Crystal structure of a member of a novel family of dioxygenases (PF10014) reveals a conserved cupin fold and active site

PF10014 is a novel family of 2‐oxyglutarate‐Fe²⁺‐dependent dioxygenases that are involved in biosynthesis of antibiotics and regulation of biofilm formation, likely by catalyzing hydroxylation of free amino acids or other related ligands. The crystal structure of a PF10014 member from Methylibium petroleiphilum at 1.9 Å resolution shows strong structural similarity to cupin dioxygenases in overall fold and active site, despite very remote homology. However, one of the β‐strands of the cupin catalytic core is replaced by a loop that displays conformational isomerism that likely regulates the active site. Proteins 2014; 82:164–170. © 2013 Wiley Periodicals, Inc.     

Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids

We determined the enzymatic characteristics of an industrially important biocatalyst, α-ketoglutarate-dependent l-isoleucine dioxygenase (IDO), which was found to be the enzyme responsible for the generation of (2S,3R,4S)-4-hydroxyisoleucine in Bacillus thuringiensis 2e2. Depending on the amino acid used as the substrate, IDO catalyzed three different types of oxidation reactions: hydroxylation, dehydrogenation, and sulfoxidation. IDO stereoselectively hydroxylated several hydrophobic aliphatic l-amino acids, as well as l-isoleucine, and produced (S)-3-hydroxy-l-allo-isoleucine, 4-hydroxy-l-leucine, (S)-4-hydroxy-l-norvaline, 4-hydroxy-l-norleucine, and 5-hydroxy-l-norleucine. The IDO reaction product of l-isoleucine, (2S,3R,4S)-4-hydroxyisoleucine, was again reacted with IDO and dehydrogenated into (2S,3R)-2-amino-3-methyl-4-ketopentanoate, which is also a metabolite found in B. thuringiensis 2e2. Interestingly, IDO catalyzed the sulfoxidation of some sulfur-containing l-amino acids and generated l-methionine sulfoxide and l-ethionine sulfoxide. Consequently, the effective production of various modified amino acids would be possible using IDO as the biocatalyst.     

The evolution of three types of indoleamine 2,3 dioxygenases in fungi with distinct molecular and biochemical characteristics

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-degrading enzyme and known as a mammalian immunosuppressive molecule. In fungi, the primary role of IDO is to supply nicotinamide adenine dinucleotide (NAD(+)) via the kynurenine pathway. We previously reported that the koji-mold, Aspergillus oryzae has two IDO genes, IDOα and IDOβ. In the present study, we found that A. oryzae also has the third IDO, IDOγ. These three-types of IDOs are widely distributed among the Pezizomycotina fungi, although the black truffle, Tuber melanosporum has only one corresponding gene to IDOα/IDOβ. The yeast, Saccharomyces cerevisiae has a single IDO gene. Generally, Pezizomycotina IDOα showed similar enzymatic properties to the yeast IDO, suggesting that the IDOα is a functional homologue of the S. cerevisiae IDO. In contrast to IDOα, the K(m) value of IDOβ is higher. However, the reaction velocity of IDOβ is very fast, resulting in comparable or higher catalytic efficiency than IDOα. Thus IDOβ may functionally substitute for IDOα in fungal L-Trp metabolism. The enzymatic activity of IDOγ was comparatively very low with the values of enzymatic parameters comparable to vertebrate IDO2 enzymes. IDOα and IDOβ have similar gene structures, suggesting that they were generated by gene duplication which occurred rather early in Pezizomycotina evolution, although the timing of the duplication remains debatable. In contrast, the phylogenetic trees suggest that IDOγs form an evolutionarily distinct group of IDO enzymes, with a closer relationship to group I bacterial IDOs than other fungal IDOs. The ancestor of the IDOγ family is likely to have diverged from other eukaryotic IDOs at a very early stage of eukaryotic evolution.     

