Alternative pathways utilize or circumvent putrescine for biosynthesis of putrescine-containing rhizoferrin.

The siderophore rhizoferrin (N1,N4-dicitrylputrescine) is produced in fungi and bacteria to scavenge iron. Putrescine-producing bacterium Ralstonia pickettii synthesizes rhizoferrin and encodes a single nonribosomal peptide synthetase-independent siderophore (NIS) synthetase. From biosynthetic logic, we hypothesized that this single enzyme is sufficient for rhizoferrin biosynthesis. We confirmed this by expression of R. pickettii NIS synthetase in Escherichia coli, resulting in rhizoferrin production. This was further confirmed in vitro using the recombinant NIS synthetase, synthesizing rhizoferrin from putrescine and citrate. Heterologous expression of homologous lbtA from Legionella pneumophila, required for rhizoferrin biosynthesis in that species, produced siderophore activity in E. coli. Rhizoferrin is also synthesized by Francisella tularensis and Francisella novicida, but unlike R. pickettii or L. pneumophila, Francisella species lack putrescine biosynthetic pathways because of genomic decay. Francisella encodes a NIS synthetase FslA/FigA and an ornithine decarboxylase homolog FslC/FigC, required for rhizoferrin biosynthesis. Ornithine decarboxylase produces putrescine from ornithine, but we show here in vitro that FigA synthesizes N-citrylornithine, and FigC is an N-citrylornithine decarboxylase that together synthesize rhizoferrin without using putrescine. We co-expressed F. novicida figA and figC in E. coli and produced rhizoferrin. A 2.1 Å X-ray crystal structure of the FigC N-citrylornithine decarboxylase reveals how the larger substrate is accommodated and how active site residues have changed to recognize N-citrylornithine. FigC belongs to a new subfamily of alanine racemase-fold PLP-dependent decarboxylases that are not involved in polyamine biosynthesis. These data reveal a natural product biosynthetic workaround that evolved to bypass a missing precursor and re-establish it in the final structure.

A polyamine-independent role for S-adenosylmethionine decarboxylase.

The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.

A QM/MM Study of Nitrite Binding Modes in a Three-Domain Heme-Cu Nitrite Reductase.

Copper-containing nitrite reductases (CuNiRs) play a key role in the global nitrogen cycle by reducing nitrite (NO2-) to nitric oxide, a reaction that involves one electron and two protons. In typical two-domain CuNiRs, the electron is acquired from an external electron-donating partner. The recently characterised Rastonia picketti (RpNiR) system is a three-domain CuNiR, where the cupredoxin domain is tethered to a heme c domain that can function as the electron donor. The nitrite reduction starts with the binding of NO2- to the T2Cu centre, but very little is known about how NO2- binds to native RpNiR. A recent crystallographic study of an RpNiR mutant suggests that NO2- may bind via nitrogen rather than through the bidentate oxygen mode typically observed in two-domain CuNiRs. In this work we have used combined quantum mechanical/molecular mechanical (QM/MM) methods to model the binding mode of NO2- with native RpNiR in order to determine whether the N-bound or O-bound orientation is preferred. Our results indicate that binding via nitrogen or oxygen is possible for the oxidised Cu(II) state of the T2Cu centre, but in the reduced Cu(I) state the N-binding mode is energetically preferred.

A complete bioconversion cascade for dehalogenation and denitration by bacterial flavin-dependent enzymes.

