Structural and functional properties of Bp-LAAO,a new l-amino acid oxidase isolated from Bothrops pauloensis snake venom

An l-amino acid oxidase (Bp-LAAO) from Bothrops pauloensis snake venom was highly purified using sequential chromatography steps on CM-Sepharose, Phenyl-Sepharose CL-4B, Benzamidine Sepharose and C18 reverse-phase HPLC. Purified Bp-LAAO showed to be a homodimeric acidic glycoprotein with molecular weight around 65 kDa under reducing conditions in SDS-PAGE. The best substrates for Bp-LAAO were l-Met, l-Leu, l-Phe and l-Ile and the enzyme showed a strong reduction of its catalytic activity upon l-Met and l-Phe substrates at extreme temperatures. Bp-LAAO showed leishmanicidal, antitumoral and bactericidal activities dose dependently. Bp-LAAO induced platelet aggregation in platelet-rich plasma and this activity was inhibited by catalase. Bp-LAAO-cDNA of 1548 bp codified a mature protein with 516 amino acid residues corresponding to a theoretical isoelectric point and molecular weight of 6.3 and 58 kDa, respectively. Additionally, structural and phylogenetic studies identified residues under positive selection and their probable location in Bp-LAAO and other snake venom LAAOs (svLAAOs). Structural and functional investigations of these enzymes can contribute to the advancement of toxinology and to the elaboration of novel therapeutic agents.     

Biochemical,functional and structural characterization of Akbu-LAAO: A novel snake venom l-amino acid oxidase from Agkistrodon blomhoffii ussurensis

An l-amino acid oxidase (Akbu-LAAO) was isolated from the venom of Agkistrodon blomhoffii ussurensis snake using DEAE Sephadex A-50 ion-exchange, Sephadex G-75 gel filtration, and high performance liquid chromatographies. The homogeneity and molecular mass of Akbu-LAAO were analyzed by SDS-PAGE and MALDI-TOF spectrometry. The sequences of ten peptides from Akbu-LAAO were established by HPLC-nESI-MS/MS analysis. Protein sequence alignment indicated that i) that Akbu-LAAO is a new snake venom LAAO, and ii) Akbu-LAAO shares homology with several LAAOs from the venoms of Calloselasma rhodost, Agkistrodon halys, Daboia russellii siamensis, and Trimeresurus stejnegeri. Akbu-LAAO is a homodimer with a molecular mass of ∼124.4 kDa. It reacts optimally with its enzymatic substrate, Leu, at pH 4.7 with a K_m of 2.1 mM. ICP-AES measurements showed that Akbu-LAAO contains four Zn²⁺ per dimer that are unessential for the hydrolytic activity of the enzyme. The emission fluorescence intensity of Akbu-LAAO decreases by 61% on removal of Zn²⁺ indicating that the zinc probably helps maintain the structural integrity of the enzyme. The addition of exogenous metal ions, including Mg²⁺, Mn²⁺, Ca²⁺, Ce³⁺, Nd³⁺, Co²⁺ and Tb³⁺, increases the l-Leu hydrolytic activity of the enzyme. Akbu-LAAO shows apparent anti-aggregation effects on human and rabbit platelets. It exhibits a strong bacteriostasis effect on Staphylococcus aureus, eighteen fold that of cephalosporin C under the same conditions. Taken together, the biochemical, proteomic, structural and functional characterizations reveal that Akbu-LAAO is a novel LAAO with promise for biotechnological and medical applications.     

Oral supplementation with a combination of l-citrulline and l-arginine rapidly increases plasma l-arginine concentration and enhances NO bioavailability

Background

Chronic supplementation with l-citrulline plus l-arginine has been shown to exhibit anti-atherosclerotic effects. However, the short-term action of this combination on the nitric oxide (NO)–cGMP pathway remains to be elucidated. The objective of the present study was to investigate the acute effects of a combination of oral l-citrulline and l-arginine on plasma l-arginine and NO levels, as well as on blood circulation.

Methods

Rats or New Zealand white rabbits were treated orally with l-citrulline, or l-arginine, or a combination of each at half dosage. Following supplementation, plasma levels of l-arginine, NO_x, cGMP and changes in blood circulation were determined sequentially.

Results

l-Citrulline plus l-arginine supplementation caused a more rapid increase in plasma l-arginine levels and marked enhancement of NO bioavailability, including plasma cGMP concentrations, than with dosage with the single amino acids. Blood flow in the central ear artery in rabbits was also significantly increased by l-citrulline plus l-arginine administration as compared with the control.

