structures. A single nonproteinogenic pipecolic acid residue is installed into the scaffold by a nonribosomal peptide synthetase that also performs the subsequent macrocyclization step at the carbonyl group of this amino acid. It has been assumed that pipecolic acid is generated from lysine by the cyclodeaminases RapL/FkbL. Herein we report the heterologous overexpression and purification of RapL and validate its ability to convert L-lysine to L-pipecolic acid by a cyclodeamination reaction that involves redox catalysis. RapL also accepts L-ornithine as a substrate, albeit with a significantly reduced catalytic efficiency. Turnover is presumed to encompass a reversible oxidation at the alpha-amine, internal cyclization, and subsequent re-reduction of the cyclic delta1-piperideine-2-carboxylate intermediate. As isolated, RapL has about 0.17 equiv of tightly bound NAD+, suggesting that the enzyme is incompletely loaded when overproduced in E. coli. In the presence of exogenous NAD+, the initial rate is elevated 8-fold with a Km of 2.3 microM for the cofactor, consistent with some release and rebinding of NAD+ during catalytic cycles. Through the use of isotopically labeled substrates, we have confirmed mechanistic details of the cyclodeaminase reaction, including loss of the alpha-amine and retention of the hydrogen atom at the alpha-carbon. In addition to the characterization of a critical enzyme in the biosynthesis of a medically important class of natural products, this work represents the first in vitro characterization of a lysine cyclodeaminase, a member of a unique group of enzymes which utilize the nicotinamide cofactor in a catalytic manner.

Two beta-alanyl-CoA:ammonia lyases in Clostridium propionicum.

The fermentation of beta-alanine by Clostridium propionicum proceeds via activation to the CoA-thiol ester, followed by deamination to acryloyl-CoA, which is also an intermediate in the fermentation of l-alanine. By shifting the organism from the carbon and energy source alpha-alanine to beta-alanine, the enzyme beta-alanyl-CoA:ammonia lyase is induced 300-fold (approximately 30% of the soluble protein). The low basal lyase activity is encoded by the acl1 gene, whereas the almost identical acl2 gene (six amino acid substitutions) is responsible for the high activity after growth on beta-alanine. The deduced beta-alanyl-CoA:ammonia lyase proteins are related to putative beta-aminobutyryl-CoA ammonia lyases involved in lysine fermentation and found in the genomes of several anaerobic bacteria. beta-Alanyl-CoA:ammonia lyase 2 was purified to homogeneity and characterized as a heteropentamer composed of 16 kDa subunits. The apparent K(m) value for acryloyl-CoA was measured as 23 +/- 4 microm, independent of the concentration of the second substrate ammonia; k(cat)/K(m) was calculated as 10(7) m(-1) x s(-1). The apparent K(m) for ammonia was much higher, 70 +/- 5 mm at 150 microm acryloyl-CoA with a much lower k(cat)/K(m) of 4 x 10(3) m(-1) x s(-1). In the reverse reaction, a K(m) of 210 +/- 30 microM was obtained for beta-alanyl-CoA. The elimination of ammonia was inhibited by 70% at 100 mm ammonium chloride. The content of beta-alanyl-CoA:ammonia lyase in beta-alanine grown cells is about 100 times higher than that required to sustain the growth rate of the organism. It is therefore suggested that the enzyme is needed to bind acryloyl-CoA, in order to keep the toxic free form at a very low level. A formula was derived for the calculation of isomerization equilibra between L-alanine/beta-alanine or D-lactate/3-hydroxypropionate.

Crystallization and X-ray diffraction analysis of ornithine cyclodeaminase from Pseudomonas putida.

Ornithine cyclodeaminase (OCD) is a member of the micro-crystallin protein family, the biological activity of which is the conversion of L-ornithine to L-proline and ammonia. In order to elucidate the functional groups of this enzyme that are involved in catalysis, the crystallization of OCD from Pseudomonas putida was undertaken. Using microbatch-under-oil screening at the high-throughput crystallization laboratory (HTC) at the Hauptman-Woodward Medical Research Institute Inc. (HWI Buffalo, NY, USA), numerous crystallization conditions were rapidly identified. Several conditions could be reproduced on a larger scale as vapor-diffusion experiments in-house. The best diffraction-quality crystals were obtained from solutions of 40%(v/v) 2-methyl-2,4-pentanediol buffered at pH 6.0 with 0.1 M MES and diffracted X-rays to 1.68 A resolution. Crystals belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 70.0, b = 78.3, c = 119.4 A. The V(M) was 2.1 A(3) Da(-1), corresponding to 42% solvent, which is consistent with two 38.5 kDa molecules per asymmetric unit. The structure determination is under way using experimental phasing methods.