Halogenated phenol and nitrophenols are toxic compounds that are widely accumulated in the environment. Enzymes in the had operon from the bacterium Ralstonia pickettii DTP0602 have the potential for application as biocatalysts in the degradation of many of these toxic chemicals. HadA monooxygenase previously was identified as a two-component reduced FAD (FADH-)-utilizing monooxygenase with dual activities of dehalogenation and denitration. However, the partner enzymes of HadA, that is, the flavin reductase and quinone reductase that provide the FADH- for HadA and reduce quinone to hydroquinone, remain to be identified. In this report, we overexpressed and purified the flavin reductases, HadB and HadX, to investigate their functional and catalytic properties. Our results indicated that HadB is an FMN-dependent quinone reductase that converts the quinone products from HadA to hydroquinone compounds that are more stable and can be assimilated by downstream enzymes in the pathway. Transient kinetics indicated that HadB prefers NADH and menadione as the electron donor and acceptor, respectively. We found that HadX is an FAD-bound flavin reductase, which can generate FADH- for HadA to catalyze dehalogenation or denitration reactions. Thermodynamic and transient kinetic experiments revealed that HadX prefers to bind FAD over FADH- and that HadX can transfer FADH- from HadX to HadA via free diffusion. Moreover, HadX rapidly catalyzed NADH-mediated reduction of flavin and provided the FADH- for a monooxygenase of a different system. Combination of all three flavin-dependent enzymes, i.e. HadA/HadB/HadX, reconstituted an effective dehalogenation and denitration cascade, which may be useful for future bioremediation applications.

Kinetic Mechanism of the Dechlorinating Flavin-dependent Monooxygenase HadA.

The accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.

RpA, an extracellular protease similar to the metalloprotease of serralysin family, is required for pathogenicity of Ralstonia pickettii.

AIM: To investigate the biochemical and functional properties of an extracellular protease, RpA, in Ralstonia pickettii WP1 isolated from water supply systems. METHODS AND RESULTS: An extracellular protease was identified and characterized from R. pickettii WP1. A mutant strain WP1M2 was created from strain WP1 by mini-Tn5 transposition. The culture filtrates from WP1M2 had a lower cytotoxic effect than the parental WP1 on several mammalian cell lines. Cloning and sequence analysis revealed the Tn5 transposon inserted at a protease gene (rpA) which is 81% homologous to prtA and aprX genes of Pseudomonas fluorescens. The rpA gene encodes a 482-residue protein showing sequence similarity to metalloproteases of the serralysin family. The RpA protein was expressed in Escherichia coli using a pET expression vector and purified as a 55 kDa molecular weight protein. Furthermore, the protease activity of RpA was inhibited by protease inhibitor and heat treatment. CONCLUSIONS: The in vitro cytotoxic activity of R. pickettii culture filtrates was attributed to RpA protease. SIGNIFICANCE AND IMPACT OF THE STUDY: An extracellular protease, RpA, was identified from R. pickettii WP1 isolated from water supply system. The RpA metalloproteases is required for the pathogenicity of R. pickettii to mammalian cell lines.

Emergence of VIM-2 metallo-beta-lactamase producing Ralstonia pickettii clinical isolate in India.

A multidrug-resistant clinical isolate of Ralstonia pickettii from a woman was analysed. Modified Hodge test was positive for carbapenemase production. Conjugation experiment revealed the presence of conjugative plasmid of >140 Kb size typed as IncN type. This is the first report of emergence blaVIM-2 in R. pickettii in India.

Effects of mutation at position 285 of Ralstonia pickettii T1 poly[(R)-3-hydroxybutyrate] depolymerase on its activities.

Asn at position 285 (N285) in the catalytic domain of poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 most likely participates in the cleavage of ester bonds as revealed by our previous evolutionary engineering study using the error-prone polymerase chain reaction (PCR) method. To exhaustively examine the effects of mutations at that position, we conducted site-directed saturation mutagenesis at that position and the resultant mutant enzymes (N285X) were evaluated in p-nitrophenyl ester (pNPCn) hydrolysis and PHB degradation. Kinetic studies demonstrated that the PHB-degrading activities of N285X were reciprocally related to their pNPCn-hydrolyzing activities, with the exception of N285A and N285G, and that His residue could functionally substitute for Asn285 on PHB degradation.

Transcriptional repression of the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1 by a tetR-like gene.