Molecular evolution of bacterial indoleamine 2,3-dioxygenase

Indoleamine 2,3-dioxygenase (IDO) and tryptophan 2,3-dioxygenase (TDO) are tryptophan-degrading enzymes that catalyze the first step in L-Trp catabolism via the kynurenine pathway. In mammals, TDO is mainly expressed in the liver and primarily supplies nicotinamide adenine dinucleotide (NAD⁺). TDO is widely distributed from mammals to bacteria. Active IDO enzymes have been reported only in vertebrates and fungi. In mammals, IDO activity plays a significant role in the immune system while in fungal species, IDO is constitutively expressed and supplies NAD⁺, like mammalian TDO. A search of genomic databases reveals that some bacterial species also have a putative IDO gene. A phylogenetic analysis clustered bacterial IDOs into two groups, group I or group II bacterial IDOs. The catalytic efficiencies of group I bacterial IDOs were very low and they are suspected not to contribute significantly to L-Trp metabolism. The bacterial species bearing the group I bacterial IDO are scattered across a few phyla and no phylogenetically close relationship is observed between them. This suggests that the group I bacterial IDOs might be acquired by horizontal gene transmission that occurred in each lineage independently. In contrast, group II bacterial IDOs showed rather high catalytic efficiency. Particularly, the enzymatic characteristics (K_m, V_max and inhibitor selectivity) of the Gemmatimonas aurantiaca IDO are comparable to those of mammalian IDO1, although comparison of the IDO sequences does not suggest a close evolutionary relationship. In several bacteria, TDO and the kynureninase gene (kynU) are clustered on their chromosome suggesting that these genes could be transcribed in an operon. Interestingly, G. aurantiaca has no TDO, and the IDO is clustered with kynU on its chromosome. Although the G. aurantiaca also has NadA and NadB to synthesize a quinolinic acid (a precursor of NAD⁺) via the aspartate pathway, the high activity of the G. aurantiaca IDO flanking the kynU gene suggests its IDO has a function similar to eukaryotic enzymes.     

Biotransformation of D-methionine into L-methionine in the cascade of four enzymes

D-Methionine was converted to L-methionine in a reaction system where four enzymes were used. D-amino acid oxidase (D-AAO) from Arthrobacter protophormiae was used for the complete conversion of D-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was added to prevent 2-oxo-4-methylthiobutyric acid decarboxylation. In the second reaction step, L-phenylalanine dehydrogenase (L-PheDH) from Rhodococcus sp. was used to convert 2- oxo-4-methylthiobutyric acid to L-methionine, and formate dehydrogenase (FDH) from Candida boidinii was added for NADH regeneration. Enzyme kinetics of all enzymes was analyzed in detail. Mathematical models for separate reactions steps, as well as for the complete system were developed and validated in the batch reactor experiments. Complete conversion of D-methionine to L-methionine was achieved. Considering that both enzymes act on different substrates, such a system could be easily employed for the synthesis of other amino acids from D-isomer, as well as from the racemate of a certain amino acid (DL-amino acid).     

Postnatal amino acid uptake by the rat small intestine. Changes in membrane transport systems for amino acids associated with maturation of jejunal morphology.

The uptake of a number of amino acids by the developing small intestine of the rat was investigated in vitro. L-valine, L-leucine, L-methionine, L-phenylalanine, L-arginine and L-lysine were all taken up by active transport and concentrated within the jejunal mucosa. GABA was not actively transported by the jejunum. The kinetics of carrier transport of amino acids was determined from birth to maturity. The Michaelis constant (Km) of the L-leucine, L-methionine, L-arginine and l-lysine transport systems was found to be low postnatally and increased with age, particularly after the time of weaning. The rate of l-leucine, L-methionine, L-phenylalanine and L-lysine transport (Vmax) was high postnatally but decreased after weaning. Neutral amino acids were transported at higher rates than basic amino acids. l-arginine was poorly transported by the jejunum. The specificity of transport systems for amino acids was investigated in inhibition studies. Amino acid transport systems appeared to be polyfunctional in the postnatal period but were more specific in post-weaned animals. The changes in kinetics and specificity of amino acid transport in the small intestine are discussed with reference to their possible functional significance and to the maturational changes in the jejunum, particularly with the appearance of a functionally distinct absorptive cell lining the intestinal villi during the third postnatal week (the time of weaning).     