Conclusion

Our data show for the first time that a combination of oral l-citrulline and l-arginine effectively and rapidly augments NO-dependent responses at the acute stage. This approach may have clinical utility for the regulation of cardiovascular function in humans.     

Structure of N-acetyl-l-glutamate synthase/kinase from Maricaulis maris with the allosteric inhibitor l-arginine bound

Maricaulis maris N-acetylglutamate synthase/kinase (mmNAGS/K) catalyzes the first two steps in l-arginine biosynthesis and has a high degree of sequence and structural homology to human N-acetylglutamate synthase, a regulator of the urea cycle. The synthase activity of both mmNAGS/K and human NAGS are regulated by l-arginine, although l-arginine is an allosteric inhibitor of mmNAGS/K, but an activator of human NAGS. To investigate the mechanism of allosteric inhibition of mmNAGS/K by l-arginine, we have determined the structure of the mmNAGS/K complexed with l-arginine at 2.8 Å resolution. In contrast to the structure of mmNAGS/K in the absence of l-arginine where there are conformational differences between the four subunits in the asymmetric unit, all four subunits in the l-arginine liganded structure have very similar conformations. In this conformation, the AcCoA binding site in the N-acetyltransferase (NAT) domain is blocked by a loop from the amino acid kinase (AAK) domain, as a result of a domain rotation that occurs when l-arginine binds. This structural change provides an explanation for the allosteric inhibition of mmNAGS/K and related enzymes by l-arginine. The allosterically regulated mechanism for mmNAGS/K differs significantly from that for Neisseria gonorrhoeae NAGS (ngNAGS). To define the active site, several residues near the putative active site were mutated and their activities determined. These experiments identify roles for Lys356, Arg386, Asn391 and Tyr397 in the catalytic mechanism.     

Configuration of l-γ-ethylideneglutamic acid from Guilandina crista

Using an NMR shift reagent, it has been established that the configuration about the double bond in naturally occurring l-γ-ethylideneglutamic acid is cis.     

Isolation and identification of l-cystathionine and l-selenocystathionine from the foliage of Astragalus pectinatus

l-Cystathionine and l-selenocystathionine have been isolated from the foliage of Astragalus pectinatus. In addition to these two amino acids, some S-methylcysteine and trace amounts of Se-methyl-selenocysteine were also detected in the foliage extracts. The seeds of A pectinatus were found to contain significant amounts of all four of these amino acids plus the γ-glutamyl peptides of S-methylcysteine and Se-methylselenocysteine.     

l-Lactic acid production by Bacillus subtilis MUR1 in continuous culture

A novel UV-induced mutant strain of recombinant Bacillus subtilis MUR1 was used for the production of l-LA in continuous cultures with a variety of culture conditions. The maximal productivity of 17.6 g/L/h was obtained with a l-LA concentration of 44.1 g/L at the dilution rate of 0.4 h⁻¹. The highest concentration of l-LA (77.1 g/L) was produced at the dilution rate of 0.05 h⁻¹. This study showed that the maximum l-LA productivity of B. subtilis MUR1 which can only last for a very short period of time during the exponential phase in fed-batch cultures, can be extended indefinitely at steady state in continuous cultures. l-LA production increased with the increase of yeast extract concentrations in the medium. Moreover, temperature, agitation rate and various glucose concentrations in the feed were compared in continuous cultures. Different nitrogen sources (lysine, glutamine, ammonium sulphate and corn steep liquor) were studied to partly or completely replace yeast extract in the medium, most of them showed positive effects on l-LA production and cell growth. The l-LA productivities from continuous cultures in this study are higher than the productivity of current microbial industrial processes which use Lactobacillus to produce l-LA.     

Structure and Engineering of l-Arabinitol 4-Dehydrogenase from Neurospora crassa

l-Arabinitol 4-dehydrogenase (LAD) catalyzes the conversion of l-arabinitol into l-xylulose with concomitant NAD⁺ reduction. It is an essential enzyme in the development of recombinant organisms that convert l-arabinose into fuels and chemicals using the fungal l-arabinose catabolic pathway. Here we report the crystal structure of LAD from the filamentous fungus Neurospora crassa at 2.6 Å resolution. In addition, we created a number of site-directed variants of N. crassa LAD that are capable of utilizing NADP⁺ as cofactor, yielding the first example of LAD with an almost completely switched cofactor specificity. This work represents the first structural data on any LAD and provides a molecular basis for understanding the existing literature on the substrate specificity and cofactor specificity of this enzyme. The engineered LAD mutants with altered cofactor specificity should be useful for applications in industrial biotechnology.     

