Crystal structure of the catalytic domain of PigE: A transaminase involved in the biosynthesis of 2-methyl-3-n-amyl-pyrrole (MAP) from Serratia sp. FS14

Prodigiosin, a tripyrrole red pigment synthesized by Serratia and some other microbes through a bifurcated biosynthesis pathway, MBC (4-methoxy-2,2′-bipyrrole-5-carbaldehyde) and MAP (2-methyl-3-n-amyl-pyrrole) are synthesized separately and then condensed by PigC to form prodigiosin. MAP is synthesized sequentially by PigD, PigE and PigB. PigE catalyzes the transamination of an amino group to the aldehyde group of 3-acetyloctanal, resulting in an aminoketone, which spontaneously cyclizes to form H₂MAP. Here we report the crystal structure of the catalytic domain of PigE which involved in the biosynthesis of prodigiosin precursor MAP for the first time to a resolution of 2.3 Å with a homodimer in the asymmetric unit. The monomer of PigE catalytic domain is composed of three domains with PLP as cofactor: a small N-terminal domain connecting the catalytic domain with the front part of PigE, a large PLP-binding domain and a C-terminal domain. The residues from both monomers build the PLP binding site at the interface of the dimer which resembles the other PLP-dependent enzymes. Structural comparison of PigE with Thermus thermophilus AcOAT showed a higher hydrophobic and smaller active site of PigE, these differences may be the reason for substrate specificity.     

Biocatalytic asymmetric amination of carbonyl functional groups – a synthetic biology approach to organic chemistry

Transaminases catalyze amino transfer reactions from amino donors such as amino acids or amines to keto acids or ketones to give chiral amino acid or amines in optically pure form. α-Amino acid dehydrogenases catalyze the asymmetric reductive amination of α-keto acids using ammonia as amino donor to furnish L -amino acids. The distinct features and synthetic application of these two enzymes are reviewed in an effort to illustrate their promising and challenging aspects in serving as approaches to the direct asymmetric synthesis of optically pure amines from the corresponding keto compounds, a formidable problem in organic chemistry.     

Use of the anti-Prelog stereospecific alcohol dehydrogenase from Leifsonia and Pseudomonas for producing chiral alcohols

The asymmetric reduction of ketones is one of the most promising processes for producing chiral alcohols. However, dehydrogenases or reductases that can catalyze the reduction of ketones to give anti-Prelog chiral alcohols have been limited to some NADP⁺/NADPH-dependent enzymes. Recently, we reported a novel NAD⁺/NADH-dependent alcohol dehydrogenase (ADH) from Leifsonia sp. and Pseudomonas ADH homologs from soil metagenomes. Moreover, we have established an efficient hydrogen-transfer bioreduction process with 2-propanol as a hydrogen donor using Leifsonia ADH. This review focuses on the recent development of novel ADHs for producing industrially useful anti-Prelog chiral alcohols from various ketones.     

The effect of three α-keto acids on 3-mercaptopyruvate sulfurtransferase activity

3-Mercaptopyruvate sulfurtransferase catalyzes the transfer of sulfur from 3-mercaptopyruvate to several possible acceptor molecules, one of which is cyanide. Because the transsulfuration of cyanide is the primary in vivo mechanism of detoxification, 3-mercaptopyruvate sulfurtransferase may function in the enzymatic detoxification of cyanide in vivo. Three α-keto acids (α-ketobutyrate, α-ketoglutarate, and pyruvate) have previously been demonstrated to be cyanide antidotes in vivo, and it has been suggested that this is due to the nonenzymatic binding of cyanide by the α-keto acid. However, it has also been proposed that α-keto acids may increase the activity of enzymes involved in the transsulfuration of cyanide. Thus, the effect of these three α-keto acids on the enzyme 3-mercaptopyruvate sulfurtransferase was examined. All three α-keto acids inhibited 3-mercaptopyruvate sulfurtransferase in a concentration-dependent manner and were determined to be uncompetitive inhibitors of MST with respect to 3-mercaptopyruvate. The inhibitor constant K_i was estimated by two methods for each inhibitor and ranged from 4.3 to 6.3 mM. The I₅₀, which is the inhibitor concentration that produces 50% inhibition, was calculated for all three α-keto acids and ranged between 9.5 and 13.7 mM. These observations add further support to the hypothesis that the mechanisms of the α-keto acid antidotes is the nonenzymatic binding of cyanide, not stimulation of enzymes involved in the transsulfuration of cyanide to thiocyanate. © 1996 John Wiley & Sons, Inc.     

