Computational Elucidation of Phylogenetic, Functional and Structural Features of Methioninase from Pseudomonas, Escherichia, Clostridium and Citrobacter Strains.

BACKGROUND: L-Methioninase (EC 4.4.1.11; MGL) is a pyridoxal phosphate (PLP)-dependent enzyme that is produced by a variety of bacteria, fungi, and plants. L-methioninase, especially from Pseudomonas and Citrobacter sp., is considered as the efficient therapeutic enzyme, particularly in cancers such as glioblastomas, medulloblastoma, and neuroblastoma that are more sensitive to methionine starvation. OBJECTIVE: The low stability is one of the main drawbacks of the enzyme; in this regard, in the current study, different features of the enzyme, including phylogenetic, functional, and structural from Pseudomonas, Escherichia, Clostridium, and Citrobacter strains were evaluated to find the best bacterial L-Methioninase. METHODS: After the initial screening of L-Methioninase sequences from the above-mentioned bacterial strains, the three-dimensional structures of enzymes from Escherichia fergusonii, Pseudomonas fluorescens, and Clostridium homopropionicum were determined through homology modeling via GalaxyTBM server and refined by GalaxyRefine server. RESULTS AND CONCLUSION: Afterwards, PROCHECK, verify 3D, and ERRAT servers were used for verification of the obtained models. Moreover, antigenicity, allergenicity, and physico-chemical analysis of enzymes were also carried out. In order to get insight into the interaction of the enzyme with other proteins, the STRING server was used. The secondary structure of the enzyme is mainly composed of random coils and alpha-helices. However, these outcomes should further be validated by wet-lab investigations.

Development of a Biosensor for Crotonobetaine-CoA Ligase Screening Based on the Elucidation of Escherichia coli Carnitine Metabolism.

l-Carnitine is essential in the intermediary metabolism of eukaryotes and is involved in the ß-oxidation of medium- and long-chain fatty acids; thus, it has applications for medicinal purposes and as a dietary supplement. In addition, l-carnitine plays roles in bacterial physiology and metabolism, which have been exploited by the industry to develop biotechnological carnitine production processes. Here, on the basis of studies of l-carnitine metabolism in Escherichia coli and its activation by the transcriptional activator CaiF, a biosensor was developed. It expresses a fluorescent reporter gene that responds in a dose-dependent manner to crotonobetainyl-CoA, which is an intermediate of l-carnitine metabolism in E. coli and is proposed to be a coactivator of CaiF. Moreover, a dual-input biosensor for l-carnitine and crotonobetaine was developed. As an application of the biosensor, potential homologues of the betaine:CoA ligase CaiC from Citrobacter freundii, Proteus mirabilis, and Arcobacter marinus were screened and shown to be functionally active CaiC variants. These variants and the developed biosensor may be valuable for improving l-carnitine production processes.

Exploring the substrate specificity of Cytochrome P450cin.

Cytochromes P450 are enzymes that catalyse the oxidation of a wide variety of compounds that range from small volatile compounds, such as monoterpenes to larger compounds like steroids. These enzymes can be modified to selectively oxidise substrates of interest, thereby making them attractive for applications in the biotechnology industry. In this study, we screened a small library of terpenes and terpenoid compounds against P450cin and two P450cin mutants, N242A and N242T, that have previously been shown to affect selectivity. Initial screening indicated that P450cin could catalyse the oxidation of most of the monoterpenes tested; however, sesquiterpenes were not substrates for this enzyme or the N242A mutant. Additionally, both P450cin mutants were found to be able to oxidise other bicyclic monoterpenes. For example, the oxidation of (R)- and (S)-camphor by N242T favoured the production of 5-endo-hydroxycamphor (65-77% of the total products, dependent on the enantiomer), which was similar to that previously observed for (R)-camphor with N242A (73%). Selectivity was also observed for both (R)- and (S)-limonene where N242A predominantly produced the cis-limonene 1,2-epoxide (80% of the products following (R)-limonene oxidation) as compared to P450cin (23% of the total products with (R)-limonene). Of the three enzymes screened, only P450cin was observed to catalyse the oxidation of the aromatic terpene p-cymene. All six possible hydroxylation products were generated from an in vivo expression system catalysing the oxidation of p-cymene and were assigned based on 1H NMR and GC-MS fragmentation patterns. Overall, these results have provided the foundation for pursuing new P450cin mutants that can selectively oxidise various monoterpenes for biocatalytic applications.