Formation of l-quebrachitol from d-bornesitol in leaves of Acer pseudoplatanus

d-Bornesitol and l-quebrachitol have been found in the leaves of Acer pseudoplatanus L. The results of incorporation studies using labeled myo-inositol-¹⁴C, l-inositol-¹⁴C and d-bornesitol-¹⁴C indicate that l-quebrachitol is produced by epimerization of d-bornesitol. In Artemisia vulgaris, however, the precursor of l-quebrachitol is l-inositol.     

Characterization of novel 2-oxoglutarate dependent dioxygenases converting l-proline to cis-4-hydroxy-l-proline

Hydroxyprolines are valuable chiral building blocks for organic synthesis of pharmaceuticals. Several microorganisms producing l-proline trans-4- and cis-3-hydroxylase were discovered and these enzymes were applied to the industrial production of trans-4- and cis-3-hydroxy-l-proline, respectively. Meanwhile, other hydroxyproline isomers, cis-4- and trans-3-hydroxy-l-proline, were not easily available because the corresponding hydroxylase have not been discovered. Herein we report novel l-proline cis-4-hydroxylases converting free l-proline to cis-4-hydroxy-l-proline. Two genes encoding uncharacterized proteins from Mesorhizobium loti and Sinorhizobium meliloti were cloned and overexpressed in Escherichia coli, respectively. The functions of purified proteins were investigated in detail, and consequently we detected l-proline cis-4-hydroxylase activity in both proteins. Likewise l-proline trans-4-, cis-3-hydroxylase and prolyl hydroxylase, these enzymes belonged to a 2-oxoglutarate dependent dioxygenase family and required a non-heme ferrous ion. Although their reaction mechanisms were similar to other hydroxylases, the amino acid sequence homology was not observed (less than 40%).     

X-ray structures of Bacillus pallidusd-arabinose isomerase and its complex with l-fucitol

d-Arabinose isomerase (d-AI), also known as l-fucose isomerase (l-FI), catalyzes the aldose–ketose isomerization of d-arabinose to d-ribulose, and l-fucose to l-fuculose. Bacillus pallidus (B. pallidus) d-AI can catalyze isomerization of d-altrose to d-psicose, as well as d-arabinose and l-fucose. Three X-ray structures of B. pallidusd-AI in complexes with 2-methyl-2,4-pentadiol, glycerol and an inhibitor, l-fucitol, were determined at resolutions of 1.77, 1.60 and 2.60 Å, respectively. B. pallidusd-AI forms a homo-hexamer, and one subunit has three domains of almost equal size; two Rossmann fold domains and a mimic of the (β/α) barrel fold domain. A catalytic metal ion (Mn²⁺) was found in the active site coordinated by Glu342, Asp366 and His532, and an additional metal ion was found at the channel for the passage of a substrate coordinated by Asp453. The X-ray structures basically supported the ene-diol mechanism for the aldose–ketose isomerization by B. pallidusd-AI, as well as Escherichia coli (E. coli) l-FI, in which Glu342 and Asp366 facing each other at the catalytic metal ion transfer a proton from C2 to C1 and O1 to O2, acting as acid/base catalysts, respectively. However, considering the ionized state of Asp366, the catalytic reaction also possibly occurs through the negatively charged ene-diolate intermediate stabilized by the catalytic metal ion. A structural comparison with E. colil-FI showed that B. pallidusd-AI possibly interconverts between “open” and “closed” forms, and that the additional metal ion found in B. pallidusd-AI may help to stabilize the channel region.     

Biological activity of l-2-azetidinecarboxylic acid,isolated from Polygonatum odoratum var. pluriflorum, against several algae

The biological activities of an aqueous fraction extracted from Polygonatum odoratum var. pluriflorum Owhi and of l-2-azetidinecarboxylic acid (AZC), purified from the extract, on the growth of several types of algae were tested. The aqueous fraction was prepared by methanol extraction of P. odoratum var. pluriflorum rhizomes followed by reverse partitioning with butanol. The aqueous extraction inhibited growth of the green alga Chlorella vulgaris by less than 10% at a concentration of 1000 mg L⁻¹. However, growth of the blue-green alga Microcystis aeruginosa was inhibited by 22.0%, 67.9%, and 87.1%, respectively, at 3.1, 6.2, and 12.5 mg extract L⁻¹. AZC was isolated from the aqueous extract and was shown to be the major active substance inhibiting algal growth. AZC concentrations higher than 25 μM inhibited growth, while at 400 μM, growth of the green algae C. vulgaris and Scenedesmus spp. was inhibited by 71.2% and 70.4%, respectively. In contrast, growth of the blue-green algae Anabaena affinis and M. aeruginosa was inhibited at concentrations greater than 1.6 and 0.2 μM, respectively, whereas 92% control required concentrations of 6.3 and 1.6 μM, respectively. AZC also suppressed the growth of the red-tide microalga Cochlodinium polykrikoides by 86.9% and 100% at concentrations of 6.3 and 12.5 μM, respectively. Azetidine and 2-azetidinone showed little activity on the tested algae. The results demonstrate that AZC selectively inhibits algal growth at low concentrations. The green algae C. vulgaris and Scenedesmus spp. were tolerant, whereas M. aeruginosa, A. affinis, and C. polykrikoides were relatively sensitive. Thus, extract and AZC, prepared from P. odoratum rhizomes, showed a potential as natural selective algicide for the control of harmful algae in laboratory assay.     