Enzyme-catalyzed side reactions with molecular oxygen may contribute to cell signaling and neurodegenerative diseases

A link between neurodegeneration and well-characterized enzymatic and non-enzymatic reactions that produce reactive oxygen species (ROS) from O₂ is well established. Several enzymes that contain pyridoxal 5′-phosphate (PLP) or thiamine diphosphate (ThDP) catalyze side reactions (paracatalytic reactions) in the presence of ambient O₂. These side reactions produce oxidants such as hydrogen peroxide [H₂O₂] or extremely reactive peracids [RC(O)OOH]. We hypothesize that although these enzymes normally produce oxidants at low or undetectable levels, changes in substrate levels or disease-induced structural alterations may enhance interactions with O₂, thereby generating higher levels of reactive oxidants. These oxidants may damage the enzymes producing them, alter nearby macromolecules and/or destroy important metabolites/coenzymes. We propose that paracatalytic reactions with O₂ catalyzed by PLP-dependent decarboxylases and by ThDP-dependent enzymes within the α-keto acid dehydrogenase complexes may contribute to normal cellular signaling and to cellular damage in neurodegenerative diseases. Special issue dedicated to John P. Blass.     

Differences in Protein Structure and Similarities in Catalytic Function of Two l-Stereoselective Carbonyl Reductases from Bakers’ Yeast

We purified and studied two l-stereoselective carbonyl reductases from bakers’ yeast (Saccharomyces cerevisiae). One catalyzed exclusively the enantioselective reduction of carbonyl compounds such as β-keto esters and the other acted on α-acetoxy ketones and β-keto esters. The enzymes had identical molecular weights and catalyzed the l-stereoselective reduction of various carbonyl compounds with similar substrate specificity, but they were different proteins coded by different genes.     

Incorporation of methionine into prodigiosin

Addition of proline to suspensions of nonpigmented, nonproliferating cells of Serratia marcescens induced biosynthesis of the pigment, prodigiosin. If methionine was included with proline, 4 times as much prodigiosin was formed, although the amount synthesized in the presence of methionine alone was nil. Uniformly ¹⁴C-labelled proline and methionine were incorporated into prodigiosin to about 30% the extent of their incorporation into cellular protein. Experiments with [carboxy-¹⁴C]-, and [Me-¹⁴C] methionine established that isotope from the methyl group was utilized preferentially for biosynthesis of prodigiosin.     

Ficin-catalyzed asymmetric aldol reactions of heterocyclic ketones with aldehydes

Ficin from fig tree latex displayed a promiscuous activity to catalyze the direct asymmetric aldol reactions of heterocyclic ketones with aromatic aldehydes. Ficin showed good substrate adaptability to different heterocyclic ketones containing nitrogen, oxygen or sulfur. The enantioselectivities up to 81% ee and diastereoselectivities up to 86:14 (anti/syn) were achieved under the optimized reaction conditions.     

Crystal Structures of Phosphoketolase: THIAMINE DIPHOSPHATE-DEPENDENT DEHYDRATION MECHANISM*

Thiamine diphosphate (ThDP)-dependent enzymes are ubiquitously present in all organisms and catalyze essential reactions in various metabolic pathways. ThDP-dependent phosphoketolase plays key roles in the central metabolism of heterofermentative bacteria and in the pentose catabolism of various microbes. In particular, bifidobacteria, representatives of beneficial commensal bacteria, have an effective glycolytic pathway called bifid shunt in which 2.5 mol of ATP are produced per glucose. Phosphoketolase catalyzes two steps in the bifid shunt because of its dual-substrate specificity; they are phosphorolytic cleavage of fructose 6-phosphate or xylulose 5-phosphate to produce aldose phosphate, acetyl phosphate, and H₂O. The phosphoketolase reaction is different from other well studied ThDP-dependent enzymes because it involves a dehydration step. Although phosphoketolase was discovered more than 50 years ago, its three-dimensional structure remains unclear. In this study we report the crystal structures of xylulose 5-phosphate/fructose 6-phosphate phosphoketolase from Bifidobacterium breve. The structures of the two intermediates before and after dehydration (α,β-dihydroxyethyl ThDP and 2-acetyl-ThDP) and complex with inorganic phosphate give an insight into the mechanism of each step of the enzymatic reaction.     