Ornithine decarboxylase activity is inhibited by the polyamine precursor amino acids at the protein stability level in Caco-2 cells

High concentrations of certain amino acids are known to affect hormonal secretion, immune function, electrolyte balance or metabolic functions. However, there is a lack of knowledge regarding the molecular mechanisms responsible for these effects. We showed that, as well as spermidine transport, the activity of ornithine decarboxylase (ODC), the first and rate-limiting enzyme in polyamine biosynthesis, is decreased in human colon adenocarcinoma cells, Caco-2, following a 4-h supplementation with one of the two polyamine precursor amino acids, L-arginine or L-methionine. Dose-response assays indicated that the inhibitory effect of supplemental L-methionine was stronger than that of supplemental L-arginine. However, it was transient, being even replaced by ODC induction after 8 h, whereas the inhibitory effect of L-arginine lasted for at least 8 h. Unlike L-cysteine, neither L-methionine nor L-arginine could inhibit ODC activity in a crude acellular preparation of the enzyme. The inhibition of ODC activity in cells exposed to L-methionine or L-arginine was due to a decreased abundance of ODC protein without change at the mRNA level and each of these amino acids could counteract ODC induction by a glycine supplement. Contrary to the latter, supplemental L-methionine or L-arginine induced a marked decrease in ODC half-life, concomitantly with an increase in the activity of antizyme, an ODC inhibitory protein. Thus, depending on their nature, amino acids can up- or downregulate ODC activity at the protein stability level.     

Upregulation of interferon-induced indoleamine 2,3-dioxygenase in human macrophage cultures by lipopolysaccharide, muramyl tripeptide, and interleukin-1

The tryptophan decyclizing enzyme indoleamine 2,3-dioxygenase (IDO) was induced in human monocyte-derived macrophages (MDM) treated with human recombinant interferon-β (IFN-β) or interferon-γ (IFN-γ). Treated cells exhibited dose-dependent increases in IDO when assayed 48 hr after treatment. Cells exposed to IFN-γ were observed to exhibit consistently higher peak levels of IDO when compared with cells incubated in the presence of IFN-β. When IFN-β-treated cells were incubated in the presence of specified amounts of bacterial lipopolysaccharide (LPS) or liposome-encapsulated muramyl tripeptide (MTP), peak IDO activity increased such that enzyme activity was comparable to maximal activity observed with IFN-γ-treated cells. LPS and MTP also upregulated IFN-γ-mediated IDO activity when suboptimal amounts of IFN-γ were used. When macrophages were costimulated with various concentrations of human recombinant interleukin 1α (IL-1α), along with either maximum-stimulating amounts of IFN-β or suboptimal amounts of IFN-γ, IDO activity was upregulated in a manner similar to results obtained using the microbial products as stimuli. While neither IL-1α or IL-1β was detected in culture supernatants from macrophages treated with either LPS or MTP (alone or in combination with IFN), IL-1α was detected in cell lysates of macrophages treated with these upregulators. Although neutralizing antibody to IL-1α abolished the upregulatory effect of exogenous IL-1α, it had no effect on upregulation by LPS or MTP. This suggests that although LPS and MTP may induce production of cell-associated IL-1α, upregulation of IDO activity by these agents is independent of IL-1α production and may be mediated through distinct pathways.     

Redefining the PF06864 Pfam Family Based on Burkholderia pseudomallei PilO2Bp S-SAD Crystal Structure

Type IV pili are surface-exposed filaments and bacterial virulence factors, represented by the Tfpa and Tfpb types, which assemble via specific machineries. The Tfpb group is further divided into seven variants, linked to heterogeneity in the assembly machineries. Here we focus on PilO2_Bp, a protein component of the Tfpb R64 thin pilus variant assembly machinery from the pathogen Burkholderia pseudomallei. PilO2_Bp belongs to the PF06864 Pfam family, for which an improved definition is presented based on newly derived Hidden Markov Model (HMM) profiles. The 3D structure of the N-terminal domain of PilO2_Bp (N-PilO2_Bp), here reported, is the first structural representative of the PF06864 family. N-PilO2_Bp presents an actin-like ATPase fold that is shown to be present in BfpC, a different variant assembly protein; the new HMM profiles classify BfpC as a PF06864 member. Our results provide structural insight into the PF06864 family and on the Type IV pili assembly machinery.     

Crystal structure of SAM-dependent O-methyltransferase from pathogenic bacterium Leptospira interrogans

The S-adenosylmethionine (SAM)-dependent O-methyltransferase from Leptospira interrogans (LiOMT) expressed by gene LA0415 belongs to the Methyltransf_3 family (Pfam PF01596). In this family all of the five bacterial homologues with known function are reported as SAM-dependent O-methylstransferases involved in antibiotic production. The crystal structure of LiOMT in complex with S-adenosylhomocysteine reported here is the first bacterial protein structure in this family. The LiOMT structure shows a conserved SAM-binding region and a probable metal-dependent catalytic site. The molecules of LiOMT generate homodimers by N-terminal swapping, which assists the pre-organization of the substrate-binding site. Based on the sequence and structural analysis, it is implied by the catalytic and substrate-binding site that the substrate of LiOMT is a phenolic derivative, which probably has a large ring-shaped moiety.     