Chitosan and chitin oligomers increase phenylalanine ammonia-lyase and tyrosine ammonia-lyase activities in soybean leaves.

Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) and tyrosine ammonia-lyase (TAL, 4.3.1.), the key enzymes of the phenylpropanoid pathway, are inducible in response to biotic (such as chitin from fungal cell walls) and abiotic cues. Application of chitin and chitosan to soybean leaf tissues caused increased activity of PAL and TAL enzymes. The elevation of enzyme activity was dependent on the chain length of the oligomers and time after treatment. The hexamer of chitin and pentamer of chitosan produced the maximum activities at 36 h after treatment as compared to controls. Total phenolic content of soybean leaves increased following chitosan and chitin oligomer treatments, showing a positive correlation between enzyme activity and total phenolic content.

Crystallization and preliminary X-ray diffraction studies on recombinant diaminopropionate ammonia lyase from Escherichia coli.

Diaminopropionate (DAP) ammonia lyase (a PLP-dependent enzyme; EC 4.3.1.15) catalyzes the alpha,beta-elimination reaction of both L- and D-alpha,beta-diaminopropionate to form pyruvate and ammonia. Escherichia coli DAP ammonia lyase gene was cloned and overexpressed in E. coli and the protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion technique. Crystals of two different morphologies were obtained, one of which belonged to the tetragonal space group P4(1)2(1)2 (or P4(3)2(1)2), with unit-cell parameters a = b = 86.01, c = 209.56 A, and the other to the monoclinic space group P2(1), with unit-cell parameters a = 87.78, b = 94.35, c = 96.02 A, beta = 109.73 degrees. The tetragonal crystals diffracted X-rays to 3.0 A resolution, while diffraction from the monoclinic form extended to 2.5 A. Complete X-ray diffraction data sets have been collected for both crystal forms.

Production of diamino propionic acid ammonia lyase by a new strain of Salmonella typhimurium PU011.

BACKGROUND: Seeds of the legume plant Lathyrus sativus, which is grown in arid and semi arid tropical regions, contain Diamino Propionic acid (DAP). DAP is a neurotoxin, which, when consumed, causes a disease called Lathyrism. Lathryrism may manifest as Neurolathyrism or Osteolathyrism, in which the nervous system, and bone formation respectively, are affected. DAP ammonia lyase is produced by a few microorganisms such as Salmonella typhi, Salmonella typhimurium and Pseudomonas, and is capable of detoxifying DAP. RESULTS: S. typhimurium PU011, a non-virulent bacterial strain isolated in our lab, was found to produce DAP ammonia lyase enzyme when grown in minimal medium containing DAP. There was a direct correlation between biomass yield and enzyme activity, until 16 h post inoculation in minimal medium containing DAP. Following ammonium sulphate precipitation and passing through Sephadex G100, CM-Sephadex and DEAE-Sephacel for crude enzyme extract preparation, about 68-fold enzyme purity was obtained. The purified enzyme gave maximum activity at pH 8.0 and was stable up to 45 degrees C. The Km value for the substrate was found to be 0.685 mM, calculated from a Line Weaver Burk plot. CONCLUSION: A new bacterial strain, S.typhimurium PU 011, which is capable of producing DAP ammonia lyase, was isolated.

Characterization of a bacterial tyrosine ammonia lyase, a biosynthetic enzyme for the photoactive yellow protein.

During genome sequence analysis of Rhodobacter capsulatus, nearby open reading frames were found that encode a photoactive yellow protein (PYP) and a hypothetical biosynthetic enzyme for its chromophore, a tyrosine ammonia lyase (TAL). We isolated the TAL gene, overproduced the recombinant protein in Escherichia coli, and after purification analyzed the enzyme for its activity. The catalytic efficiency for tyrosine was shown to be approximately 150 times larger than for phenylalanine, suggesting that the enzyme could in fact be involved in biosynthesis of the PYP chromophore. To our knowledge it is the first time this type of enzyme has been found in bacteria.