Asymmetric synthesis of α-amino acids via homologation of Ni(II) complexes of glycine Schiff bases. Part 3: Michael addition reactions and miscellaneous transformations

The major goal of this review is a critical discussion of the literature data on asymmetric synthesis of α-amino acids via Michael addition reactions involving Ni(II)-complexes of amino acids. The material covered is divided into two conceptually different groups dealing with applications of: (a) Ni(II)-complexes of glycine as C-nucleophiles and (b) Ni(II)-complexes of dehydroalanine as Michael acceptors. The first group is significantly larger and consequently subdivided into four chapters based on the source of stereocontrolling element. Thus, a chiral auxiliary can be used as a part of nucleophilic glycine Ni(II) complex, Michael acceptor or both, leading to the conditions of matching vs. mismatching stereochemical preferences. The particular focus of the review is made on the practical aspects of the methodology under discussion and mechanistic considerations.     

Hydrolase-catalyzed Michael addition of 1,3-dicarbonyl compounds to α,β-unsaturated compounds in organic solvent

A novel strategy to perform Michael additions between 1,3-dicarbonyl compounds and α,β-unsaturated compounds was developed by the catalysis of hydrolase. We found that 11 hydrolase could catalyze the enzymatic Michael addition reaction to form the carbon–carbon bond. In 2-methyl-2-butanol d-aminoacylase showed high Michael addition activity. The influence of substrate and Michael acceptor structure on Michael addition was evaluated systematically. Some control experiments demonstrated that the active site of d-aminoacylase was responsible for the enzymatic Michael addition reaction. This novel Michael addition activity of hydrolase is of practical significance in expanding the application of enzymes and in the evolution of new biocatalysts.     

Glycosyltransferase activities of Ehrlich ascites tumor cells: Detection,isolation, and characterization using oligosaccharide-Synsorb beads

Detergent extracts of Ehrlich tumor cell membranes exhibit a host of glycosyltransferase activities which have been investigated using oligosaccharides immobilized to Synsorb beads as acceptors. Glycosidase digestions in combination with methylation analysis of the insoluble products have demonstrated the presence of an α(1,3)-galactosyltransferase and a β(1,3)-N-acetylglucosaminyltransferase, enzymes that utilize N-acetyllactosamine as their acceptor substrate. The two enzymes are presumably involved in the biosynthesis of α-d-galactosyl-terminated poly-N-acetyllactosamine glycans that occur on the surface of Ehrlich cells. In addition, a β-galactosyltransferase acting on N-acetylglucosamine and a separate β-N-acetylglucosaminyltransferase that is capable of incorporating GlcNAc into the trisaccharide β-d-GlcNAc(1,3)-β-d-Gal(1,4)-β-d-Glc-Synsorb have been identified. The Ehrlich cell α- and β-galactosyltransferases have been separated by chromatography on β-GlcNAc-Synsorb beads. In the presence of MnCl₂ and UDP the β-galactosyltransferase is specifically adsorbed to the monosaccharide column whereas the α-galactosyltransferase passes through unretarded.     

Investigation of one-enzyme systems in the ω-transaminase-catalyzed synthesis of chiral amines

ω-Transaminase (TA) catalyzed asymmetric syntheses of amines were carried out in the one enzyme systems with wild-type enzymes (S)-TA from Pseudomonas aeruginosa, (S)-TA from Paracoccus denitrificans and (R)-TA from Aspergillus terreus. The scope of amine donors and aromatic carbonyl substrates was thoroughly explored. Among the range of potential amino donors, 2-propylamine, 2-butylamine and 1-phenylethylamine were found as promising candidates, which gave superior conversions in the amination reactions compared to other donors. Various prochiral aromatic ketones were accepted as substrates by the investigated enzymes. In most cases, good to excellent conversions (up to 98%) to the amine products with excellent e.e.-values (>99.9% for (S) or (R)) were obtained by the action of a single enzyme and an appropriate amino donor. (S)-TA from Paracoccus denitrificans was found to accept bulky ketones, e.g. 1-indanone, α- and β-tetralone or 2-acetonaphthone, in the asymmetric amination. In some cases the enantiomeric excesses in the amination reactions were dependent on the amino donor. Moreover, the influence of the pH, temperature and cosolvents on the outcome of reactions was additionally investigated.     