Ralstonia pickettii T1 secretes a poly(3-hydroxybutyrate) (PHB) depolymerase (PhaZ) and a 3-hydroxybutyrate (3HB)-oligomer hydrolase, and extracellularly degrades PHB to produce 3HB. However, it is not clear how the expression of phaZ is regulated. In this study, the mechanism by which phaZ expression is controlled in R. pickettii T1 was examined using a mutant made by the random insertion of a transposon, Tn5. The mutant produced a larger amount of PhaZ than the wild type in nutrient broth or a minimal salt (SM) medium supplemented with succinate. However, there was essentially no difference in the activity or amount of PhaZ in the culture supernatant between the wild type and mutant when the two were grown on 3HB. The gene disrupted by the insertion of Tn5 (epdR) was cloned and its nucleotide sequence was determined. In a BLAST search, epdR showed a high degree of similarity to genes for TetR transcriptional regulators of several bacteria. The introduction of epdR into the wild type and mutant grown on the three media described above decreased the amount of PhaZ. These results indicated EpdR to be involved in the repression of phaZ in R. pickettii T1. A quantitative RT-PCR analysis indicated that mRNA levels corresponded with the activity detected and the amounts of PhaZ in the wild type and mutant. Furthermore, the amount of epdR transcript was inversely proportional to the amount of phaZ transcript. In addition, the existence of a positive element acting on phaZ expression was suggested, because in the mutant lacking EpdR, the amount of phaZ transcript varied in cells grown in SM-3HB, SM-succinate or nutrient broth. Based on the above results, a model for the regulation of PhaZ expression in R. pickettii T1 is proposed.

Structures of protein-protein complexes involved in electron transfer.

Electron transfer reactions are essential for life because they underpin oxidative phosphorylation and photosynthesis, processes leading to the generation of ATP, and are involved in many reactions of intermediary metabolism. Key to these roles is the formation of transient inter-protein electron transfer complexes. The structural basis for the control of specificity between partner proteins is lacking because these weak transient complexes have remained largely intractable for crystallographic studies. Inter-protein electron transfer processes are central to all of the key steps of denitrification, an alternative form of respiration in which bacteria reduce nitrate or nitrite to N2 through the gaseous intermediates nitric oxide (NO) and nitrous oxide (N2O) when oxygen concentrations are limiting. The one-electron reduction of nitrite to NO, a precursor to N2O, is performed by either a haem- or copper-containing nitrite reductase (CuNiR) where they receive an electron from redox partner proteins a cupredoxin or a c-type cytochrome. Here we report the structures of the newly characterized three-domain haem-c-Cu nitrite reductase from Ralstonia pickettii (RpNiR) at 1.01 Å resolution and its M92A and P93A mutants. Very high resolution provides the first view of the atomic detail of the interface between the core trimeric cupredoxin structure of CuNiR and the tethered cytochrome c domain that allows the enzyme to function as an effective self-electron transfer system where the donor and acceptor proteins are fused together by genomic acquisition for functional advantage. Comparison of RpNiR with the binary complex of a CuNiR with a donor protein, AxNiR-cytc551 (ref. 6), and mutagenesis studies provide direct evidence for the importance of a hydrogen-bonded water at the interface in electron transfer. The structure also provides an explanation for the preferential binding of nitrite to the reduced copper ion at the active site in RpNiR, in contrast to other CuNiRs where reductive inactivation occurs, preventing substrate binding.

Directed evolution of poly[(R)-3-hydroxybutyrate] depolymerase using cell surface display system: functional importance of asparagine at position 285.

Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase from Ralstonia pickettii T1 (PhaZRpiT1) consists of three functional domains to effectively degrade solid PHB materials, and its catalytic domain catalyzes the ester bond cleavage of the substrate. We performed the directed evolution of PhaZRpiT1 targeted at the catalytic domain in combination with the cell surface display method to effectively screen for mutants with improved p-nitrophenyl butyrate (pNPC4) activity. Mutated PhaZRpiT1 genes generated by error-prone PCR were fused to the oprI gene to display them as fusion proteins on Escherichia coli cell surface. Some cells displaying the mutant enzymes showed a two- to fourfold increase in pNPC4 hydrolysis activity relative to cells displaying wild-type enzyme. These mutant genes were recombined by a staggered extension process and the recombined enzymes were displayed to result in a five- to eightfold higher pNPC4 hydrolysis activity than the wild type. To further evaluate the mutation effects, unfused and undisplayed enzymes were prepared and applied to the hydrolysis of p-nitrophenyl esters having different chain lengths (pNPCn; n = 2-6) and PHB degradation. One specific second-generation mutant showed an approximately tenfold increase in maximum rate for pNPC3 hydrolysis, although its PHB degradation efficiency at 1 µg/mL of enzyme concentration was approximately 3.5-fold lower than that of the wild type. Gene analysis showed that N285D or N285Y mutations were found in six of the seven improved second-generation mutants, indicating that Asn285 probably participates in the regulation of substrate recognition and may be more favorable for PHB degradation process than other amino acid residues.

Characterization of a novel copper-haem c dissimilatory nitrite reductase from Ralstonia pickettii.

NiRs (nitrite reductases) convert nitrite into NO in the denitrification process. RpNiR (Ralstonia pickettii NiR), a new type of dissimilatory Cu-containing NiR with a C-terminal haem c domain from R. pickettii, has been cloned, overexpressed in Escherichia coli and purified to homogeneity. The enzyme has a subunit molecular mass of 50515 Da, consistent with sequence data showing homology to the well-studied two-domain Cu NiRs, but with an attached C-terminal haem c domain. Gel filtration and combined SEC (size-exclusion chromatography)-SAXS (small angle X-ray scattering) analysis shows the protein to be trimeric. The metal content of RpNiR is consistent with each monomer having a single haem c group and the two Cu sites being metallated by Cu(2+) ions. The absorption spectrum of the oxidized as-isolated recombinant enzyme is dominated by the haem c. X-band EPR spectra have clear features arising from both type 1 Cu and type 2 Cu centres in addition to those of low-spin ferric haem. The requirements for activity and low apparent K(m) for nitrite are similar to other CuNiRs (Cu-centre NiRs). However, EPR and direct binding measurements of nitrite show that oxidized RpNiR binds nitrite very weakly, suggesting that substrate binds to the reduced type 2 Cu site during turnover. Analysis of SEC-SAXS data suggests that the haem c domains in RpNiR form extensions into the solvent, conferring a high degree of conformational flexibility in solution. SAXS data yield R(g) (gyration radius) and D(max) (maximum particle diameter) values of 43.4 Å (1 Å=0.1 nm) and 154 Å compared with 28 Å and 80 Å found for the two-domain CuNiR of Alcaligenes xylosoxidans.

Gradually accumulating beneficial mutations to improve the thermostability of N-carbamoyl-D-amino acid amidohydrolase by step-wise evolution.

To further enhance repeated batch reactions with immobilized N-carbamoyl-D-amino acid amidohydrolase (DCase), which can be used for the industrial production of D-amino acids, the stability of high soluble mutant DCase-M3 from Ralstonia pickettii CGMCC1596 was improved by step-wise evolution. In our previous report, six thermostability-related sites were identified by error-prone PCR. Based on the above result, an improved mutant B5 (Q12L/Q23L/H248Q/T262A/T263S) was obtained through two rounds of DNA shuffling, showing a 10°C increase in the T (50) (defined as the temperature at which heat treatment for 15 min reduced the initial activity by 50%) compared with the parental enzyme DCase-M3. Furthermore, several thermostability-related sites (Met(31), Asn(93), Gln(207), Asn(242), Glu(266), Thr(271), Ala(273)) on B5 were identified using amino acid consensus approach based on sequence alignment of homologous DCases. These sites were further investigated by iterative saturation mutagenesis (ISM), and a combinational mutant D1 (Q12L/Q23L/Q207E/N242G/H248Q/T262A/T263S/E266D/T271I/A273P) that enhanced the T(50) by about 16°C over DCase-M3 was obtained. Oxidative stability assay showed that the most heat-resisting mutant displayed only a slight increase in resistance to hydrogen peroxide. Comparative characterization showed that D1 not only maintained its characteristic high solubility but also shared similar k(cat) and K(m) values and optimum reaction pHs with the parental enzyme. The significantly improved mutants in the immobilized form are expected to be applied in the industrial production of D-p-hydroxyphenylglycine.