Gene cloning, purification, and characterization of 2,3-diaminopropionate ammonia-lyase from Escherichia coli.

2,3-Diaminopropionate ammonia-lyase (DAPAL), which catalyzes alpha,beta-elimination of 2,3-diaminopropionate regardless of its stereochemistry, was purified from Salmonella typhimurium. We cloned the Escherichia coli ygeX gene encoding a putative DAPAL and purified the gene product to homogeneity. The protein obtained contained pyridoxal 5'-phosphate and was composed of two identical subunits with a calculated molecular weight of 43,327. It catalyzed the alpha,beta-elimination of both D- and L-2,3-diaminopropionate. The results confirmed that ygeX encoded DAPAL. The enzyme acted on D-serine, but its catalytic efficiency was only 0.5% that with D-2,3-diaminopropionate. The enzymologic properties of E. coli DAPAL resembled those of Salmonella DAPAL, except that L-serine, D-and L-beta-Cl-alanine were inert as substrates of the enzyme from E. coli. DAPAL had significant sequence similarity with the catalytic domain of L-threonine dehydratase, which is a member of the fold-type II group of pyridoxal phosphate enzymes, together with D-serine dehydratase and mammalian serine racemase.

Purification and characterization of beta-methylaspartase from Fusobacterium varium.

Beta-methylaspartase (EC 4.3.1.2) was purified 20-fold in 35% yield from Fusobacterium varium, an obligate anaerobe. The purification steps included heat treatment, fractional precipitation with ammonium sulfate and ethanol, gel filtration, and ion exchange chromatography on DEAE-Sepharose. The enzyme is dimeric, consisting of two identical 46 kDa subunits, and requires Mg2+ (Km = 0.27+/-0.01 mM) and K+ (Km = 3.3+/-0.8 mM) for maximum activity. Beta-methylaspartase-catalyzed addition of ammonia to mesaconate yielded two diastereomeric amino acids, identified by HPLC as (2S,3S)-3-methylaspartate (major product) and (2S,3R)-3-methylaspartate (minor product). Optimal activity for the deamination of (2S,3S)-3-methylaspartate (Km = 0.51+/-0.04 mM) was observed at pH 9.7. The N-terminal protein sequence (30 residues) of the F. varium enzyme is 83% identical to the corresponding sequence of the clostridial enzyme.

Overexpression, purification, crystallization and data collection of 3-methylaspartase from Clostridium tetanomorphum.

3-Methylaspartase (E.C. 4.3.1.2) catalyses the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid as well as a slower syn elimination from the (2S,3R)-epimer, L-erythro-3-methylaspartic acid. The anti-elimination reaction occurs in the second step of the catabolic pathway for glutamic acid in Clostridium tetanomorphum. The reverse reaction is of particular interest because the addition of ammonia to substituted fumaric acids is highly stereoselective and gives highly functionalized amino acids. The mechanism of the transformation is unusual and of considerable interest. 3-Methylaspartase from C. tetanomorphum has been overexpressed and purified from Escherichia coli. Crystals of the enzyme have been obtained by sitting-drop vapour diffusion. Two native data sets have been collected, one in-house on a rotating-anode generator to 3.2 A and one at the European Synchrotron Radiation Facility to 2.0 A. A 2.1 A data set has been collected on a crystal of selenomethionine protein. Combining the data sets identify the space group as P2(1)2(1)2, with unit-cell parameters a = 110.3, b = 109.9, c = 67.2 A, alpha = beta = gamma = 90 degrees. The asymmetric unit contains two monomers with 42% solvent. A self-rotation function indicates the presence of a twofold axis, consistent with a biological dimer.

Crystallization and preliminary X-ray analysis of the formiminotransferase domain from the bifunctional enzyme formiminotransferase-cyclodeaminase.

Formiminotransferase-cyclodeaminase (E.C. 2.1.2.5-E.C. 4.3.1.4) is a bifunctional enzyme involved in the histidine-degradation pathway which exhibits specificity for polyglutamylated folate substrates. The first function of the enzyme transfers the formimino group of formiminoglutamate to the N5 position of tetrahydrofolate, while the second function catalyses the cyclodeamination of the formimino group, yielding N5,10-methenyl-tetrahydrofolate, with efficient channeling of the intermediate between these activities. Initial studies have shown that the enzyme consists of eight identical subunits of 62 kDa each, arranged as a circular tetramer of dimers. It is this formation which results in two different dimeric interfaces, which are necessary for the two different activities. The identical subunits have been shown to consist of two domains, each of which can be obtained as dimers. The formiminotransferase domain has been crystallized in the presence of the substrate analogue folinic acid. The crystals belong to space group P212121, with unit-cell dimensions a = 64.4, b = 103.7, c = 122.3 A. Both a native data set and a mercurial derivative data set have been collected to 2.8 A resolution.