Glyceraldehyde-3-phosphate dehydrogenase from Citrobacter sp. S-77 is post-translationally modified by CoA (protein CoAlation) under oxidative stress.

Protein CoAlation (S-thiolation by coenzyme A) has recently emerged as an alternative redox-regulated post-translational modification by which protein thiols are covalently modified with coenzyme A (CoA). However, little is known about the role and mechanism of this post-translational modification. In the present study, we investigated CoAlation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from a facultative anaerobic Gram-negative bacterium Citrobacter sp. S-77 (Cb GAPDH). GAPDH is a key glycolytic enzyme whose activity relies on the thiol-based redox-regulated post-translational modifications of active-site cysteine. LC-MS/MS analysis revealed that CoAlation of Cb GAPDH occurred in vivo under sodium hypochlorite (NaOCl) stress. The purified Cb GAPDH was highly sensitive to overoxidation by H2O2 and NaOCl, which resulted in irreversible enzyme inactivation. By contrast, treatment with coenzyme A disulphide (CoASSCoA) or H2O2/NaOCl in the presence of CoA led to CoAlation and inactivation of the enzyme; activity could be recovered after incubation with dithiothreitol, glutathione and CoA. CoAlation of the enzyme in vitro was confirmed by mass spectrometry. The presence of a substrate, glyceraldehyde-3-phosphate, fully protected Cb GAPDH from inactivation by CoAlation, suggesting that the inactivation is due to the formation of mixed disulphides between CoA and the active-site cysteine Cys149. A molecular docking study also supported the formation of mixed disulphide without steric constraints. These observations suggest that CoAlation is an alternative mechanism to protect the redox-sensitive thiol (Cys149) of Cb GAPDH against irreversible oxidation, thereby regulating enzyme activity under oxidative stress.

Efficient expression and characterization of a cold-active endo-1, 4-β -glucanase from Citrobacter farmeri by co-expression of Myxococcus xanthus protein S

Background: Cold-active endo-1, 4-β-glucanase (EglC) can decrease energy costs and prevent product denaturation in biotechnological processes. However, the nature EglC from C. farmeri A1 showed very low activity (800 U/L). In an attempt to increase its expression level, C. farmeri EglC was expressed in Escherichia coli as an N-terminal fusion to protein S (ProS) from Myxococcus xanthus. Results: A novel expression vector, pET(ProS-EglC), was successfully constructed for the expression of C. farmeri EglC in E. coli. SDS-PAGE showed that the recombinant protein (ProS-EglC) was approximately 60 kDa. The activity of ProS-EglC was 12,400 U/L, which was considerably higher than that of the nature EglC (800 U/L). ProS-EglC was active at pH 6.5-pH 8.0, with optimum activity at pH 7.0. The recombinant protein was stable at pH 3.5-pH 6.5 for 30 min. The optimal temperature for activity of ProS-EglC was 30°C-40°C. It showed greater than 50% of maximum activity even at 5°C, indicating that the ProS-EglC is a cold-active enzyme. Its activity was increased by Co2+ and Fe2+, but decreased by Cd2+, Zn2+, Li+, methanol, Triton-X-100, acetonitrile, Tween 80, and SDS. Conclusions: The ProS-EglC is promising in application of various biotechnological processes because of its cold-active characterizations. This study also suggests a useful strategy for the expression of foreign proteins in E. coli using a ProS tag.

CYP101J2, CYP101J3, and CYP101J4, 1,8-Cineole-Hydroxylating Cytochrome P450 Monooxygenases from Sphingobium yanoikuyae Strain B2.

