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The worldwide spread of multidrug-resistant Enterobacterales is a serious threat to public health. Here, we compared the MICs of plazomicin, amikacin, gentamicin, and tobramycin against 303 multinational multidrug-resistant Gram-negative bacilli. We followed Clinical and Laboratory Standards Institute (CLSI) guidelines and applied CLSI breakpoints as well as those of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) for amikacin, gentamicin, and tobramycin and of the U.S. Food and Drug Administration for plazomicin. Overall, the highest percentage of susceptible isolates (80.2%) was demonstrated for plazomicin, which had the lowest MIC 50 (1 μg/ml) of the aminoglycosides studied. Of the 42 isolates resistant to plazomicin, 34 had MICs of ≥128 μg/ml, with 33 of the 34 having MICs of >128 μg/ml for amikacin, gentamicin, and tobramycin. Among the 42 bla NDM -positive isolates, 35.7% were plazomicin susceptible, with the percentage of isolates susceptible to amikacin being 38.1% or 35.7% when applying the CLSI or EUCAST breakpoint, respectively. The 20 bla OXA-48-like -positive isolates showed 50.0% susceptibility to plazomicin. Among 35 isolates with bla CTX-M as their only characterized resistance mechanism, 68.6% were plazomicin susceptible, while the percentage susceptible to amikacin was 74.3% or 62.9% when applying the CLSI or EUCAST breakpoint, respectively. Among the 117 bla KPC -positive isolates, 94.9% were susceptible to plazomicin, whereas when the CLSI and EUCAST breakpoints were applied, 43.6% and 25.6%, respectively, were susceptible to amikacin; 56.4% and 44.4%, respectively, were susceptible to gentamicin; and 5.1% and 4.3%, respectively, were susceptible to tobramycin., (Copyright © 2020 American Society for Microbiology.)

In the aerobic methanotrophic bacteria Methylomicrobium alcaliphilum 20Z, Methylococcus capsulatus Bath, and Methylosinus trichosporium OB3b, the biochemical properties of hydroxypyruvate reductase (Hpr), an indicator enzyme of the serine pathway for assimilation of reduced C 1 -compounds, were comparatively analyzed. The recombinant Hpr obtained by cloning and heterologous expression of the hpr gene in Escherichia coli catalyzed NAD(P)H-dependent reduction of hydroxypyruvate or glyoxylate, but did not catalyze the reverse reactions of D-glycerate or glycolate oxidation. The absence of the glycerate dehydrogenase activity in the methanotrophic Hpr confirmed a key role of the enzyme in utilization of C 1 -compounds via the serine cycle. The enzyme from Ms. trichosporium OB3b realizing the serine cycle as a sole assimilation pathway had much higher special activity and affinity in comparison to Hpr from Mm. alcaliphilum 20Z and Mc. capsulatus Bath assimilating carbon predominantly via the ribulose monophosphate (RuMP) cycle. The hpr gene was found as part of gene clusters coding the serine cycle enzymes in all sequenced methanotrophic genomes except the representatives of the Verrucomicrobia phylum. Phylogenetic analyses revealed two types of Hpr: (i) Hpr of methanotrophs belonging to the Gammaproteobacteria class, which use the serine cycle along with the RuMP cycle, as well as of non-methylotrophic bacteria belonging to the Alphaproteobacteria class; (ii) Hpr of methylotrophs from Alpha- and Betaproteobacteria classes that use only the serine cycle and of non-methylotrophic representatives of Betaproteobacteria. The putative role and origin of hydroxypyruvate reductase in methanotrophs are discussed.

The ever-increasing production and use of polyvinyl alcohol (PVA) threaten our environment. Yet PVA can be assimilated by microbes in two steps: oxidation and cleavage. Here we report novel α/β-hydrolase structures of oxidized PVA hydrolase (OPH) from two known PVA-degrading organisms, Sphingopyxis sp. 113P3 and Pseudomonas sp. VM15C, including complexes with substrate analogues, acetylacetone and caprylate. The active site is covered by a lid-like β-ribbon. Unlike other esterase and amidase, OPH is unique in cleaving the CC bond of β-diketone, although it has a catalytic triad similar to that of most α/β-hydrolases. Analysis of the crystal structures suggests a double-oxyanion-hole mechanism, previously only found in thiolase cleaving β-ketoacyl-CoA. Three mutations in the lid region showed enhanced activity, with potential in industrial applications., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Ceftolozane/tazobactam is under clinical development for treatment of complicated intra-abdominal infections (IAI), complicated urinary tract infections (UTI) and ventilator-associated pneumonia. We evaluated the in vitro activity of ceftolozane/tazobactam and comparator agents tested against Gram-negative aerobic bacteria causing IAI and healthcare-associated UTI (HCA-UTI). The organisms were consecutively collected from January to December 2012 from 59 medical centers located in the United States (USA) and 15 European countries by the Program to Assess Ceftolozane/Tazobactam Susceptibility (PACTS). The collection included 809 organisms from IAI and 2474 organisms from HCA-UTI, and susceptibility testing was performed by reference broth microdilution methods as described by the Clinical and Laboratory Standards Institute (CLSI) M07-A9 document. Overall, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa were the most frequently isolated pathogens from both infection types. Ceftolozane/tazobactam was very active against E. coli (MIC50/90, 0.25/0.5 mg/L; 98.5-99.9% inhibited at an MIC of ≤8 mg/L) and retained activity against many of the multidrug-resistant (MDR; MIC50/90, 0.5/2->32 mg/L) and ESBL-phenotype strains (MIC50/90, 0.5/2-32 mg/L). Ceftolozane/tazobactam was active against most K. pneumoniae strains (MIC50/90, 0.25/16 mg/L, 88.9-89.6% inhibited at an MIC of ≤8 mg/L), but some ESBL-phenotype (MIC50/90, 4-8/>32 mg/L) and MDR (MIC50/90, 16/>32 mg/L) isolates exhibited elevated MIC values. Ceftolozane/tazobactam was the most active agent tested against P. aeruginosa (MIC50/90, 0.5/4 mg/L; 93.4-95.7% inhibited at ≤8 mg/L) and retained potency against many MDR (MIC50/90, 2-4/>32 mg/L), ceftazidime-nonsusceptible (MIC50/90, 2-4/>32 mg/L) and meropenem-nonsusceptible (MIC50/90, 2/>32 mg/L) strains. Ceftolozane/tazobactam was also active against Klebsiella oxytoca (MIC50/90, ≤0.12-0.25/0.5-1 mg/L), Enterobacter spp. (MIC50/90, 0.25-0.5/4-8 mg/L), Citrobacter spp. (MIC50/90, 0.25/2-32 mg/L), Proteus mirabilis (MIC50/90, 0.5/0.5 mg/L), indole-positive Proteae (MIC50/90, 0.25/0.5-1 mg/L), and Serratia spp. (MIC50/90, 0.5/1-2 mg/L). In summary, ceftolozane/tazobactam demonstrated potent in vitro activity when tested against contemporary aerobic Gram-negative pathogens causing IAI and HCA-UTI in USA and European medical centers., (Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.)