Degradation and adsorption characteristics of PHB depolymerase as revealed by kinetics of mutant enzymes with amino acid substitution in substrate-binding domain.

Extracelluar Poly[(R)-3-hydroxybutyrate] (PHB) depolymerase (PhaZ(RpiT1)) from Ralstonia pickettii T1 adsorbs to PHB surface via its substrate-binding domain (SBD) to enhance PHB degradation. Our previous study combining PCR random mutagenesis with the determination of PHB degradation levels of mutant enzymes suggested that Ser, Tyr, Val, Ala, and Leu residues in SBD are probably involved in the enzymatic adsorption to and degradation of PHB. In the present study, the effects of mutations at Leu441, Tyr443, and Ser445 on PHB degradation were investigated because these residues were predicted to form a beta-sheet structure and orient in the same direction to interact possibly directly with the PHB surface. Purified L441H, Y443H, and S445C mutant enzymes were prepared, and their CD spectra and hydrolytic activities for water-soluble substrates were found to be identical to those of wild-type enzyme, indicating that these mutations have no influence on their structures and their ability to cleave the ester bond. In contrast, the PHB-degrading activity of these mutants differed from that of the wild type: L441H and Y443H enzymes had lower PHB-degrading activity than their wild-type counterpart, whereas S445C had higher activity. Kinetic analysis of PHB degradation by the mutants suggested that the hydrophobic residues at these positions are important for the enzyme adsorption to the PHB surface, and such substitutions as Y443H and S445C may more effectively disrupt the PHB surface to enhance the hydrolysis of PHB polymer chains than the wild-type enzyme. Surface plasmon resonance (SPR) analysis revealed that the three substitutions mentioned above altered the association phase rather than the dissociation phase in the enzyme adsorption to the polymer surface.

Novel Tn4371-ICE like element in Ralstonia pickettii and genome mining for comparative elements.

BACKGROUND: Integrative Conjugative Elements (ICEs) are important factors in the plasticity of microbial genomes. An element related to the ICE Tn4371 was discovered during a bioinformatic search of the Ralstonia pickettii 12J genome. This element was analysed and further searches carried out for additional elements.A PCR method was designed to detect and characterise new elements of this type based on this scaffold and a culture collection of fifty-eight Ralstonia pickettii and Ralstonia insidiosa strains were analysed for the presence of the element. RESULTS: Comparative sequence analysis of bacterial genomes has revealed the presence of a number of uncharacterised Tn4371-like ICEs in the genomes of several beta and gamma- Proteobacteria. These elements vary in size, GC content, putative function and have a mosaic-like structure of plasmid- and phage-like sequences which is typical of Tn4371-like ICEs. These elements were found after a through search of the GenBank database. The elements, which are found in Ralstonia, Delftia, Acidovorax, Bordetella, Comamonas, Acidovorax, Congregibacter, Shewanella, Pseudomonas Stenotrophomonas, Thioalkalivibrio sp. HL-EbGR7, Polaromonas, Burkholderia and Diaphorobacter sp. share a common scaffold. A PCR method was designed (based on the Tn4371- like element detected in the Ralstonia pickettii 12J genome) to detect and characterise new elements of this type. CONCLUSION: All elements found in this study possess a common scaffold of core genes but contain different accessory genes. A new uniform nomenclature is suggested for ICEs of the Tn4371 family. Two novel Tn4371-like ICE were discovered and characterised, using the novel PCR method described in two different isolates of Ralstonia pickettii from laboratory purified water.