We report the isolation and characterization of three new cytochrome P450 monooxygenases: CYP101J2, CYP101J3, and CYP101J4. These P450s were derived from Sphingobium yanoikuyae B2, a strain that was isolated from activated sludge based on its ability to fully mineralize 1,8-cineole. Genome sequencing of this strain in combination with purification of native 1,8-cineole-binding proteins enabled identification of 1,8-cineole-binding P450s. The P450 enzymes were cloned, heterologously expressed (N-terminally His6 tagged) in Escherichia coli BL21(DE3), purified, and spectroscopically characterized. Recombinant whole-cell biotransformation in E. coli demonstrated that all three P450s hydroxylate 1,8-cineole using electron transport partners from E. coli to yield a product putatively identified as (1S)-2α-hydroxy-1,8-cineole or (1R)-6α-hydroxy-1,8-cineole. The new P450s belong to the CYP101 family and share 47% and 44% identity with other 1,8-cineole-hydroxylating members found in Novosphingobium aromaticivorans and Pseudomonas putida Compared to P450cin (CYP176A1), a 1,8-cineole-hydroxylating P450 from Citrobacter braakii, these enzymes share less than 30% amino acid sequence identity and hydroxylate 1,8-cineole in a different orientation. Expansion of the enzyme toolbox for modification of 1,8-cineole creates a starting point for use of hydroxylated derivatives in a range of industrial applications. IMPORTANCE: CYP101J2, CYP101J3, and CYP101J4 are cytochrome P450 monooxygenases from S. yanoikuyae B2 that hydroxylate the monoterpenoid 1,8-cineole. These enzymes not only play an important role in microbial degradation of this plant-based chemical but also provide an interesting route to synthesize oxygenated 1,8-cineole derivatives for applications as natural flavor and fragrance precursors or incorporation into polymers. The P450 cytochromes also provide an interesting basis from which to compare other enzymes with a similar function and expand the CYP101 family. This could eventually provide enough bacterial parental enzymes with similar amino acid sequences to enable in vitro evolution via DNA shuffling.

Biochemical characterization of a bifunctional acetaldehyde-alcohol dehydrogenase purified from a facultative anaerobic bacterium Citrobacter sp. S-77.

Acetaldehyde-alcohol dehydrogenase (ADHE) is a bifunctional enzyme consisting of two domains of an N-terminal acetaldehyde dehydrogenase (ALDH) and a C-terminal alcohol dehydrogenase (ADH). The enzyme is known to be important in the cellular alcohol metabolism. However, the role of coenzyme A-acylating ADHE responsible for ethanol production from acetyl-CoA remains uncertain. Here, we present the purification and biochemical characterization of an ADHE from Citrobacter sp. S-77 (ADHE(S77)). Interestingly, the ADHE(S77) was unable to be solubilized from membrane with detergents either 1% Triton X-100 or 1% Sulfobetaine 3-12. However, the enzyme was easily dissociated from membrane by high-salt buffers containing either 1.0 M NaCl or (NH(4))(2)SO(4) without detergents. The molecular weight of a native protein was estimated as approximately 400 kDa, consisting of four identical subunits of 96.3 kDa. Based on the specific activity and kinetic analysis, the ADHES77 tended to have catalytic reaction towards acetaldehyde elimination rather than acetaldehyde formation. Our experimental observation suggests that the ADHES77 may play a pivotal role in modulating intracellular acetaldehyde concentration.

P450cin active site water: implications for substrate binding and solvent accessibility.

In P450cin, Tyr81, Asp241, Asn242, two water molecules, and the substrate participate in a complex H-bonded network. The role of this H-bonded network in substrate binding and catalysis has been probed by crystallography, spectroscopy, kinetics, isothermal titration calorimetry (ITC), and molecular dynamics. For the Y81F mutant, the substrate binds about 20-fold more weakly and Vmax decreases by about 30% in comparison to WT. The enhanced susceptibility of the heme to H2O2-mediated destruction in Y81F suggests that this mutant favors the open, low-spin conformational state. Asn242 H-bonds directly with the substrate, and replacing this residue with Ala results in water taking the place of the missing Asn side chain. This mutant exhibits a 70% decrease in activity. Crystal structures and molecular dynamics simulations of substrate-bound complexes show that the solvent has more ready access to the active site, especially for the N242A mutant. This accounts for about a 64% uncoupling of electron transfer from substrate hydroxylation. These data indicate the importance of the interconnected water network on substrate binding and on the open/closed conformational equilibrium, which are both critically important for maintaining high-coupling efficiency.