Aspartate transcarbamoylase and dihydroorotase, enzymes that catalyze the second and third step in de novo pyrimidine biosynthesis, are associated in dodecameric complexes in Aquifex aeolicus and many other organisms. The architecture of the dodecamer is ideally suited to channel the intermediate, carbamoyl aspartate from its site of synthesis on the ATC subunit to the active site of DHO, which catalyzes the next step in the pathway, because both reactions occur within a large, internal solvent-filled cavity. Channeling usually requires that the reactions of the enzymes are coordinated so that the rate of synthesis of the intermediate matches its rate of utilization. The linkage between the ATC and DHO subunits was demonstrated by showing that the binding of the bisubstrate analog, N-phosphonacetyl-L-aspartate to the ATC subunit inhibits the activity of the distal DHO subunit. Structural studies identified a DHO loop, loop A, interdigitating between the ATC domains that would be expected to interfere with domain closure essential for ATC catalysis. Mutation of the DHO residues in loop A that penetrate deeply between the two ATC domains inhibits the ATC activity by interfering with the normal reciprocal linkage between the two enzymes. Moreover, a synthetic peptide that mimics that part of the DHO loop that binds between the two ATC domains was found to be an allosteric or noncompletive ATC inhibitor (K(i) = 22 μM). A model is proposed suggesting that loop A is an important component of the functional linkage between the enzymes., (© 2013 The Protein Society.)

In Gram-negative bacteria, the hydrophobic anchor of the outer membrane lipopolysaccharide is lipid A, a saccharolipid that plays key roles in both viability and pathogenicity of these organisms. The tetraacyldisaccharide 4'-kinase (LpxK) of the diverse P-loop-containing nucleoside triphosphate hydrolase superfamily catalyzes the sixth step in the biosynthetic pathway of lipid A, and is the only known P-loop kinase to act upon a lipid substrate at the membrane. Here, we report the crystal structures of apo- and ADP/Mg(2+)-bound forms of Aquifex aeolicus LpxK to a resolution of 1.9 Å and 2.2 Å, respectively. LpxK consists of two α/β/α sandwich domains connected by a two-stranded β-sheet linker. The N-terminal domain, which has most structural homology to other family members, is responsible for catalysis at the P-loop and positioning of the disaccharide-1-phosphate substrate for phosphoryl transfer on the inner membrane. The smaller C-terminal domain, a substructure unique to LpxK, helps bind the nucleotide substrate and Mg(2+) cation using a 25° hinge motion about its base. Activity was severely reduced in alanine point mutants of conserved residues D138 and D139, which are not directly involved in ADP or Mg(2+) binding in our structures, indicating possible roles in phosphoryl acceptor positioning or catalysis. Combined structural and kinetic studies have led to an increased understanding of the enzymatic mechanism of LpxK and provided the framework for structure-based antimicrobial design.

The shelf life of poultry legs stored aerobically and the possible role of the aminopeptidase activity of gram-negative bacteria (p-nitroaniline test) as a predictor of poultry spoilage were evaluated on the basis of microbiological and sensory parameters. Chicken legs (n = 30) obtained immediately after evisceration in a local poultry processing plant were kept under aerobic refrigeration (4 +/- 1 degrees C) for 7 days. Microbiological (counts of aerobic bacteria and psychrotrophs) and sensory (odor, color, and general acceptability on a hedonic scale of 1 to 9) parameters and aminopeptidase activity (absorbance at 390 nm [A(390)]) determinations were performed after 0, 1, 3, 5, and 7 days of storage. Aerobic plate counts of 7 log CFU/g and a score of 6 for general acceptability were used as indicators of the end point of shelf life. Strong correlations (r > or = 0.76; P < 0.001) were obtained between bacterial counts, hedonic scores, and A(390) values. Samples were judged as unacceptable (shelf-life end point) after 2 and 4 days on the basis of sensory and microbiological analyses, respectively. A(390) values of 0.52 and 0.89 (corresponding to p-nitroaniline concentrations of 6.25 and 10.7 microg/ml, respectively) are proposed as the upper limits for acceptability on the basis of sensory and microbiological determinations, respectively. However, these recommendations are based on a small set of samples, and their general application is yet to be verified.

The increasing emergence of serious multidrug-resistant (MDR) Gram-negative infections has led to a new health-care crisis. These infections predominately include MDR Pseudomonas aeruginosa, extended-spectrum beta-lactamase-producing Enterobacteriaceae and MDR Acinetobacter baumannii. These organisms are present in a variety of clinical settings, but there is a distinct paucity of antibiotics to effectively treat these infections. The increasing use of broad-spectrum antibiotics and lack of good stewardship have contributed to the increase in these MDR organisms. This review focuses on the main MDR Gram-negative infections contributing to the current crisis in health care, their mechanisms of resistance and various treatment options for empiric therapy., (Copyright © 2010 S. Karger AG, Basel.)

Objective: To describe any potential effect on in vitro susceptibility to imipenem for aerobic gram-negative bacteria following the addition of ertapenem to the formulary of a large teaching hospital., Methods: Changes in imipenem susceptibilities for aerobic gram-negative bacteria were compared among clinical isolates from 2002 to 2007 using a Poisson model. Changes in the susceptibility of imipenem to P. aeruginosa, K. pneumoniae, K. oxytoxa, E. coli, and S. marcescens were compared over time using a chi-squared test for trend. Carbapenem use was measured using a defined daily dose per 1000 patient days. Change in utilization was compared for all years. The prevalence of ESBL-producing K. pneumoniae, K. oxytoxa, and E. coli over time was compared., Results: Susceptibility to imipenem did not change after the addition of ertapenem and this most notably includes P. aeruginosa (p=0.43). Additionally, an increase in the incidence of ESBL K. pneumoniae from 2002 (4%) to 2007 (18%) p<0.0001 occurred with significant increases in both imipenem and ertapenem use in that time frame (p<0.001)., Conclusion: The continued use of ertapenem over 5 years with predominant use of imipenem did not select for P. aeruginosa resistance to imipenem. The rising rate of community and healthcare associated ESBL K. pneumoniae increased total carbapenem use.

A metalloprotease secreted by the moderately halophilic bacterium Salinivibrio sp. strain AF-2004 when the culture reached the stationary growth phase. This enzyme was purified to homogeneity by acetone precipitation and subsequent Q-Sepharose anion exchange and Sephacryl S-200 gel filtration chromatography. The apparent molecular mass of the protease was 31 kDa by SDS-PAGE, whereas it was estimated as approximately 29 kDa by Sephacryl S-200 gel filtration. The purified protease had a specific activity of 116.8 mumol of tyrosine/min per mg protein on casein. The optimum temperature and salinity of the enzyme were at 55 degrees C and 0-0.5 M NaCl, although at salinities up to 4 M NaCl activity still remained. The protease was stable and had a broad pH profile (5.0-10.0) with an optimum of 8.5 for casein hydrolysis. The enzyme was strongly inhibited by phenylmethyl sulfonylfluoride (PMSF), Pefabloc SC, chymostatin and also EDTA, indicating that it belongs to the class of serine metalloproteases. The protease in solutions containing water-soluble organic solvents or alcohols was more stable than that in the absence of organic solvents. These characteristics make it an ideal choice for applications in industrial processes containing organic solvents and/or salts.

Different responses to salinity were observed for an extremely halotolerant endo-xylanase when assayed with soluble birchwood glucoronoxylan and cross-linked dyed insoluble birchwood glucoronoxylan. Shrinking of insoluble xylan particles due to increased ionic strength is proposed as the explanation. Temperature affected the xylanase activity measurement on the insoluble xylan greatly, likely due to increased enzyme accessible surface of the substrate at high temperatures.