Structural Basis for Catalysis by the Mono- and Dimetalated Forms of the dapE-Encoded N-succinyl-l,l-Diaminopimelic Acid Desuccinylase

Biosynthesis of lysine and meso-diaminopimelic acid in bacteria provides essential components for protein synthesis and construction of the bacterial peptidoglycan cell wall. The dapE operon enzymes synthesize both meso-diaminopimelic acid and lysine and, therefore, represent potential targets for novel antibacterials. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase functions in a late step of the pathway and converts N-succinyl-l,l-diaminopimelic acid to l,l-diaminopimelic acid and succinate. Deletion of the dapE gene is lethal to Helicobacter pylori and Mycobacterium smegmatis, indicating that DapE's are essential for cell growth and proliferation. Since there are no similar pathways in humans, inhibitors that target DapE may have selective toxicity against only bacteria. A major limitation in developing antimicrobial agents that target DapE has been the lack of structural information. Herein, we report the high-resolution X-ray crystal structures of the DapE from Haemophilus influenzae with one and two zinc ions bound in the active site, respectively. These two forms show different activity. Based on these newly determined structures, we propose a revised catalytic mechanism of peptide bond cleavage by DapE enzymes. These structures provide important insight into catalytic mechanism of DapE enzymes as well as a structural foundation that is critical for the rational design of DapE inhibitors.     

Structural analysis of 1l-chiro-inositol diester from Taraxacumudum

1l-1,5-Di-O-p-hydroxyphenylacetyl-chiro-inositol was isolated from the leaves of Taraxacumudum, along with seven other secondary metabolites. Identification of the inositol derivative, based on extensive spectroscopic analyses (¹H, ¹³C and 2D NMR) in two solvents, allowed the correction of previously published data and conformational studies. This is the second report on the presence of inositol esters with p-hydroxyphenylacetic acid in plants.     

Protective role of S-adenosyl-l-methionine against hydrochloric acid stress in Saccharomyces cerevisiae

S-adenosyl-l-methionine (AdoMet, 1 mM) protects the stationary phase cells of Saccharomyces cerevisiae against the killing effect of acid (10 mM HCl, pH ∼ 2). Both the acid and the acid plus AdoMet treatment for 2 h increased the plasma membrane H⁺-ATPase activity; thereafter it decreased to the basal level. AdoMet partially recovered the intracellular pH (pH_in) that dropped in presence of acid. AdoMet treatment facilitated acid induced phospholipid biosynthesis as well as membrane proliferation, which was reflected in the cellular lipid composition.     

Lactose as an inexpensive starting material for the preparation of aldohexos-5-uloses: synthesis of l-ribo and d-lyxo derivatives

Partially protected derivatives of l-ribo- and d-lyxo-aldohexos-5-ulose have been prepared starting from triacetonlactose dimethyl acetal derivatives. Key steps of the synthetic sequences are (a) the synthesis of 4′-deoxy-4′-eno- and 6′-deoxy-5′-eno lactose derivatives, and (b) the epoxidation-methanolysis of the above-mentioned enol ethers to give 1,5-bis-glycopyranosides, masked form of the target 1,5-dicarbonyl hexoses.     