Oxygen activation by P450(cin): Protein and substrate mutagenesis.

A conserved threonine found in the majority of cytochromes P450 (P450s) has been implicated in the activation of dioxygen during the catalytic cycle. P450(cin) (CYP176A) has been found to be an exception to this paradigm, where the conserved threonine has been replaced with an asparagine. Prior studies with a P450(cin) N242A mutant established that the Asn-242 was not a functional replacement for the conserved threonine but was essential for the regio- and stereocontrol of the oxidation of cineole. To explore further how P450(cin) controls the activation of the dioxygen in the absence of the conserved threonine, two concurrent lines of investigation were followed. Modification of P450(cin) indicated that the Thr-243 was not involved in controlling the protonation of the hydroperoxy species. In addition, the N242T mutant did not enhance the rate and/or efficiency of catalytic turnover of cineole by P450(cin). In parallel experiments, the substrate cineole was modified by removing the ethereal oxygen to produce camphane or 2,2-dimethylbicyclo[2.2.2]octane (cinane). An analogous experiment with P450(EryF) showed that a hydroxyl group on the substrate was vital, and in its absence catalytic turnover was effectively abolished. Catalytic turnover of P450(cin) with either of these alternative substrates (camphane or cinane) revealed that in the absence of the ethereal oxygen there was still a significant amount of coupling of the NADPH-reducing equivalents to the formation of oxidised product. Again the substrate itself was not found to be important in controlling oxygen activation, in contrast to P450(EryF), but was shown to be essential for regio- and stereoselective substrate oxidation. Thus, it still remains unclear how dioxygen activation in the catalytic turnover of cineole by P450(cin) is controlled.

Cloning, expression and purification of cindoxin, an unusual Fmn-containing cytochrome p450 redox partner.

Cytochromes P450 (P450s) belong to a superfamily of haemoproteins that catalyse a remarkable variety of oxidative transformations. P450 catalysis generally requires that cognate redox proteins transfer electrons, derived ultimately from NAD(P)H, to the P450 for oxygen activation. P450(cin) (CYP176A1) is a bacterial P450 that is postulated to allow Citrobacter braakii to live on cineole as its sole carbon source by initiating cineole biodegradation. Here we report the cloning, expression, purification and characterisation of one of its postulated redox partners, cindoxin (Cdx), which has strong similarity to the FMN domain of cytochrome P450 reductase. Cindoxin reductase (CdR), which displays strong similarity to NADPH-dependent ferredoxin reductases, was unable to be expressed in a functional form. Mass spectrometric and HPLC analyses confirmed that the flavin cofactor of cindoxin was FMN. Redox potentiometric titrations were performed with cindoxin within the range 6

Cineole biodegradation: molecular cloning, expression and characterisation of (1R)-6beta-hydroxycineole dehydrogenase from Citrobacter braakii.

The first steps in the biodegradation of 1,8-cineole involve the introduction of an alcohol and its subsequent oxidation to a ketone. In Citrobacter braakii, cytochrome P450(cin) has previously been demonstrated to perform the first oxidation to produce (1R)-6beta-hydroxycineole. In this study, we have cloned cinD from C. braakii and expressed the gene product, which displays significant homology to a number of short-chain alcohol dehydrogenases. It was demonstrated that the gene product of cinD exhibits (1R)-6beta-hydroxycineole dehydrogenase activity, the second step in the degradation of 1,8-cineole. All four isomers of 6-hydroxycineole were examined but only (1R)-6beta-hydroxycineole was converted to (1R)-6-ketocineole. The (1R)-6beta-hydroxycineole dehydrogenase exhibited a strict requirement for NAD(H), with no reaction observed in the presence of NADP(H). The enzyme also catalyses the reverse reaction, reducing (1R)-6-ketocineole to (1R)-6beta-hydroxycineole. During this study the N-terminal His-tag used to assist protein purification was found to interfere with NAD(H) binding and lower enzyme activity. This could be recovered by the addition of Ni(2+) ions or proteolytic removal of the His-tag.

