L-asparaginase genes in Escherichia coli: isolation of mutants and characterization of the ansA gene and its protein product

Mutants of Escherichia coli have been isolated which are resistant to beta-aspartyl hydroxamate, a lethal substrate of asparaginase II in fungi and a substrate for asparaginase II in E. coli. Among the many phenotypic classes observed, a single mutant (designated GU16) was found with multiple defects affecting asparaginases I and II and aspartase. Other asparaginase II-deficient mutants have also been derived from an asparaginase I-deficient mutant. The mutant strain, GU16, was unable to utilize asparagine and grew poorly on aspartate as the sole source of carbon; transformation of this strain with an E. coli recombinant plasmid library resulted in a large recombinant plasmid which complemented both these defects. Two subclones were isolated, designated pDK1 and pDK2; the former complemented the partial defect in the utilization of aspartate, although its exact function was not established. pDK2 encoded the asparaginase I gene (ansA), the coding region of which was further defined within a 1.7-kilobase fragment. The ansA gene specified a polypeptide, identified in maxicells, with a molecular weight of 43,000. Strains carrying recombinant plasmids encoding the ansA gene overproduced asparaginase I approximately 130-fold, suggesting that the ansA gene might normally be under negative regulation. Extracts from strains overproducing asparaginase I were electrophoresed, blotted, and probed with asparaginase II-specific antisera; no cross-reaction of the antisera with asparaginase I was observed, indicating that asparaginases I and II are not appreciably related immunologically. When a DNA fragment containing the ansA gene was used to probe Southern blots of restriction endonuclease-digested E. coli chromosomal DNA, no homologous sequences were revealed other than the expected ansA-containing fragments. Therefore, the genes encoding asparaginases I and II are highly sequence related.     

Cloning and nucleotide sequence of the Bacillus subtilis ansR gene, which encodes a repressor of the ans operon coding for L-asparaginase and L-aspartase.

Previous work has shown that expression of the Bacillus subtilis ans operon which codes for L-asparaginase and L-aspartase, is both increased and made insensitive to repression by NH4+ by the aspH1 mutation. In current work, the gene in which the aspH1 mutation resides has been identified and sequenced; this gene, termed ansR, is immediately upstream of, but transcribed in the opposite direction from, the ans operon. The promoter region of ansR contains -10 and -35 sequences similar to those recognized by RNA polymerase containing the major vegetative-cell sigma factor sigma A, and ansR appears to be monocistronic. The ansR gene codes for a 116-residue protein, but the aspH1 mutant allele has an additional guanine residue at codon 55, resulting in generation of a truncated polypeptide of only 58 residues. Insertional inactivation of ansR resulted in a phenotype identical to that of the aspH1 mutant. The predicted amino acid sequence of the ansR gene product (AnsR) was homologous to that of the repressor of B. subtilis prophage PBSX, and a helix-turn-helix motif, characteristic of many DNA-binding proteins, was present in the AnsR amino-terminal region. These results suggest that ansR codes for a repressor of the ans operon.     

Isolation and characterization of Rhizobium etli mutants altered in degradation of asparagine.

Rhizobium etli mutants unable to grow on asparagine as the nitrogen and carbon source were isolated. Two kinds of mutants were obtained: AHZ1, with very low levels of aspartase activity, and AHZ7, with low levels of asparaginase and very low levels of aspartase compared to the wild-type strain. R. etli had two asparaginases differentiated by their thermostabilities, electrophoretic mobilities, and modes of regulation. The AHZ mutants nodulated as did the wild-type strain and had nitrogenase levels similar to that of the wild-type strain.     

Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10?3 M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s?1, and 1.2 +/- 0.105 × 10? M?1s?1, respectively. The L-asparaginase activity was reduced in the presence of Mn2?, Zn2?, Ca2?, and Mg2? metal ions for about 52% to 31%. In addition, we found that NH??, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.     

The Rhizobium etli opt operon is required for symbiosis and stress resistance

Rhizobium etli is a Gram-negative root-colonizing soil bacterium capable of fixing nitrogen while living in symbiosis with its leguminous host Phaseolus vulgaris. A genome-wide screening for R. etli symbiotic mutants revealed a R. etli operon encoding an oligopeptide ABC-transporter (Opt), two redA homologous genes and one redB gene. Expression analysis showed this opt operon to be transcribed both under free-living and symbiotic conditions and expression levels were demonstrated to be growth-phase-dependent. Plants nodulated by R. etli opt mutants showed a reduced symbiotic nitrogen fixation activity (approximately 50% reduction). Growth experiments with opt mutants in the presence of oligopeptides as the sole nitrogen source confirmed the involvement of the opt genes in oligopeptide uptake. Further phenotypic analysis of the opt mutants revealed them to display an enhanced resistance to the oligopeptide antibiotic bacitracin, an increased susceptibility to the beta-lactam antibiotic ampicillin and a decreased osmotolerance. In conclusion, our results demonstrate that the opt operon plays a crucial role during symbiosis and stress resistance.     