Palmitoylated lipid A can both protect pathogenic bacteria from host immune defences and attenuate the activation of those same defences through the TLR4 signal transduction pathway. A palmitate chain from a phospholipid is incorporated into lipid A by an outer membrane enzyme PagP, which is an 8-stranded antiparallel beta-barrel preceded by an amino-terminal amphipathic alpha-helix. The PagP barrel axis is tilted by 25 degrees with respect to the membrane normal. An interior hydrophobic pocket in the outer leaflet-exposed half of the molecule functions as a hydrocarbon ruler that allows the enzyme to distinguish palmitate from other acyl chains found in phospholipids. Internalization of a phospholipid palmitoyl group within the barrel appears to occur by lateral diffusion from the outer leaflet through non-hydrogen-bonded regions between beta-strands. The MsbA-dependent trafficking of lipids from the inner membrane to the outer membrane outer leaflet is necessary for lipid A palmitoylation in vivo. The mechanisms by which bacteria regulate pagP gene expression strikingly reflect the corresponding pathogenic lifestyle of the bacterium. Variations on PagP structure and function can be illustrated with the known homologues from Gram-negative bacteria, which include pathogens of humans and other mammals in addition to pathogens of insects and plants. The PagP enzyme is potentially a target for the development of anti-infective agents, a probe of outer membrane lipid asymmetry, and a tool for the synthesis of lipid A-based vaccine adjuvants and endotoxin antagonists.

Beta-lactamase production by oral bacteria is common in infancy and is associated with use of antimicrobial agents in infants. The present longitudinal study aimed to examine the frequency of salivary beta-lactamase activity (SbetaA), to compare SbetaA with the presence of beta-lactamase-producing (beta+) aerobic and anaerobic species in saliva, and to estimate the impact of antimicrobial exposure on the emergence of SbetaA in healthy infants during their first year of life. At 6 months, SbetaA was detected in 46% infants; 89% SbetaA-positive infants and 55% SbetaA-negative infants harboured beta+ species at this time (OR 7.08; CI 1.31-38.34). At 12 months, SbetaA was detected in 54% infants. Exposure to antimicrobials during the first year of life increased the risk (OR 2.60; CI 0.72-9.36) of having SbetaA.

The rotenone sensitive NADH:menaquinone oxidoreductase (NDH-I or complex I) from the thermohalophilic bacterium Rhodothermus marinus has been purified and characterized. Three of its subunits react with antibodies against 78, 51, and 21.3c kDa subunits of Neurospora crassa complex I. The optimum conditions for NADH dehydrogenase activity are 50 degrees C and pH 8.1, and the enzyme presents a KM of 9 microM for NADH. The enzyme also displays NADH:quinone oxidoreductase activity with two menaquinone analogs, 1,4-naphtoquinone (NQ) and 2,3-dimethyl-1,4-naphtoquinone (DMN), being the last one rotenone sensitive, indicating the complex integrity as purified. When incorporated in liposomes, a stimulation of the NADH:DMN oxidoreductase activity is observed by dissipation of the membrane potential, upon addition of CCCP. The purified enzyme contains 13.5 +/- 3.5 iron atoms and approximately 3.7 menaquinone per FMN. At least five iron-sulfur centers are observed by EPR spectroscopy: two [2Fe-2S](2+/1+) and three [4Fe-4S](2+/1+) centers. By fluorescence spectroscopy a still unidentified chromophore was detected in R. marinus complex I.

Display of heterologous proteins on the surface of microorganisms, enabled by means of recombinant DNA technology, has become an increasingly used strategy in various applications in microbiology, biotechnology and vaccinology. Gram-negative, Gram-positive bacteria, viruses and phages are all being investigated in such applications. This review will focus on the bacterial display systems and applications. Live bacterial vaccine delivery vehicles are being developed through the surface display of foreign antigens on the bacterial surfaces. In this field, 'second generation' vaccine delivery vehicles are at present being generated by the addition of mucosal targeting signals, through co-display of adhesins, in order to achieve targeting of the live bacteria to immunoreactive sites to thereby increase immune responses. Engineered bacteria are further being evaluated as novel microbial biocatalysts with heterologous enzymes immobilized as surface exposed on the bacterial cell surface. A discussion has started whether bacteria can find use as new types of whole-cell diagnostic devices since single-chain antibodies and other type of tailor-made binding proteins can be displayed on bacteria. Bacteria with increased binding capacity for certain metal ions can be created and potential environmental or biosensor applications for such recombinant bacteria as biosorbents are being discussed. Certain bacteria have also been employed for display of various poly-peptide libraries for use as devices in in vitro selection applications. Through various selection principles, individual clones with desired properties can be selected from such libraries. This article explains the basic principles of the different bacterial display systems, and discusses current uses and possible future trends of these emerging technologies.

Carbapenemases may be defined as beta-lactamases that significantly hydrolyze at least imipenem or/and meropenem. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. Class A, clavulanic acid-inhibited carbapenemases are rare. They are either chromosomally encoded (NMC-A, Sme-1 to Sme-3, IMI-1) in Enterobacter cloacae and Serratia marcescens, or plasmid encoded, such as KPC-1 in Klebsiella pneumoniae and GES-2 in Pseudomonas aeruginosa, the latter being a point-mutant of the clavulanic acid-inhibited extended-spectrum beta-lactamase GES-1. The class B enzymes are the most clinically significant carbapenemases. They are metalloenzymes of the IMP or VIM series. They have been reported worldwide but mostly from South East Asia and Europe. Metalloenzymes, whose genes are plasmid and integron located, hydrolyze virtually all beta-lactams except aztreonam. Finally, the class D carbapenemases are increasingly reported in Acinetobacter baumannii but compromise imipenem and meropenem susceptibility only marginally. The sources of the acquired carbapenemase genes remain unknown, as does the relative importance of the spread of epidemic strains as opposed to the spread of plasmid- or integron-borne genes. Because most of these carbapenemases confer only reduced susceptibility to carbapenems in Enterobacteriaceae, they may remain underestimated as a consequence of the lack of their detection.

Xylose isomerases from Thermotoga neapolitana (TNXI) and Thermoanaerobacterium thermosulfurigenes (TTXI) share 70.4% sequence identity and are thermostable. The double mutants Trp138Phe/Val185Thr of TNXI and TTXI have higher catalytic efficiencies than TNXI and TTXI, respectively. The Trp138Phe/Val185Thr TNXI and TTXI mutants were overexpressed in Escherichia coli strain BL21(DE3) and purified. Crystals of the two proteins were grown with polyethylene glycol 8000 as the major precipitant by the hanging-drop vapour-diffusion method. Crystals of the TNXI mutant were obtained in the absence of substrate, in complex with glucose and in complex with fructose. Crystals of the TTXI mutant were obtained complexed with glucose. Diffraction data were collected at 1.9, 2.1 and 2.1 A resolution for the fructose-TNXI mutant, glucose-TNXI mutant and substrate-unbound TNXI mutant, respectively. The diffraction data for the glucose-TTXI mutant were collected at 2.0 A resolution. The crystals belong to the orthorhombic space groups C222(1) (TNXI mutant) and P2(1)2(1)2(1) (TTXI mutant). The TNXI and TTXI mutant crystals contain two and four monomers in the asymmetric unit, respectively.