FERM Domain of Moesin Desorbs the Basic-Rich Cytoplasmic Domain of l-Selectin from the Anionic Membrane Surface

Moesin and calmodulin (CaM) jointly associate with the cytoplasmic domain of l-selectin in the cell to modulate the function and ectodomain shedding of l-selectin. Using fluorescence spectroscopy, we have examined the association of moesin FERM domain with the recombinant transmembrane and cytoplasmic domains of l-selectin (CLS) reconstituted in model phospholipid liposomes. The dissociation constant of moesin FERM domain to CLS in the phosphatidylcholine liposome is about 300 nM. In contrast to disrupting the CaM association with CLS, inclusion of anionic phosphatidylserine lipids in the phosphatidylcholine liposome increased the apparent binding affinity of moesin FERM domain for CLS. Using the environmentally sensitive fluorescent probe attached to the cytoplasmic domain of CLS and the nitroxide quencher attached to the lipid bilayer, we showed that the association of moesin FERM domain induced the desorption of the basic-rich cytoplasmic domain of CLS from the anionic membrane surface, which enabled subsequent association of CaM to the cytoplasmic domain of CLS. These results have elucidated the molecular basis for the moesin/l-selectin/CaM ternary complex and suggested an important role of phospholipids in modulating l-selectin function and shedding.     

Mass spectrometric and kinetic studies on slow progression of papain-catalyzed polymerization of l-glutamic acid diethyl ester

Papain polymerizes l-glutamic acid diethyl ester (Glu-di-OEt) regioselectively, resulting in the formation of poly (γ-ethyl α-l-glutamic acid) with various degrees of polymerization of less than 13. Reaction temperatures below 20 °C were appropriate for the reaction in terms of suppression of non-enzymatic degradation of Glu-di-OEt and an increase in the peptide yield, while the reaction was preceded by a pronounced induction period. Mass spectrometric analyses of the reaction conducted at 0 °C revealed that the accumulation of the initial dimerization product, l-glutamyl-l-glutamic acid triethyl ester (Glu-Glu-tri-OEt), was limited during the induction period, and that a sequential polymer derived from a further elongation of the dimer was the tetramer, but not the trimer. Kinetic analyses of acyl transfer reactions with Glu-di-OEt and Glu-Glu-tri-OEt as acyl acceptors and Nα-benzoyl-l-arginine ethyl ester as an acyl donor affirmed that Glu-Glu-tri-OEt bound more strongly than Glu-di-OEt both to the S- and S′-subsites of papain. Therefore, what occurred during the initial stage of the polymerization was interpreted as follows: the rate of the papain-catalyzed dimerization of Glu-di-OEt was extremely slow, once Glu-Glu-tri-OEt was initially synthesized it exclusively bound to the active site of papain, and then papain utilized the dimer in polymerization effectively rather than the monomer.     

l-Glyceraldehyde 3-phosphate reductase from Escherichia coli is a heme binding protein

Recently, we reported that YghZ from Escherichia coli functions as an efficient l-glyceraldehyde 3-phosphate reductase (Gpr). Here we show that Gpr co-purifies with a b-type heme cofactor. Gpr associates with heme in a 1:1 stoichiometry to form a complex that is characterized by a K_d value of 5.8 ± 0.2 μM in the absence of NADPH and a K_d value of 11 ± 1.3 μM in the presence of saturating NADPH. The absorbance spectrum of reconstituted Gpr indicates that heme is bound in a hexacoordinate low-spin state under both oxidizing and reducing conditions. The physiological function of heme association with Gpr is unclear, as the l-glyceraldehyde 3-phosphate reductase activity of Gpr does not require the presence of the cofactor. Bioinformatics analysis reveals that Gpr clusters with a family of putative monooxygenases in several organisms, suggesting that Gpr may act as a heme-dependent monooxygenase. The discovery that Gpr associates with heme is interesting because Gpr shares 35% amino acid identity with the mammalian voltage-gated K⁺ channel β-subunit, an NADPH-dependent oxidoreductase that endows certain voltage-gated K⁺ channels with hemoprotein-like, O₂-sensing properties. To date the molecular origin of O₂ sensing by voltage-gated K⁺ channels is unknown and the results presented herein suggest a role for heme in this process.     

Expression of recombinant human antibody fragments capable of inhibiting the phospholipase and myotoxic activities of Bothrops jararacussu venom

Phospholipases A₂ are components of Bothrops venoms responsible for disruption of cell membrane integrity via hydrolysis of its phospholipids. This study used a large nonimmune human scFv library named Griffin.1 (MRC, Cambridge, UK) for selection of recombinant antibodies against antigens present in Bothrops jararacussu venom and identification of specific antibodies able to inhibit phospholipase activity. Four clones were identified as capable of inhibiting this activity in vitro. These clones were able to reduce in vivo the myotoxic activity of BthTX-I and BthTX-II PLA₂, but had no effect on the in vitro anticoagulant activity of BthTX-II. This work shows the potential of using recombinant scFv libraries in the search for antibodies that neutralize relevant venom components.