Influence of stearyl and trifluoromethylquinoline modifications of the cell penetrating peptide TP10 on its interaction with a lipid membrane

The PepFect family of cell-penetrating peptides (CPPs) was designed to improve the delivery of nucleic acids across plasma membranes. We present here a comparative study of two members of the family, PepFect3 (PF3) and PepFect6 (PF6), together with their parental CPP transportan-10 (TP10), and their interactions with lipid membranes. We show that the addition of a stearyl moiety to TP10 increases the amphipathicity of these molecules and their ability to insert into a lipid monolayer composed of zwitterionic phospholipids. The addition of negatively charged phospholipids into the monolayer results in decreased binding and insertion of the stearylated peptides, indicating modification in the balance of hydrophobic versus electrostatic interactions of peptides with lipid bilayer, thus revealing some clues for the selective interaction of these CPPs with different lipids. The trifluoromethylquinoline moieties, in PF6 make no significant contribution to membrane binding and insertion. TP10 actively introduces pores into the bilayers of large and giant unilamellar vesicles, while PF3 and PF6 do so only at higher concentrations. This is consistent with the lower toxicity of PF3 and PF6 observed in previous studies.     

Molecular cloning, sequencing and expression of human interferon-gamma-inducible indoleamine 2,3-dioxygenase cDNA

The antiproliferative action of human interferon (HuIFN)-gamma on human cells and the inhibition of intracellular pathogens, e.g. Toxoplasma gondii and Chlamydia psittaci, is at least in part due to an induction of indoleamine 2,3-dioxygenase (IDO) enzyme which degrades tryptophan, an essential amino acid. A cDNA clone (called C42) was isolated from a cDNA library made from poly(A)+ RNA obtained from HuIFN-gamma-treated human fibroblasts. Its nucleotide sequence revealed an open reading frame coding for a polypeptide of 403 amino acids, but no homology with any known gene in GenBank database was found. Evidence was obtained indicating that this cDNA codes for IDO: (i) Hybrid selected C42 specific poly(A)+ RNA from IFN-gamma-treated human cells coded for a polypeptide in vitro of approximately 42 kD (reported size of IDO, approximately 40 kD) which was immunoprecipitated by monoclonal anti-IDO antibody but not by a control antibody; and (ii) transfection of human fibroblasts with an expression plasmid containing C42 cDNA transcribed from chicken beta-actin promoter led to constitutive expression of C42 specific RNA as well as IDO activity. This cDNA clone will be useful in studying the role of IDO in the biological effects of IFN-gamma, and the regulation of IDO gene by IFN-gamma.     

Indoleamine 2,3-Dioxygenase Tissue Distribution and Cellular Localization in Mice: Implications for Its Biological Functions

Earlier studies have suggested that indoleamine 2,3-dioxygenase (IDO) has a wide tissue distribution in mammals. However, detailed information on its cellular localization and also the levels of expression in various tissues is still scarce. In the present study, we sought to determine the cellular localization of IDO and also to quantify the level of its expression in various mouse tissues by using the branched DNA signal amplification assay, Western blotting, and immunohistochemical staining. The highest levels of constitutive IDO expression were found to be selectively present in the caput of epididymis, except for its initial segment. IDO expression was also detected inside the luminal compartment and even in the stereocilia within this region. In the prostate, high levels of IDO were selectively expressed in the capsular cells. In addition, high levels of IDO expression were also selectively detected in certain types of cells in the placenta, spleen, thymus, lung, and digestive tract. Notably, the morphological features of most of the positively stained cells in these organs closely resembled those of antigen-presenting cells. Based on the tissue distribution and cellular localization characteristics of IDO, it is hypothesized that its expression may serve two main functions: one is to deplete tryptophan in an enclosed microenvironment (such as in the epididymal duct lumen) to prevent bacterial or viral infection, and the other is to produce bioactive tryptophan catabolites that would serve to suppress T-cell–mediated immune responses against self-antigens, fetal antigens, or allogeneic antigens, in different situations. (J Histochem Cytochem 58:17–28, 2010)     