The succinate dehydrogenase from the thermohalophilic bacterium Rhodothermus marinus is a member of the succinate:menaquinone oxidoreductases family. It is constituted by three subunits with apparent molecular masses of 70, 32, and 18 kDa. The optimum temperature for succinate dehydrogenase activity is 80 degrees C, higher than the optimum growth temperature of R. marinus, 65 degrees C. The enzyme shows a high affinity for both succinate (Km = 0.165 mM) and fumarate (Km = 0.10 mM). It contains the canonical iron-sulfur centers S1, S2, and S3, as well as two B-type hemes. In contrast to other succinate dehydrogenases, the S3 center has an unusually high reduction potential of +130 mV and is present in two different conformations, one of which presents an unusual EPR signal with g values at 2.035, 2.009, and 2.001. The apparent midpoint reduction potentials of the hemes, +75 and -65 mV at pH 7.5, are also higher than those reported for other enzymes. The heme with the lower potential (heme bL) presents a considerable dependence of the reduction potential with pH (redox-Bohr effect), having a pKa(OX) = 6.5 and a pKa(red) = 8.7. This behavior is consistent with the proposal that in these enzymes menaquinone reduction occurs close to heme bL, near to the periplasmic side of the membrane, and involving dissipation of the proton transmembrane gradient.

The thermostable cellulase Cel12A from Rhodothermus marinus was produced at extremely low levels when expressed in Escherichia coli and was cytotoxic to the cells. In addition, severe aggregation occurred when moderately high concentrations of the enzyme were heat-treated at 65 degrees C, the growth optimum of R. marinus. Sequence analysis revealed that the catalytic module of this enzyme is preceded by a typical linker sequence and a highly hydrophobic putative signal peptide. Two deletion mutants lacking this hydrophobic region were cloned and successfully expressed in E. coli. These results indicated that the N-terminal putative signal peptide was responsible for the toxicity of the full-length enzyme in the host organism. This was further corroborated by cloning and expressing the hydrophobic N-terminal domain in E. coli, which resulted in extensive cell lysis. The deletion mutants, made up of either the catalytic module of Cel12A or the catalytic module and the putative linker sequence, were characterised and their properties compared to those of the full-length enzyme. The specific activity of the mutants was approximately three-fold higher than that of the full-length enzyme. Both mutant proteins were highly thermostable, with half-lives exceeding 2 h at 90 degrees C and unfolding temperatures up to 103 degrees C.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains an oxygen reductase, which uses HiPIP (high potential iron-sulfur protein) as an electron donor. The structural genes encoding the four subunits of this HiPIP:oxygen oxidoreductase were cloned and sequenced. The genes for subunits II, I, III, and IV (named rcoxA to rcoxD) are found in this order and seemed to be organized in an operon of at least five genes with a terminator structure a few nucleotides downstream of rcoxD. Examination of the amino acid sequence of the Rcox subunits shows that the subunits of the R. marinus enzyme have homology to the corresponding subunits of oxidases belonging to the superfamily of heme-copper oxidases. RcoxB has the conserved histidines involved in binding the binuclear center and the low-spin heme. All of the residues proposed to be involved in proton transfer channels are conserved, with the exception of the key glutamate residue of the D-channel (E(278), Paracoccus denitrificans numbering). Analysis of the homology-derived structural model of subunit I shows that the phenol group of a tyrosine (Y) residue and the hydroxyl group of the following serine (S) may functionally substitute the glutamate carboxyl in proton transfer. RcoxA has an additional sequence for heme C binding, after the Cu(A) domain, that is characteristic of caa(3) oxidases belonging to the superfamily. Homology modeling of the structure of this cytochrome domain of subunit II shows no marked electrostatic character, especially around the heme edge region, suggesting that the interaction with a redox partner is not of an electrostatic nature. This observation is analyzed in relation to the electron donor for this caa(3) oxidase, the HiPIP. In conclusion, it is shown that an oxidase, which uses an iron-sulfur protein as an electron donor, is structurally related to the caa(3) class of heme-copper cytochrome c oxidases. The data are discussed in the framework of the evolution of oxidases within the superfamily of heme-copper oxidases.

The taxonomic status of a gram-negative, oxidase positive rod (strain S1) able to degrade 4-aminobenzenesulfonate was studied using a polyphasic approach. Chemotaxonomic investigations of quinones and polar lipids established the allocation of this strain to the beta-subclass of the Proteobacteria and revealed similarities to Hydrogenophaga palleronii. 16S rRNA sequence comparisons demonstrated that this strain clusters phylogenetically with H. palleronii and H. taeniospiralis, but clearly represents a new species. The fatty acid patterns and substrate utilization profile displayed similarity to the characteristics of the four validly published species of Hydrogenophaga, although clear differentiating characters were also observed. No close similarities between the type strains of H. palleronii and H. taeniospiralis were detected in hybridization experiments with the genomic DNAs. On basis of these results, the new species Hydrogenophaga intermedia sp. nov. is proposed, with the type strain S1T (= DSM 5680).

A novel protein-deamidating enzyme, which has potential for industrial applications, was purified from the culture supernatant of Chryseobacterium proteolyticum strain 9670(T) isolated from rice field soil in Tsukuba, Japan. The deamidating activities on carboxybenzoxy (Cbz)-Gln-Gly and caseins and protease activity were produced synchronously by the isolate. Both deamidating activities were eluted as identical peaks separated from several proteases by phenyl-Sepharose chromatography of the culture supernatant. The enzyme catalyzed the deamidation of native caseins with no protease and transglutaminase activities. Phenotypic characterization and DNA analyses of the isolate were performed to determine its taxonomy. Physiological and biochemical characteristics, 16S rRNA gene sequence analysis, and DNA-DNA relatedness data indicated that the isolate should be placed as a new species belonging to the genus Chryseobacterium. The isolate showed no growth on MacConkey agar and produced acid from sucrose. The levels of DNA-DNA relatedness between the isolate and other related strains were less than 17%. The name Chryseobacterium proteolyticum is proposed for the new species; strain 9670 is the type strain (=FERM P-17664).

Rhodothermus marimus ATCC 43812, a thermophilic bacterium isolated from marine hot springs, possesses hydrolytic activities for depolymerising substrates such as carob-galactomannan. Screening of expression libraries identified mannanase-positive clones. Subsequently, the corresponding DNA sequences were determined, eventually identifying a coding sequence specifying a 997 amino acid residue protein of 113 kDa. Analyses revealed an N-terminal domain of unknown function and a C-terminal mannanase domain of 550 amino acid residues with homology to known mannanases of glycosidase family 26. Action pattern analysis categorised the R. marinus mannanase as an endo-acting enzyme with a requirement for at least five sugar moieties for effective catalytic activity. When expressed in Escherichia coli, purified gene product with catalytic activity was mainly found as two protein fragments of 45 kDa and 50 kDa. The full-length protein of 113 kDa was only detected in crude extracts of R. marinus, while truncated protein-containing fractions of the original source resulted in a major active protein of 60 kDa. Biochemical analysis of the mannanase revealed a temperature and pH optimum of 85 degrees C and pH 5.4, respectively. Purified, E. coil-produced protein fragments showed high heat stability, retaining more than 70% and 25% of the initial activity after 1 h incubation at 70 degrees C and 90 degrees C, respectively. In contrast, R. marinus-derived protein retained 87% activity after 1 h at 90 degrees C. The enzyme hydrolysed carob-galactomannan (locust bean gum) effectively and to a smaller extent guar gum, but not yeast mannan.