Metabolic pathway optimization for biosynthesis of 1,2,4-butanetriol from xylose by engineered Escherichia coli

1,2,4-Butanetriol (BT) and related derivatives have been widely used in many fields, especially in the military and in medicine. In this paper, we systematically optimized the BT biosynthetic pathway. We first investigated the activities of various NADH dependent aldehyde reductases (ALRs), which catalyze the fourth reaction in the four-step pathway for BT production from xylose in E. coli, and found that a combination of multiple endogenous enzymes catalyzed aldehyde reduction in the BT production bioprocess and that YqhD in E. coli was a main ALR for BT production. In addition, ADH2 from Saccharomyces cerevisiae can effectively catalyze 3,4-dihydroxybutanal to BT. Also, YjhG was identified as the major xylonate dehydratase and was co-overexpressed with YqhD, resulting in an improvement of BT production by 30%. Moreover, we identified and eliminated the competing branch pathway by inactivating 2-keto acid reductases (yiaE). Finally, the combination of these approaches led to BT production of 5.1 g/L. In summary, our study provides insights into the biosynthetic pathway for BT production, demonstrates an effective strategy to enhance BT production, and paves the way toward in-depth research on BT biosynthesis.     

Separation of L-phenylalanine: pyruvate transaminase and L-phenylalanine: alpha-ketoglutarate transaminase by sephadex-electrophoresis.

By means of a Sephadex-electrophoresis column, L-phenylalanine: pyruvate transaminase (PPT) was separated from L-phenylalanine: α-ketoglutarate transaminase (PKT) from rat liver. These enzymes differed in heat lability $\text{in vitro}$ and in their inducibility by glucagon $\text{in vivo}$. PPT was heat-stable and was induced by chronic glucagon injection. On the other hand, PKT was heat-labile and was not induced by glucagon under the experimental conditions used. These studies provide evidence that distinct enzymes catalyze the transamination of phenylalanine with pyruvate or with α-ketoglutarate as the amino acceptor.     

On the decarboxylation of α-keto acids by isolated chloroplasts

The mechanism of the decarboxylation of α-keto acids by isolated chloroplasts has been studied with the aid of superoxide dismutase and catalase. Using photosynthetic and enzymatic systems, which are known to catalyze peroxidic oxidations, we have been able to demonstrate that both the superoxide free radical ion and H₂O₂ are necessary for maximal rates of decarboxylation. In isolated chloroplasts, an auto-oxidizable electron acceptor as well as an electron donor for Photosystem I are absolute requirements for the decarboxylation. H₂O₂ seems to be the primary oxidant in the decarboxylation of pyruvate or glyoxylate by isolated chloroplasts. A secondary rate of decarboxylation is superimposed on the primary one, mediated by superoxide free radical ion. Mn²⁺ stimulates the decarboxylation probably via intermediarily-formed Mn³⁺ in a reaction, which is neither inhibited by catalase nor by superoxide dismutase. A decarboxylation of pyruvate or glyoxylate by isolated chloroplasts in the presence of NADP⁺ is initiated, as soon as the available NADP⁺ is fully reduced. In this case, the open-chain electron transport seems to switch from NADP⁺ to oxygen as the terminal electron acceptor.     