Characterization of a novel 3-hydroxybutyrate dehydrogenase from Ralstonia pickettii T1.

We previously reported that the activities of two 3-hydroxybutyrate dehydrogenases (BDH1 and BDH2) were greatly influenced by culture conditions when Ralstonia pickettii T1, a strain growing on extracellular poly-3-hydroxybutyrate (PHB), was grown on different carbon sources such as 3HB and succinate. In this study, knockout mutants of bdh1 or bdh2 were constructed and characterized under different culture conditions. In addition, a novel BDH (BDH3) was found in bdh2 mutants, and bdh3 was cloned. Apparent kinetic parameters for the substrates of BDH3 indicated that the enzyme is suitable for the oxidation reaction of 3-hydroxybutyrate (3HB) to acetoacetate. In Western blotting, it was clear that BDH3 is produced only in cells grown on 3HB or PHB as a carbon source, while BDH1 and BDH2 are produced in cells grown on various carbon sources such as sugars, amino acids, organic acids, 3HB, and PHB. Both the bdh1 and bdh2 mutants lagged behind the wild type in growth rates when the cells were cultured with 3HB, citrate, succinate, or nutrient broth. A test of sensitivity to diamide as an oxidative stress revealed that the lack of BDH1 or BDH2 caused a decline in the capacity to neutralize the stress. These results suggested that BDH1 and BDH2 are needed to regulate the cytoplasmic redox state as well as to utilize 3HB, while BDH3 is specialized to utilize 3HB. The expression of bdh3 may be coordinately regulated with a gene encoding putative 3HB permease.

Improving the thermostability of N-carbamyl-D-amino acid amidohydrolase by error-prone PCR.

To facilitate the easier production of D-amino acids using N-carbamyl-D-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 degrees C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of D-amino acids.

Crystalline-structure-dependent enzymatic degradation of polymorphic poly(3-hydroxypropionate).

The crystalline structure dependence of enzymatic degradation behavior was investigated for the polymorphic poly(3-hydroxypropionate) (P3HP), which has a basic backbone chemical structure of bacterial poly(3-hydroxyalkanoate)s (P3HAs). The P3HP films consisting of the beta-, gamma-, and/or delta-form crystal were cast or melt-crystallized as reported previously (Macromolecules 2005, 38, 6455; Macromolecules 2006, 39, 194-203) by controlling the molecular weight, crystallization temperature, and/or temperature of the melt. Their thermal properties, crystalline structures, morphologies, and (13)C solid spin-lattice relaxation dynamics were characterized by the differential scanning calorimetry, the wide-angle X-ray diffraction, the small-angle X-ray scattering (SAXS), and the (13)C solid-state NMR spectra (SNMR), respectively. Both the crystallinities and the lamellar thicknesses of P3HP films were found to decrease roughly in the order of beta-form > (or approximately) gamma-form > delta-form. From previous work, which indicates that the P3HA enzymatic degradation depends only on the degree of crystallinity and the lamellar thickness, their enzymatic degradation rates are then expected to increase in the order of beta-form < (or approximately) gamma-form < delta-form. Unexpectedly, their experimental P3HP enzymatic degradation rates in the presence of P3HA depolymerase isolated from Ralstonia pickettii T1 increase in the reverse order, i.e., delta-form < gamma-form < beta-form. The weight loss rate of the delta-form film is almost 1 order of magnitude smaller than that of the fastest degraded beta-form film. It is then strongly indicated that the crystalline structure plays a strikingly decisive role in the enzymatic degradation of P3HP. In particular, only when the conformation of crystalline chain accords with that of the bacterial poly(3-hydroxybutyrate) (P3HB) sample, i.e., the 2 1 helix conformation, is the P3HP sample degraded as slow as the P3HB sample. The inherent reason responsible for the unique P3HP enzymatic degradation behavior has been further clarified by comparing the molecular interaction and dynamics of polymorphic P3HP crystals.