The thermohalophilic bacterium Rhodothermus marinus expresses a caa(3)-type dioxygen reductase as one of its terminal oxidases. The subunit I amino acid sequence shows the presence of all the essential residues of the D- and K-proton channels, defined in most heme-copper oxidases, with the exception of the key glutamate residue located in the middle of the membrane dielectric (E278 in Paracoccus denitrificans). On the basis of homology modeling studies, a tyrosine residue (Y256, R. marinus numbering) has been proposed to act as a functional substitute [Pereira, M. M., Santana, M., Soares, C. M., Mendes, J., Carita, J. N., Fernandes, A. S., Saraste, M., Carrondo, M. A., and Teixeira, M. (1999) Biochim. Biophys. Acta 1413, 1-13]. Here, R. marinus caa(3) oxidase was reconstituted in liposomes and shown to operate as a proton pump, translocating protons from the cytoplasmic side of the bacterial inner membrane to the periplasmatic space with a stoichiometry of 1H(+)/e(-), as in the case in heme-copper oxidases that contain the glutamate residue. Possible mechanisms of proton transfer in the D-channel with the participation of the tyrosine residue are discussed. The observation that the tyrosine residue is conserved in several other members of the heme-copper oxidase superfamily suggests a common alternative mode of action for the D-channel.

The enzymatic hydrolysis of polysaccharides by the 1, 3(4)-beta-glucanase (LamR) from Rhodothermus marinus has been explored. The enzyme cleaves the 1,3-beta-linkages of 3-O-substituted glucose units in 1,3-beta-glucans such as laminarin and curdlan, and also the 1,4-beta-linkages of 3-O-substituted beta-glucose in beta-glucans such as lichenin and 1,3-1, 4-beta-glucan from the cell walls of barley endosperm. The polysaccharide substrates (laminarin, curdlan and barley beta-glucan) were characterised using NMR spectroscopy. The reaction of LamR with its substrates was followed by recording one-dimensional and two-dimensional 1H-NMR and 13C-NMR spectra at suitable time intervals after addition of the enzyme. It is shown that hydrolysis occurs with retention of the anomeric configuration and that LamR performs transglycosylation to generate both 1, 3-beta-glycosidic and 1,4-beta glycosidic linkages. The transglycosylation results in, e.g. formation of the trisaccharide 4-O-glucosyl-laminaribiose from exclusively 1,3-beta-oligoglucosides. When barley 1,3-1,4-beta-glucan was incubated with LamR the beta-1, 4-linkages of 3-O-substituted beta-glycosyl residues were rapidly hydrolysed. Simultaneously de novo formation of 1,3-beta-glycosidic linkages was observed which, however, were cleaved during prolonged incubations. It is shown that a laminaribiosyl unit is the minimum requirement for formation of an enzyme-substrate complex and subsequent hydrolysis/transglycosylation.

The two N-terminally repeated carbohydrate-binding modules (CBM4-1 and CBM4-2) encoded by xyn10A from Rhodothermus marinus were produced in Escherichia coli and purified by affinity chromatography. Binding assays to insoluble polysaccharides showed binding to insoluble xylan and to phosphoric-acid-swollen cellulose but not to Avicel or crystalline cellulose. Binding to insoluble substrates was significantly enhanced by the presence of Na(+) and Ca(2+) ions. The binding affinities for soluble polysaccharides were tested by affinity electrophoresis; strong binding occurred with different xylans and beta-glucan. CBM4-2 displayed a somewhat higher binding affinity than CBM4-1 for both soluble and insoluble substrates but both had similar specificities. Binding to short oligosaccharides was measured by NMR; both modules bound with similar affinities. The binding of the modules was shown to be dominated by enthalpic forces. The binding modules did not contribute with any significant synergistic effects on xylan hydrolysis when incubated with a Xyn10A catalytic module. This is the first report of family 4 CBMs with affinity for both insoluble xylan and amorphous cellulose.

The biosynthetic reaction scheme for the compatible solute mannosylglycerate in Rhodothermus marinus is proposed based on measurements of the relevant enzymatic activities in cell-free extracts and in vivo (13)C labeling experiments. The synthesis of mannosylglycerate proceeded via two alternative pathways; in one of them, GDP mannose was condensed with D-glycerate to produce mannosylglycerate in a single reaction catalyzed by mannosylglycerate synthase, in the other pathway, a mannosyl-3-phosphoglycerate synthase catalyzed the conversion of GDP mannose and D-3-phosphoglycerate into a phosphorylated intermediate, which was subsequently converted to mannosylglycerate by the action of a phosphatase. The enzyme activities committed to the synthesis of mannosylglycerate were not influenced by the NaCl concentration in the growth medium. However, the combined mannosyl-3-phosphoglycerate synthase/phosphatase system required the addition of NaCl or KCl to the assay mixture for optimal activity. The mannosylglycerate synthase enzyme was purified and characterized. Based on partial sequence information, the corresponding mgs gene was identified from a genomic library of R. marinus. In addition, the mgs gene was overexpressed in Escherichia coli with a high yield. The enzyme had a molecular mass of 46,125 Da, and was specific for GDP mannose and D-glycerate. This is the first report of the characterization of a mannosylglycerate synthase.

Haloalkane dehalogenase (DhlA) converts haloalkanes to their corresponding alcohols and halide ions. The rate-limiting step in the reaction of DhlA is the release of the halide ion. The kinetics of halide release have been analyzed by measuring halide binding with stopped-flow fluorescence experiments. At high halide concentrations, halide import occurs predominantly via the rapid formation of a weak initial collision complex, followed by transport of the ion to the active site. To obtain more insight in this collision complex, we determined the X-ray structure of DhlA in the presence of bromide and investigated the kinetics of mutants that were constructed on the basis of this structure. The X-ray structure revealed one bromide ion firmly bound in the active site and two bromide ions weakly bound on the surface of the enzyme. One of the weakly bound ions is close to Thr197 and Phe294, near the entrance of the earlier proposed tunnel for substrate import. Kinetic analysis of bromide import by the Thr197Ala and Phe294Ala mutants of DhlA at high halide concentration showed that the rate constants for halide binding no longer displayed a wild-type-like parabolic increase with increasing bromide concentrations. This is in agreement with an elimination or a decrease in affinity of the surface-located halide-binding site. Likewise, chloride binding kinetics of the mutants indicated significant differences with wild-type enzyme. The results indicate that Thr197 and Phe294 are involved in the formation of an initial collision complex for halide import in DhlA and provide experimental evidence for the role of the tunnel in substrate and product transport.