Biosynthesis of the red antibiotic, prodigiosin, in Serratia: identification of a novel 2-methyl-3-n-amyl-pyrrole (MAP) assembly pathway, definition of the terminal condensing enzyme, and implications for undecylprodigiosin biosynthesis in Streptomyces

The biosynthetic pathway of the red-pigmented antibiotic, prodigiosin, produced by Serratia sp. is known to involve separate pathways for the production of the monopyrrole, 2-methyl-3-n-amyl-pyrrole (MAP) and the bipyrrole, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC) which are then coupled in the final condensation step. We have previously reported the cloning, sequencing and heterologous expression of the pig cluster responsible for prodigiosin biosynthesis in two Serratia sp. In this article we report the creation of in-frame deletions or insertions in every biosynthetic gene in the cluster from Serratia sp. ATCC 39006. The biosynthetic intermediates accumulating in each mutant have been analysed by LC-MS, cross-feeding and genetic complementation studies. Based on these results we assign specific roles in the biosynthesis of MBC to the following Pig proteins: PigI, PigG, PigA, PigJ, PigH, PigM, PigF and PigN. We report a novel pathway for the biosynthesis of MAP, involving PigD, PigE and PigB. We also report a new chemical synthesis of MAP and one of its precursors, 3-acetyloctanal. Finally, we identify the condensing enzyme as PigC. We reassess the existing literature and discuss the significance of the results for the biosynthesis of undecylprodigiosin by the Red cluster in Streptomyces coelicolor A3(2).     

Molecular Phylogeny and Functional Genomics of β-Galactoside α2,6-Sialyltransferases That Explain Ubiquitous Expression of st6gal1 Gene in Amniotes

Sialyltransferases are key enzymes in the biosynthesis of sialoglycoconjugates that catalyze the transfer of sialic residue from its activated form to an oligosaccharidic acceptor. β-Galactoside α2,6-sialyltransferases ST6Gal I and ST6Gal II are the two unique members of the ST6Gal family described in higher vertebrates. The availability of genome sequences enabled the identification of more distantly related invertebrates'' st6gal gene sequences and allowed us to propose a scenario of their evolution. Using a phylogenomic approach, we present further evidence of an accelerated evolution of the st6gal1 genes both in their genomic regulatory sequences and in their coding sequence in reptiles, birds, and mammals known as amniotes, whereas st6gal2 genes conserve an ancestral profile of expression throughout vertebrate evolution.     

SCOPE AND LIMITATIONS OF THE USE OF NICOTINOPROTEIN ALCOHOL DEHYDROGENASE FOR THE COENZYME-FREE PRODUCTION OF ENANTIOPURE FINE-CHEMICALS

Nicotinoprotein alcohol dehydrogenases are enzymes that contain non-dissociable NAD(P)(H) in the active site. The suitability of a nicotinoprotein alcohol dehydrogenase as coenzyme-independent alternative to classic alcohol dehydrogenases for enantioselective synthetic applications was studied. To this end the NADH-containing nicotinoprotein, np-ADH, from Rhodococcus erythropolis DSM 1069 was used as a model enzyme in different types of conversion: asymmetric synthesis, kinetic resolution and racemization. The enzyme was found to catalyze the asymmetric reduction of ketones using cheap reductants, such as ethanol, with high stereoselectivity, but the reaction was too slow to obtain good yields. Kinetic resolutions of racemic alcohols failed due to dismutation of the aldehyde that was used as cosubstrate. Racemization of a secondary alcohol via the corresponding ketone could not be achieved, which was due to an unidentified side reaction. This evaluation shows that, for developing biotransformations of industrial interest using nicotinoprotein alcohol dehydrogenases, the attention should be focused on enzymes with a higher reactivity towards prochiral ketones and secondary alcohols.     

Green Asymmetric Synthesis: β‐Amino Alcohol‐Catalyzed Direct Asymmetric Aldol Reactions in Aqueous Micelles

The ability of chiral β‐amino alcohols to catalyze the direct asymmetric aldol reaction was evaluated for the first time in aqueous micellar media. A family of cheap and easily accessible β‐amino alcohols, obtained in one step from naturally occurring amino acids, was shown to successfully catalyze the asymmetric aldol reaction between a series of ketones and aromatic aldehydes. These aldol reactions furnished the corresponding β‐hydroxy ketones with up to 93% isolated yield and 89% ee. (S)‐2‐phenylglycinol and Triton X‐100 proved to be the best organocatalyst and surfactant, respectively. Chirality 25:119–125, 2013. © 2012 Wiley Periodicals, Inc.