Methionine gamma-lyase: mechanistic deductions from the kinetic pH-effects. The role of the ionic state of a substrate in the enzymatic activity.

We have studied and compared the pH-dependencies of the main kinetic parameters for the alpha,gamma-elimination reactions of methionine gamma-lyase (MGL) of Citrobacter intermedius with natural substrate, l-methionine, with its phosphinic analogue, and for alpha,beta-elimination reaction with S-methyl-l-cysteine. From the pH-dependency of k(cat)/K(m) for the reaction with l-methionine we have concluded that MGL is selective with respect to the zwitterionic form of its natural substrate. For the reaction of MGL with 1-amino-3-methylthiopropylphosphinic acid the pK(a) of the substrate's amino group, equal to 7.55, is not reflected in the pH-profile of k(cat)/K(m). Consequently, the enzyme does not manifest well-defined selectivity with respect to the zwitterion and anion ionic forms of the substrate. The ascending limbs of pH-dependencies of k(cat)/K(m) for reactions with l-methionine and S-methyl-l-cysteine are controlled by a single pK(a) equal to 7.1-7.2, while for the reaction with 1-amino-3-methylthiopropylphosphinic acid two equal pK(a)s of 6.2 were found in the respective pH-profile. The descending limbs of pH-dependencies of k(cat)/K(m) for the reactions with S-methyl-l-cysteine and racemic 1-amino-3-methylthiopropylphosphinic acid are very similar and are controlled by two acidic groups having average pK(a) values of 8.7. On the basis of these results we suggest a mechanism of catalytic action of MGL. According to this mechanism Tyr 113, in its conjugated base form, acts as an acceptor of the proton from the amino group of the substrate upon its binding in the active site. Elimination of the leaving thiol groups during both alpha,gamma- and alpha,beta-elimination reactions is assisted by the acidic groups of Tyr 113 and Tyr 58. Both tyrosyl residues are able to fulfill this catalytic function with different efficiencies depending on the type of elimination reaction. Tyr 113 residue plays the determining role in the alpha,gamma-elimination, and Tyr 58 - in the alpha,beta-elimination process.

Use of several inducer and substrate antibiotic combinations in a disk approximation assay format to screen for AmpC induction in patient isolates of Pseudomonas aeruginosa, Enterobacter spp., Citrobacter spp., and Serratia spp.

Two-hundred consecutive, single patient isolates of Enterobacter spp., Serratia spp., Citrobacter spp., and Pseudomonas aeruginosa were evaluated for AmpC production using a variety of inducer-substrate antibiotic combinations in a disk approximation format. The combinations examined included cefoxitin-piperacillin, imipenem-cefotaxime, imipenem-ceftazidime, imipenem-piperacillin-tazobactam, and imipenem-cefoxitin. All isolates were also screened for the presence of extended-spectrum beta-lactamase (ESBL) activity. In total, 85.5% of isolates were shown to be inducible for the production of AmpC by one or more inducer/substrate combinations and 11% of all isolates were stably derepressed for the expression of AmpC. Of all of the combinations, imipenem/piperacillin-tazobactam provided the greatest sensitivity (97.1%). All combinations were 100% specific when a positive test was observed. Given this background among these organisms in our institution, it is reasonable to develop an antibiotic reporting strategy that favors the selection of agents for therapy of these organisms that do not serve as labile substrates of AmpC.

Crystallization and preliminary X-ray diffraction study of the class A beta-lactamase SED-1 and its mutant SED-G238C from Citrobacter sedlakii.

SED-1, a class A beta-lactamase from Citrobacter sedlakii, is a CTX-M-type extended-spectrum beta-lactamase that has the ability to hydrolyze expanded-spectrum cephalosporins such as cefotaxime. SED-1 and a SED mutant in which Gly238 has been replaced by a cysteine, forming a disulfide bridge with the other Cys residue located at position 69 (SED-G238C), have been crystallized. The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 188.09, b = 73.65, c = 105.41 A, beta = 121.67 degrees for SED-1 and a = 187.64, b = 73.2, c = 103.89 A, beta = 121.89 degrees for the SED-G238C mutant. X-ray diffraction data were collected to maximum resolutions of 2.4 A for SED-1 and 2.0 A for SED-G238C.

Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii, Escherichia fergusonii and Enterobacter cancerogenus.

To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced. These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E. coli recombinant strains. The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases. The AmpC-type enzymes of C. murliniae, C. braakii and C. werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii. The AmpC-type enzyme of E. cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E. fergusonii shared 96% amino acid sequence identity with that of E. coli K12. The ampC genes, except for E. fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains. This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization.

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at approximately 2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450(cin), it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K(D) = 0.7 microm), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.

Influence of sulfobetaines on the stability of the Citrobacterdiversus ULA-27 beta-lactamase.

The activity and stability of beta-lactamase from Citrobacter diversus ULA-27 have been investigated in the presence of different ionic and zwitterionic surfactants. All the sulfobetaine surfactants tested allow the enzyme to retain its full activity, but the best stabilizing effect is greatly dependent on their structure. Very little variations on the monomer headgroup can significantly reduce enzyme deactivation or speed up the loss of activity with respect to buffer alone. The whole hydrophobic/hydrophilic balance on the headgroup seems to have a determining role in preserving beta-lactamase activity and structure. The presence of zwitterionic surfactants stabilizes the protein conformation toward denaturation by urea and low-temperature inactivation. Similar experiments were performed in the presence of other two zwitterionic surfactants, an amine oxide, dimethylmyristylamine oxide (DMMAO) and a carboxybetaine, cetyldimethylammonium methanecarboxylate (CB1-16). The former stabilizes the enzyme even better than the sulfobetaines, the latter quickly deactivates it. Therefore, the factors responsible for beta-lactamase stabilization are dependent not only on the zwitterionic nature of the surfactant headgroup but also specific interactions between the surfactant and the protein may be important.

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.

The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows.

A Citrobacter sp. originally isolated from metal-polluted soil accumulates heavy metals via metalphosphate deposition utilizing inorganic phosphate liberated via PhoN phosphatase activity. Further strain development was limited by the non-transformability of this environmental isolate. Recombinant Escherichia coli DH5 alpha bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp. with respect to removal of uranyl ion (UO2(2+)) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO2PO4). Subsequently, HUO2PO4-laden cells removed Ni2+ from dilute aqueous flows via intercalation of Ni2+ into the HUO2PO4 lattice. Despite comparable acid phosphatase activity in all three strains, the E. coli DH5 alpha (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E. coli DH5 alpha (phoC) construct was greatly inferior in both respects. Expression of phosphatase activity alone is not the only factor that permits efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases, which are otherwise considered to be related in many respects.

Bioaccumulation of nickel by intercalation into polycrystalline hydrogen uranyl phosphate deposited via an enzymatic mechanism.

A Citrobacter sp. accumulates uranyl ion (UO2(2+)) as crystalline HUO2PO4.4H2O (HUP), using enzymatically generated inorganic phosphate. Ni was not removed by this mechanism, but cells already loaded with HUP removed Ni2+ by intercalative ion-exchange, forming Ni(UO2PO4)2.7H2O, as concluded by x-ray diffraction (XRD) and proton induced x-ray emission (PIXE) analyses. The loaded biomass became saturated with Ni rapidly, with a molar ratio of Ni:U in the cellbound deposit of approx. 1:6; Ni penetration was probably surface-localized. Cochallenge of the cells with Ni2+ and UO2(2+), and glycerol 2-phosphate (phosphate donor for phosphate release and metal bioprecipitation) gave sustained removal of both metals in a flow through bioreactor, with more extensively accumulated Ni. We propose 'Microbially Enhanced Chemisorption of Heavy Metals' (MECHM) to describe this hybrid mechanism of metal bioaccumulation via intercalation into preformed, biogenic crystals, and note also that MECHM can promote the removal of the transuranic radionuclide neptunium, which is difficult to achieve by conventional methods.