We purified from the culture supernatant of Alteromonas sp. strain O-7 and characterized a transglycosylating enzyme which synthesized beta-(1-->6)-(GlcNAc)2, 2-acetamido-6-O-(2-acetamido-2-deoxy-beta-D-glucopyranosyl)-2- deoxyglucopyranose from beta-(1-->4)-(GlcNAc)2. The gene encoding a novel transglycosylating enzyme was cloned into Escherichia coli, and its nucleotide sequence was determined. The molecular mass of the deduced amino acid sequence of the mature protein was determined to be 99,560 Da which corresponds very closely with the molecular mass of the cloned enzyme determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular mass of the cloned enzyme was much larger than that of enzyme (70 kDa) purified from the supernatant of this strain. These results suggest that the native enzyme was the result of partial proteolysis occurring in the N-terminal region. The enzyme showed significant sequence homology with several bacterial beta-N-acetylhexosaminidases which belong to family 20 glycosyl hydrolases. However, this novel enzyme differs from all reported beta-N-acetylhexosaminidases in its substrate specificity. To clarify the role of the enzyme in the chitinolytic system of the strain, the effect of beta-(1-->6)-(GlcNAc)2 on the induction of chitinase was investigated. beta-(1-->6)-(GlcNAc)2 induced a level of production of chitinase similar to that induced by the medium containing chitin. On the other hand, GlcNAc, (GlcNAc)2, and (GlcNAc)3 conversely repressed the production of chitinase to below the basal level of chitinase activity produced constitutively in medium without a carbon source.

The respiratory chain of the thermohalophilic bacterium Rhodothermus marinus contains a novel complex III and a high potential iron-sulfur protein (HiPIP) as the main electron shuttle (Pereira et al., Biochemistry 38 (1999) 1268-1275 and 1276-1283). In this paper, one of the terminal oxidases expressed in this bacterium is extensively characterised. It is a caa3-type oxidase, isolated with four subunits (apparent molecular masses of 42, 19 and 15 kDa and a C-haem containing subunit of 35 kDa), which has haems of the A(s) type. This oxidase is capable of using TMPD and horse heart cytochrome c as substrates, but has a higher turnover with HiPIP, being the first example of a HiPIP:oxygen oxidoreductase. The oxidase has unusually low reduction potentials of 260 (haem C), 255 (haem A) and 180 mV (haem A3). Subunit I of R. marinus caa3 oxidase has an overall significant homology with the subunits I of the COX type oxidases, namely the metal binding sites and most residues considered to be functionally important for proton uptake and pumping (K- and D-channels). However, a major difference is present: the putative essential glutamate (E278 in Paraccocus denitrificans) of the D-channel is missing in the R. marinus oxidase. Homology modelling of the R. marinus oxidase shows that the phenol group of a tyrosine residue may occupy a similar spatial position as the glutamate carboxyl, in relation to the binuclear centre. Moreover, sequence comparisons reveal that several enzymes lacking that glutamate have a conserved substitution pattern in helix VI: -YSHPXV- instead of -XGHPEV-. These observations are discussed in terms of the mechanisms for proton uptake and it is suggested that, in these enzymes, tyrosine may play the role of the glutamate in the proton channel.

A Rhodothermus marinus gene, hemB, coding for 5-aminolevulinic acid (ALA) dehydratase (ALAD) has been cloned and sequenced. The reading frame of the hemB gene is 1020 base pairs encoding a protein of 340 amino acids with a calculated molecular mass of 37.4 kDa. The amino acid sequence shows homology with eubacterial and eukaryotic ALA dehydratases. A putative metal-binding site of the protein shows strongest homology with corresponding sites from plant ALA dehydratases that require Mg2+ for activity. It differs with respect to only one amino acid out of 20 from a corresponding site in pea ALAD.

The gram-negative marine bacterium Pseudoalteromonas atlantica produces extracellular polysaccharide (EPS) that is important in biofilm formation by this bacterium. Insertion and precise excision of IS492 at a locus essential for extracellular polysaccharide production (eps) controls phase variation of EPS production in P. atlantica. Examination of IS492 transposition in P. atlantica by using a PCR-based assay revealed a circular form of IS492 that may be an intermediate in transposition or a terminal product of excision. The DNA sequence of the IS492 circle junction indicates that the ends of the element are juxtaposed with a 5-bp spacer sequence. This spacer sequence corresponds to the 5-bp duplication of the chromosomal target sequence found at all IS492 insertion sites on the P. atlantica chromosome that we identified by using inverse PCR. IS492 circle formation correlated with precise excision of IS492 from the P. atlantica eps target sequence when introduced into Escherichia coli on a plasmid. Deletion analyses of the flanking host sequences at the eps insertion site for IS492 demonstrated that the 5-bp duplicated target sequence is essential for precise excision of IS492 and circle formation in E. coli. Excision of IS492 in E. coli also depends on the level of expression of the putative transposase, MooV. A regulatory role for the circular form of IS492 is suggested by the creation of a new strong promoter for expression of mooV by the joining of the ends of the insertion sequence element at the circle junction.

Methylobacterium sp. strain CM4 is a strictly aerobic methylotrophic proteobacterium growing with chloromethane as the sole carbon and energy source. Genetic evidence and measurements of enzyme activity in cell-free extracts have suggested a multistep pathway for the conversion of chloromethane to formate. The postulated pathway is initiated by a corrinoid-dependent methyltransferase system involving methyltransferase I (CmuA) and methyltransferase II (CmuB), which transfer the methyl group of chloromethane onto tetrahydrofolate (H4folate) [Vannelli et al. (1999) Proc. Natl Acad. Sci. USA 96, 4615-4620]. We report the overexpression in Escherichia coli and the purification to apparent homogeneity of methyltransferase II. This homodimeric enzyme, with a subunit molecular mass of 33 kDa, catalyzed the conversion of methylcobalamin and H4folate to cob(I)alamin and methyl-H4folate with a specific activity of 22 nmol x min-1 x (mg protein)-1. The apparent kinetic constants for H4folate were: Km = 240 microM, Vmax = 28.5 nmol x min-1 x (mg protein)-1. The reaction appeared to be first order with respect to methylcobalamin at concentrations up to 2 mM, presumably reflecting the fact that methylcobalamin is an artificial substitute for the methylated methyltransferase I, the natural substrate of the enzyme. Tetrahydromethanopterin, a coenzyme also present in Methylobacterium, did not serve as a methyl group acceptor for methyltransferase II. Purified methyltransferase II restored chloromethane dehalogenation by a cell free extract of a strain CM4 mutant defective in methyltransferase II.

The reactions of wild-type trimethylamine dehydrogenase (TMADH) and of a His-172-->Gln (H172Q) mutant were studied by rapid-mixing stopped-flow spectroscopy over the pH range 6.0-10.5, to address the potential role of His-172 in abstracting a proton from the substrate in a 'carbanion' mechanism for C-H bond cleavage. The pH-dependence of the limiting rate for flavin reduction (klim) was studied as a function of pH for the wild-type enzyme with perdeuterated trimethylamine as substrate. The use of perdeuterated trimethylamine facilitated the unequivocal identification of two kinetically influential ionizations in the enzyme-substrate complex, with macroscopic pKa values of 6.5+/-0.2 and 8.4+/-0.1. A plot of klim/Kd revealed a bell-shaped curve and two kinetically influential ionizations with macroscopic pKa values of 9.4+/-0.1 and 10.5+/-0.1. Mutagenesis of His-172, a potential active-site base and a component of a novel Tyr-His-Asp triad in the active site of TMADH, revealed that the pKa of 8.4+/-0.1 for the wild-type enzyme-substrate complex represents ionization of the imidazolium side-chain of His-172. H172Q TMADH retains catalytic competence throughout the pH range investigated. At pH 10.5, and in contrast with the wild-type enzyme, flavin reduction in H172Q TMADH is biphasic. The fast phase is dependent on the trimethylamine concentration and exhibits a kinetic isotope effect of about 3; C-H bond cleavage is thus partially rate-limiting. In contrast, the slow phase does not show hyperbolic dependence on substrate concentration, and the observed rate shows no dependence on isotope, revealing that C-H bond cleavage is not rate-limiting. The analysis of H172Q TMADH, together with data recently acquired for the Y169F mutant of TMADH, reveals that C-H bond breakage is not initiated via abstraction of a proton from the substrate by an active-site base. The transfer of reducing equivalents to flavin via a carbanion mechanism is therefore unlikely.