Secretion pathway for the poly(3-hydroxybutyrate) depolymerase in Ralstonia pickettii T1.

The extracellular poly(3-hydroxybutyrate) depolymerase from Ralstonia pickettii T1 has been purified, its function and character investigated in detail, and its gene cloned and sequenced. However, the mechanism by which this enzyme is secreted has not been elucidated. A mutant unable to degrade poly(3-hydroxybutyrate), N17, was obtained with the random insertion of a mini-transposon, Tn5. Western analysis using antiserum against the poly(3-hydroxybutyrate) depolymerase of Ralstonia pickettii T1, revealed that N17 accumulated the poly(3-hydroxybutyrate) depolymerase in the periplasm and cytoplasm, and did not secrete the enzyme into the external medium. It was also found that 3-hydroxybutyrate-oligomer hydrolase was secreted but inactive. The disrupted gene in N17, depO, was analyzed by Southern hybridization and its nucleotide sequence was determined. One complete open reading frame was found in the cloned 2.3-kbp DNA fragment. From a BLAST search, this gene product was found to be homologous to PulO of Ralstonia eutropha JMP134 (60% identity) and XcpA of Pseudomonas aeruginosa (60% identity). These proteins are prepilin peptidase/N-metyltransferases, a component of the Type II secretion pathway. DepO also had the four cysteines highly conserved in most prepilin peptidases at the same positions. The transcript of depO was examined by Northern hybridization using depO as a probe. In the total RNA of Ralstonia pickettii T1 in the early stationary phase, a band at 2.6-kb was detected, suggesting depO to be a functional gene. In this study, it was found that poly(3-hydroxybutyrate) depolymerase was secreted by the Type II pathway.

Genomic analysis reveals widespread occurrence of new classes of copper nitrite reductases.

Recently, the structure of a Cu-containing nitrite reductase (NiR) from Hyphomicrobium denitrificans (HdNiR) has been reported, establishing the existence of a new family of Cu-NiR where an additional type 1 Cu (T1Cu) containing cupredoxin domain is located at the N-terminus (Nojiri et al. in Proc. Natl. Acad. Sci. USA 104:4315-4320, 2007). HdNiR retains the well-characterised coupled T1Cu-type 2 Cu (T2Cu) core, where the T2Cu catalytic site is also built utilising ligands from neighbouring monomers. We have undertaken a genome analysis and found the wide occurrence of these NiRs, with members clustering in two groups, one showing an amino acid sequence similarity of around 80% with HdNiR, and a second group, including the NiR from the extremophile Acidothermus cellulolyticus, clustering around 50% similarity to HdNiR. This is reminiscent of the difference observed between the blue (Alcaligenes xylosoxidans) and green (Achromobacter cycloclastes and Alcaligenes faecalis) NiRs which have been extensively studied and may indicate that these also form two distinct subclasses of the new family. Genome analysis also showed the presence of Cu-NiRs with a C-terminal extension of 160-190 residues containing a class I cytochrome c domain with a characteristic beta-sheet extension. Currently no structural information exists for any member of this family. Genome analysis suggests the widespread occurrence of these novel NiRs with representatives in the alpha, beta and gamma subclasses of the Proteobacteria and in two species of the fungus Aspergillus. We selected the enzyme from Ralstonia pickettii for comparative modelling and produced a plausible structure highlighting an electron transfer mode in which the cytochrome c haem at the C-terminus can come within 16-A reach of the T1Cu centre of the T1Cu-T2Cu core.