1H NMR studies of substrate hydrogen exchange reactions catalyzed by L-methionine gamma-lyase

Hydrogen exchange reactions of various L-amino acids catalyzed by L-methionine gamma-lyase (EC 4.4.1.11) have been studied. The enzyme catalyzes the rapid exchange of the alpha- and beta-hydrogens of L-methionine and S-methyl-L-cysteine with deuterium from the solvent. The rate of alpha-hydrogen exchange was about 40 times faster than that of the enzymatic elimination reaction of the sulfur-containing amino acids. The enzyme also catalyzes the exchange reaction of alpha- and beta-hydrogens of the following straight-chain L-amino acids which are not susceptible to elimination: norleucine, norvaline, alpha-aminobutyrate, and alanine. The exchange rates of the alpha-hydrogen and the total beta-hydrogens of L-alanine and L-alpha-aminobutyrate with deuterium followed first-order kinetics. For L-norvaline, L-norleucine, S-methyl-L-cysteine, and L-methionine, the rate of alpha-hydrogen exchange followed first-order kinetics, but the rate of total beta-hydrogen exchange decreased due to a primary isotope effect at the alpha-position. One beta-hydrogen of S-methyl-L-cysteine was exchanged faster than the other, although both the beta-hydrogens were exchanged completely with deuterium ultimately. L-Phenylalanine and L-tryptophan slowly underwent alpha-hydrogen exchange. The pro-R hydrogen of glycine was deuterated stereospecifically. None of the following amino acids were susceptible to the enzymatic hydrogen exchange: D isomers of the above amino acids, branched chain L-amino acids, acidic L-amino acids, and basic L-amino acids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification and characterization of a putative active site for peptide methionine sulfoxide reductase (MsrA) and its substrate stereospecificity

Peptide methionine sulfoxide reductases (MsrA) from many different organisms share a consensus amino acid sequence (GCFWG) that could play an important role in their active site. Site-directed single substitution of each of these amino acids except glycines in the yeast MsrA resulted in total loss of enzyme activity. Nevertheless, all the recombinant MsrA mutants and native proteins had a very similar circular dichroism spectrum. The demonstration that either treatment with iodoacetamide or replacement of the motif cysteine with serine leads to inactivation of the enzyme underscores the singular importance of cysteine residues in the activity of MsrA. The recombinant yeast MsrA was used for general characterization of the enzyme. Its K(m) value was similar to the bovine MsrA and appreciably lower than the K(m) of the bacterial enzyme. Also, it was shown that the enzymatic activity increased dramatically with increasing ionic strength. The recombinant yeast MsrA activity and the reduction activity of free methionine sulfoxide(s) were stereoselective toward the L-methionine S-sulfoxide and S-methyl p-tolyl sulfoxide. It was established that a methionine auxotroph yeast strain could grow on either form of L-methionine sulfoxide.     

Ligation of B7-1/B7-2 by human CD4+ T cells triggers indoleamine 2,3-dioxygenase activity in dendritic cells

Human monocyte-derived dendritic cells (DCs) are capable of expressing the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO), which allows them to suppress Ag-driven proliferation of T cells in vitro. In DCs that express IDO, the activity of the enzyme is tightly regulated, with the protein being constitutively expressed, but functional activity requiring an additional set of triggering signals supplied during Ag presentation. We now show that triggering of functional IDO obligately requires ligation of B7-1/B7-2 molecules on the DCs by CTLA4/CD28 expressed on T cells. When this interaction was disrupted, IDO remained in the inactive state, and the DCs were unable to inhibit T cell proliferation. Inhibition could be fully restored by direct Ab-mediated cross-linking of B7-1/B7-2. Although both CD4(+) and CD8(+) T cells were susceptible to inhibition once IDO was induced, the ability to trigger functionally active IDO was strictly confined to the CD4(+) subset. Thus, the ability of CD4(+) T cells to induce IDO activity in DCs allowed the CD4(+) population to dominantly inhibit proliferation of the CD8(+) population via the bridge of a conditioned DC. We hypothesize that IDO activation via engagement of B7-1/B7-2 molecules on DCs, specifically, engagement by CTLA4 expressed on regulatory CD4(+) T cells, may function as a physiologic regulator of T cell responses in vivo.