Crystals of the 35 kDa protein haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 diffract to 1.15 A resolution at cryogenic temperature using synchrotron radiation. Blocked anisotropic least-squares refinement with SHELXL gave a final conventional R factor of 10.51% for all reflections in the 15-1.15 A resolution range. The estimated r.m.s. errors of the model are 0.026 and 0.038 A for protein atoms and all atoms, respectively. The structure comprises all 310 amino acids, with 28 side chains and two peptide bonds in multiple conformations, two covalently linked Pb atoms, 601 water molecules, seven glycerol molecules, one sulfate ion and two chloride ions. Water molecules accounting for alternative solvent structure are modelled with a fixed occupancy of 0.5. The structure is described in detail and compared with previously reported dehalogenase structures refined at 1.9-2.3 A resolution. An analysis of the protein's geometry and stereochemistry reveals eight mean values of bond lengths and angles which deviate significantly from the Engh & Huber parameters, a wide spread in the main-chain omega torsion angle around its ideal value of 180 (6) degrees and a role for C-HcO interactions in satisfying the hydrogen-bond acceptor capacity of main-chain carbonyl O atoms in the central beta-sheet.

Cytochrome cH is the electron donor to the oxidase in methylotrophic bacteria. Its amino acid sequence suggests that it is a typical Class 1 cytochrome c, but some features of the sequence indicated that its structure might be of special interest. The structure of oxidized cytochrome cH has been solved to 2.0 A resolution by X-ray diffraction. It has the classical tertiary structure of the Class 1 cytochromes c but bears a closer gross resemblance to mitochondrial cytochrome c than to the bacterial cytochrome c2. The left-hand side of the haem cleft is unique; in particular, it is highly hydrophobic, the usual water is absent, and the "conserved" Tyr67 is replaced by tryptophan. A number of features of the structure demonstrate that the usual hydrogen bonding network involving water in the haem channel is not essential and that other mechanisms may exist for modulation of redox potentials in this cytochrome.

A total of 59 bacteria samples from Antarctic sea water were collected and screened for their ability to produce alpha-amylase. The highest activity was recorded from an isolate identified as an Alteromonas species. The purified alpha-amylase shows a molecular mass of about 50,000 Da and a pI of 5.2. The enzyme is stable from pH 7.5 to 9 and has a maximal activity at pH 7.5. Compared with other alpha-amylases from mesophiles and thermophiles, the "cold enzyme" displays a higher activity at low temperature and a lower stability at high temperature. The psychrophilic alpha-amylase requires both Cl- and Ca2+ for its amylolytic activity. Br- is also quite efficient as an allosteric effector. The comparison of the amino acid composition with those of other alpha-amylases from various organisms shows that the cold alpha-amylase has the lowest content in Arg and Pro residues. This could be involved in the principle used by the psychrophilic enzyme to adapt its molecular structure to the low temperature of the environment.

Enzymes catalyzing the hydrolysis of highly toxic organophosphorus compounds (OPs) are classified as organophosphorus acid anhydrolases (OPAA; EC 3.1.8.2). Recently, the genes encoding OPAA from two species of Alteromonas were cloned and sequenced. Sequence and biochemical analyses of the cloned genes and enzymes have established Alteromonas OPAAs to be prolidases (E.C. 3.4.13.9), a type of dipeptidase hydrolyzing dipeptides with a prolyl residue in the carboxyl-terminal position (X-Pro). Alteromonas prolidases hydrolyze a broad range of G-type chemical warfare (CW) nerve agents. Efforts to over-produce a prolidase from A. sp.JD6.5 with the goal of developing strategies for long-term storage and decontamination have been successfully achieved. Large-scale production of this G-agent degrading enzyme is now feasible with the availability of an over-producing recombinant cell line. Use of this enzyme for development of a safe and non-corrosive decontamination system is discussed.

The reductive half-reaction of trimethylamine dehydrogenase with its physiological substrate trimethylamine has been examined by stopped-flow spectroscopy over the pH range 6.0-11.0, with attention focusing on the fastest of the three kinetic phases of the reaction, the flavin reduction/substrate oxidation process. As in previous work with the slow substrate diethylmethylamine, the reaction is found to consist of three well resolved kinetic phases. The observed rate constant for the fast phase exhibits hyperbolic dependence on the substrate concentration with an extrapolated limiting rate constant (klim) greater than 1000 s-1 at pH above 8.5, 10 degrees C. The kinetic parameter klim/Kd for the fast phase exhibits a bell-shaped pH dependence, with two pKa values of 9.3 +/- 0.1 and 10. 0 +/- 0.1 attributed to a basic residue in the enzyme active site and the ionization of the free substrate, respectively. The sigmoidal pH profile for klim gives a single pKa value of 7.1 +/- 0. 2. The observed rate constants for both the intermediate and slow phases are found to decrease as the substrate concentration is increased. The steady-state kinetic behavior of trimethylamine dehydrogenase with trimethylamine has also been examined, and is found to be adequately described without invoking a second, inhibitory substrate-binding site. The present results demonstrate that: (a) substrate must be protonated in order to bind to the enzyme; (b) an ionization group on the enzyme is involved in substrate binding; (c) an active site general base is involved, but not strictly required, in the oxidation of substrate; (d) the fast phase of the reaction with native enzyme is considerably faster than observed with enzyme isolated from Methylophilus methylotrophus that has been grown up on dimethylamine; and (e) a discrete inhibitory substrate-binding site is not required to account for excess substrate inhibition, the kinetic behavior of trimethylamine dehydrogenase can be readily explained in the context of the known properties of the enzyme.

The substrate specificities and product inhibition patterns of haloalkane dehalogenases from Xanthobacter autotrophicus GJ10 (XaDHL) and Rhodococcus rhodochrous (RrDHL) have been compared using a pH-indicator dye assay. In contrast to XaDHL, RrDHL is efficient toward secondary alkyl halides. Using steady-state kinetics, we have shown that halides are uncompetitive inhibitors of XaDHL with 1, 2-dichloroethane as the varied substrate at pH 8.2 (Cl-, Kii = 19 +/- 0.91; Br-, Kii = 2.5 +/- 0.19 mM; I-, Kii = 4.1 +/- 0.43 mM). Because they are uncompetitive with the substrate, halide ions do not bind to the free form of the enzyme; therefore, halide ions cannot be the last product released from the enzyme. The Kii for chloride was pH dependent and decreased more than 20-fold from 61 mM at pH 8.9 to 2.9 mM at pH 6.5. The pH dependence of 1/Kii showed simple titration behavior that fit to a pKa of approximately 7.5. The kcat was maximal at pH 8.2 and decreased at lower pH. A titration of kcat versus pH also fits to a pKa of approximately 7.5. Taken together, these data suggest that chloride binding and kcat are affected by the same ionizable group, likely the imidazole of a histidyl residue. In contrast, halides do not inhibit RrDHL. The Rhodococcus enzyme does not contain a tryptophan corresponding to W175 of XaDHL, which has been implicated in halide ion binding. The site-directed mutants W175F and W175Y of XaDHL were prepared and tested for halide ion inhibition. Halides do not inhibit either W175F or W175Y XaDHL.