L-Asparaginase activity in Leptosphaeria michotii. Isolation and properties of two forms of the enzyme

The properties of L-asparaginase (EC 3.5.1.1) in Leptosphaeria michotii (West) Sacc., which has previously been shown to have an activity rhythm, were analyzed. Two forms of L-asparaginase were isolated from acetic acid and ammonium sulfate fractionations followed by DEAE-Sephacel chromatography. The activity of L-asparaginase changed rhythmically with the same period as that of crude extracts, but the rhythms of the two enzyme forms were out of phase. The two asparaginase forms differed in their isoelectric points and the substrate concentrations for attaining half-maximal velocity; non-Michaelis-Menten kinetics for hydrolysis of L-asparagine were observed. Analyses of asparaginase form II by polyacrylamide gel electrophoresis showed that four proteins, irrespective of the phase of the activity rhythm at which the enzyme was extracted, could be detected: asparaginase oligomer (M_r 130 000 to 140 000), its dimer, an aggregate (M_r 500 000 to 600 000) having a low asparaginase activity, and a protein (M_r 60 000) without asparaginase activity; the same proteins were found in asparaginase form I. These results indicate that L. michotii asparaginase could be implicated in a protein complex.     

The first characterized asparaginase from a basidiomycete, Flammulina velutipes

Flammulina velutipes enjoys high popularity as an edible mushroom in Asian cuisines. Investigating the secretion of peptidases in nutrient media enriched with gluten, an enzyme was noticed that catalyzed the deamidation of L-asparagine and L-glutamine. The enzyme was purified to electrophoretic homogeneity by foaming and SEC. PAGE analysis revealed a protein of about 85 kDa with 13 kDa subunits indicating a hexameric protein. Degenerated primers were deduced from peptide fragments and the complete coding sequence of 372 bp was determined. The gene of Flammulina velutipes asparaginase (FvNase) over expressed in E. coli achieved an L-asparagine-hydrolyzing activity of 16 U/mL in crude extract, which was five times higher than its L-glutamine-hydrolyzing ability. The enzyme showed a pH-optimum at pH 7, remarkable tolerance towards elevated temperature and sodium chloride concentration in both the native and recombinant form, and no significant homology to any conserved domains of published asparaginases or glutaminases.     

Arabidopsis mutants lacking asparaginases develop normally but exhibit enhanced root inhibition by exogenous asparagine

Asparaginase catalyzes the degradation of L-asparagine to L-aspartic acid and ammonia, and is implicated in the catabolism of transported asparagine in sink tissues of higher plants. The Arabidopsis genome includes two genes, ASPGA1 and ASPGB1, belonging to distinct asparaginase subfamilies. Conditions of severe nitrogen limitation resulted in a slight decrease in seed size in wild-type Arabidopsis. However, this response was not observed in a homozygous T-DNA insertion mutant where ASPG genes had been inactivated. Under nitrogen-sufficient conditions, the ASPG mutant had elevated levels of free asparagine in mature seed. This phenotype was observed exclusively under conditions of low illumination, when a low ratio of carbon to nitrogen was translocated to the seed. Mutants deficient in one or both asparaginases were more sensitive than wild-type to inhibition of primary root elongation and root hair emergence by L-asparagine as a single nitrogen source. This enhanced inhibition was associated with increased accumulation of asparagine in the root of the double aspga1-1/-b1-1 mutant. This indicates that inhibition of root growth is likely elicited by asparagine itself or an asparagine-derived metabolite, other than the products of asparaginase, aspartic acid or ammonia. During germination, a fusion between the ASPGA1 promoter and beta-glucuronidase was expressed in endosperm cells starting at the micropylar end. Expression was initially high throughout the root and hypocotyl, but became restricted to the root tip after three days, which may indicate a transition to nitrogen-heterotrophic growth.     

Diminished redundancy of outer membrane factor proteins in rhizobiales: a nodT homolog is essential for free-living Rhizobium etli

Rhizobium etli is a gram-negative soil bacterium that induces nitrogen-fixing nodules on common bean roots (Phaseolus vulgaris). R. etli encodes two genes homologous to nodT of Rhizobium leguminosarum. nodTch is chromosomal and forms an operon with new genes resembling a multi-drug efflux pump of the resistance-nodulation-cell division (RND) family. nodTch is the last gene of this operon and can also be independently transcribed; the gene product is located in the bacterial outer membrane. Cell survival requires nodTch under all conditions tested. A second nodT gene, nodTpc, is encoded by plasmid c; it is constitutively transcribed but does not complement the essential function encoded by nodTch. NodT proteins belong to the outer membrane efflux proteins of the TolC superfamily. The number of duplications in the tolC gene family positively correlates with genome size in gram-negative bacteria. Nonetheless, some alpha-proteobacteria, including R. etli, encode fewer outer membrane factor exporters than expected suggesting further roles in addition to detoxification.     