Cloning, nucleotide sequencing, and expression of the 3-methylaspartate ammonia-lyase gene from Citrobacter amalonaticus strain YG-1002.

The gene coding for 3-methylaspartate ammonia-lyase (3-methylaspartase, MAL, EC 4.3.1.2) from Citrobacter amalonaticus strain YG-1002 (TPU 6323) was cloned onto plasmid pBluescript II KS(+), and the nucleotide sequence of the 1239-bp open reading frame (ORF), consisting of 413 codons, was identified as the mal gene coding for MAL. The predicted polypeptide has 62.5% identity with MAL from the obligate anaerobe, Clostridium tetanomorphum NCIMB 11547. ORF1, which showed 58.6% and 58.8% identities with subunit E of the glutamate mutases of C. tetanomorphum and Clostridium cochlearium respectively, was found in the upstream region of the mal gene. An expression plasmid pMALCA3 (5.4 kb), in which the mal gene was expressed under control of the lac promoter on the vector, was constructed. With feeding of 1 mM isopropyl beta-D-thiogalactopyranoside, the amount of the enzyme in a cell-free extract of the transformant, E. coli JM109/pMALCA3, was elevated to 51,800 units/l culture, which is about 50-fold that of C. amalonaticus strain YG-1002. It was calculated that the enzyme comprised over 40% of the total extractable cellular proteins. The enzyme produced by the E. coli transformant was purified in a crystalline form and shown to be identical to that of the wild-type strain with respect to specific activity, molecular mass, subunit structure, enzymological properties, and N-terminal amino acid sequences.

3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae.

The enzyme 3-methylaspartase (3-methylaspartate ammonia-lyase, EC 4. 3.1.2) was found in the cells of enteric bacteria, especially in the genera Citrobacter and Morganella, that were grown under anoxic and oxygen-limited conditions. The enzymes were purified to homogeneity from the cell-free extracts of 18 active strains and had similar enzymological properties such as action on columns, specific activity, molecular weight, subunit structure, and N-terminal amino acid sequence similarity. The production of the enzyme was dependent on the limitation of oxygen during growth and was arrested by aeration. The addition of external electron acceptors such as dimethylsulfoxide could support cell growth and production of the enzyme. Activities of glutamate mutase (EC 5.4.99.1) and (S)-citramalate hydrolyase (EC 4.2.1.34), key enzymes of the mesaconate pathway of (S)-glutamate fermentation in the genus Clostridium, were detected in the cells of the active strains grown under oxygen-limited conditions. Based on the results, the mesaconate pathway is proposed to explain the (S)-glutamate fermentation process observed in Enterobacteriaceae, and 3-methylaspartase could be a marker enzyme for this pathway.

3-Methylaspartate ammonia-lyase from a facultative anaerobe, strain YG-1002.

3-Methylaspartase was purified 24-fold and crystallized from the crude extract of the cells of a facultative anaerobic bacterium from soil, strain YG-1002. The molecular mass of the native enzyme was about 84 kDa and that of the subunit was about 42 kDa. The pH optimum for the deamination reaction of (2S, 3S)-3-methylaspartic acid and those for the amination reaction of mesaconic acid were 9.7 and 8.5; its optimum temperature was 50 degrees C. The enzyme was stable at pH 5.5-11.0 and up to 50 degrees C. The enzyme required both divalent and monovalent cations such as Mg2+ and K+. The enzyme was inhibited by sulfhydryl reagents, metal-chelating reagents and some divalent cations. The enzyme catalyzed the reversible amination/deamination reactions between several 3-substituted (S)-aspartic acids and their corresponding fumaric acid derivatives. The enzyme preferentially acted on (2S, 3S)-3-methylaspartic acid and mesaconic acid in the deamination and the amination reactions respectively. The enzyme showed high similarities in several enzymological properties and N-terminal amino acid sequence with 3-methylaspartase from an obligate anaerobic bacterium Clostridium tetanomorphum.