Cloning, sequence analysis, and expression of ansB from Pseudomonas fluorescens, encoding periplasmic glutaminase/asparaginase

A gene (ansB) encoding a class II glutaminase/asparaginase has been cloned from Pseudomonas fluorescens and characterized by DNA sequencing, promoter analysis and heterologous expression in Escherichia coli. We show that ansB is monocistronic and depends on the alternate sigma factor sigma 54 for expression. A second open reading frame located downstream of ansB is highly homologous to a number of bacterial genes that encode secreted endonucleases of unknown function.     

The Rhizobium etli gene iscN is highly expressed in bacteroids and required for nitrogen fixation

Sequence analysis of the rpoN (2)- fixA intergenic region in the genome of Rhizobium etli CNPAF512 has uncovered three genes involved in nitrogen fixation, namely nifU, nifS and nifW. These genes are preceded by an ORF that is highly conserved among nitrogen-fixing bacteria. It encodes a putative gene product of 105 amino acids, belonging to the HesB-like protein family. A phylogenetic analysis of members of the HesB-like protein family showed that the R. etli HesB-like protein clusters with polypeptides encoded by ORFs situated upstream of the nifUS nitrogen fixation regions in the genomes of other diazotrophs. The R. etli ORF that encodes the HesB-like protein was designated iscN. iscN is co-transcribed with nifU and nifS, and is preferentially expressed under free-living microaerobic conditions and in bacteroids. Expression is regulated by the alternative sigma factor RpoN and the enchancer-binding protein NifA. A R. etli iscN mutant displays a reduction in nitrogen fixation capacity of 90% compared to the wild-type strain. This Nif(-) phenotype could be complemented by the introduction of intact copies of R. etli iscN.     

Structural and functional insights into Erwinia carotovora L-asparaginase

Bacterial L-asparaginases are enzymes that catalyze the hydrolysis of l-asparagine to aspartic acid. For the past 30 years, these enzymes have been used as therapeutic agents in the treatment of acute childhood lymphoblastic leukemia. Their intrinsic low-rate glutaminase activity, however, causes serious side-effects, including neurotoxicity, hepatitis, coagulopathy, and other dysfunctions. Erwinia carotovora asparaginase shows decreased glutaminase activity, so it is believed to have fewer side-effects in leukemia therapy. To gain detailed insights into the properties of E. carotovora asparaginase, combined crystallographic, thermal stability and cytotoxic experiments were performed. The crystal structure of E. carotovoral-asparaginase in the presence of L-Asp was determined at 2.5 A resolution and refined to an R cryst of 19.2 (R free = 26.6%) with good stereochemistry. Cytotoxicity measurements revealed that E. carotovora asparaginase is 30 times less toxic than the Escherichia coli enzyme against human leukemia cell lines. Moreover, denaturing experiments showed that E. carotovora asparaginase has decreased thermodynamic stability as compared to the E. coli enzyme and is rapidly inactivated in the presence of urea. On the basis of these results, we propose that E. carotovora asparaginase has limited potential as an antileukemic drug, despite its promising low glutaminase activity. Our analysis may be applicable to the therapeutic evaluation of other asparaginases as well.     

Regulation and symbiotic role of nirK and norC expression in Rhizobium etli

Rhizobium etli CFN42 is unable to use nitrate for respiration and lacks nitrate reductase activity as well as the nap or nar genes encoding respiratory nitrate reductase. However, genes encoding proteins closely related to denitrification enzymes, the norCBQD gene cluster and a novel nirKnirVnnrRnnrU operon are located on pCFN42f. In this study, we carried out a genetic and functional characterization of the reductases encoded by the R. etli nirK and norCB genes. By gene fusion expression analysis in free-living conditions, we determined that R. etli regulates its response to nitric oxide through NnrR via the microaerobic expression mediated by FixKf. Interestingly, expression of the norC and nirK genes displays a different level of dependence for NnrR. A null mutation in nnrR causes a drastic drop in the expression of norC, while nirK still exhibits significant expression. A thorough analysis of the nirK regulatory region revealed that this gene is under both positive and negative regulation. Functional analysis carried out in this work demonstrated that reduction of nitrite and nitric oxide in R. etli requires the reductase activities encoded by the norCBQD and nirK genes. Levels of nitrosylleghemoglobin complexes in bean plants exposed to nitrate are increased in a norC mutant but decreased in a nirK mutant. The nitrate-induced decline in nitrogenase-specific activity observed in both the wild type and the norC mutant was not detected in the nirK mutant. This data indicate that bacterial nitrite reductase is an important contributor to the formation of NO in bean nodules in response to nitrate.