The thermal stability of the cold-active alpha-amylase (AHA) secreted by the Antarctic bacterium Alteromonas haloplanctis has been investigated by intrinsic fluorescence, circular dichroism, and differential scanning calorimetry. It was found that this heat-labile enzyme is the largest known multidomain protein exhibiting a reversible two-state unfolding, as demonstrated by the recovery of DeltaHcal values after consecutive calorimetric transitions, a DeltaHcal/DeltaHeff ratio close to unity, and the independence of unfolding thermodynamic parameters of scan rates. By contrast, the mesophilic alpha-amylases investigated here (from porcine pancreas, human salivary glands, yellow meal beetle, Bacillus amyloliquefaciens, and Bacillus licheniformis) unfold irreversibly according to a non-two-state mechanism. Unlike mesophilic alpha-amylases, the melting point of AHA is independent of calcium and chloride binding while the allosteric and structural functions of these ions are conserved. The thermostability of AHA at optimal conditions is characterized by a Tm of 43.7 degrees C, a DeltaHcal of 238 kcal mol-1, and a DeltaCp of 8.47 kcal mol-1 K-1. These values were used to calculate the Gibbs free energy of unfolding over a wide range of temperatures. This stability curve shows that (a) the specific DeltaGmax of AHA [22 cal (mol of residue)-1] is 4 times lower than that of mesophilic alpha-amylases, (b) group hydration plays a crucial role in the enzyme flexibility at low temperatures, (c) the temperature of cold unfolding closely corresponds to the lower limit of bacterial growth, and (d) the recombinant heat-labile enzyme can be expressed in mesophilic hosts at moderate temperatures. It is also argued that the cold-active alpha-amylase has evolved toward the lowest possible conformational stability of its native state.

A recombinant form of His-tagged kappa-carrageenase from Pseudoalteromonas carrageenovora has been expressed, purified and crystallized. Crystals have been obtained by the vapour-diffusion method using polyethylene glycol (Mr = 4000) as a precipitant. These crystals belong to the space group P212121, with unit-cell parameters a = 58.2, b = 62.8, c = 77.9 A, and diffract to 2.2 A resolution on a rotating-anode X-ray source.

A Hydrogenophaga pseudoflava strain was able to synthesize poly(3-hydroxybutyric acid-co-4-hydroxybutyric acid) [P(3HB-co-4HB)] having a high level of 4-hydroxybutyric acid monomer unit (4HB) from gamma-butyrolactone. In a two-step process in which the first step involved production of cells containing a minimum amount of poly(3-hydroxybutyric acid) [P(3HB)] and the second step involved polyester accumulation from the lactone, approximately 5 to 10 mol% of the 3-hydroxybutyric acid (3HB) derived from the first-step culture was unavoidably reincorporated into the polymer in the second cultivation step. Reincorporation of the 3HB units produced from degradation of the first-step residual P(3HB) was confirmed by high-resolution 13C nuclear magnetic resonance spectroscopy. In order to synthesize 3HB-free poly(4-hydroxybutyric acid) [P(4HB)] homopolymer, a three-stage cultivation technique was developed by adding a nitrogen addition step, which completely removed the residual P(3HB). The resulting polymer was free of 3HB. However, when the strain was grown on gamma-butyrolactone as the sole carbon source in a synthesis medium, a copolyester of P(3HB-co-4HB) containing 45 mol% 3HB was produced. One-step cultivation on gamma-butyrolactone required a rather long induction time (3 to 4 days). On the basis of the results of an enzymatic study performed with crude extracts, we suggest that the inability of cells to produce 3HB in the multistep culture was due to a low level of 4-hydroxybutyric acid (4HBA) dehydrogenase activity, which resulted in a low level of acetyl coenzyme A. Thus, 3HB formation from gamma-butyrolactone is driven by a high level of 4HBA dehydrogenase activity induced by long exposure to gamma-butyrolactone, as is the case for a one-step culture. In addition, intracellular degradation kinetics studies showed that P(3HB) in cells was completely degraded within 30 h of cultivation after being transferred to a carbon-free mineral medium containing additional ammonium sulfate, while P(3HB-co-4HB) containing 5 mol% 3HB and 95 mol% 4HB was totally inert in interactions with the intracellular depolymerases. Intracellular inertness could be a useful factor for efficient synthesis of the P(4HB) homopolymer and of 4HB-rich P(3HB-co-4HB) by the strain used in this study.

Recently it was found that Methylobacterium extorquens AM1 contains both tetrahydromethanopterin (H4MPT) and tetrahydrofolate (H4F) as carriers of C1 units. In this paper we report that the aerobic methylotroph contains a methenyl H4MPT cyclohydrolase (0.9 U x mg-1 cell extract protein) and a methenyl H4F cyclohydrolase (0.23 U x mg-1). Both enzymes, which were specific for their substrates, were purified and characterized and the encoding genes identified via the N-terminal amino acid sequence. The purified methenyl H4MPT cyclohydrolase with a specific activity of 630 U x mg-1 (Vmax = 1500 U x mg-1; Km = 30 microm) was found to be composed of two identical subunits of molecular mass 33 kDa. Its sequence was approximately 40% identical to that of methenyl H4MPT cyclohydrolases from methanogenic archaea. The methenyl H4F cyclohydrolase with a specific activity of 100 U x mg-1 (Vmax = 330 U x mg-1; Km = 80 microm) was found to be composed of two identical subunits of molecular mass 22 kDa. Its sequence was not similar to that of methenyl H4MPT cyclohydrolases or to that of other methenyl H4F cyclohydrolases. Based on the specific activities in cell extract and from the growth properties of insertion mutants it is suggested that the methenyl H4MPT cyclohydrolase might have a catabolic, and the methenyl-H4F cyclohydrolase an anabolic function in the C1-unit metabolism of M. extorquens AM1.

A cluster of three closely linked chitinase genes organized in the order chiA, chiB and chiC, with the same transcriptional direction, and two unlinked genes, chiP and chiQ, involved in chitin degradation in Pseudoalteromnas sp. strain S91 were cloned, sequenced and characterized. The deduced amino acid sequences revealed that ChiA, ChiB and ChiC exhibited similarities to chitinases belonging to family 18 of the glycosyl hydrolases while ChiP and ChiQ belonged to family 20. ChiP and ChiQ showed different enzymic activities against fluorescent chitin analogues, but neither was able to degrade colloidal chitin. ChiA possessed chitinase activity but did not bind chitin; ChiB bound chitin but had no chitinase activity; ChiC possessed strong chitinase activity and also bound chitin. Production of ChiC in S91 appeared to be controlled by chiA expression, since insertion of a transposon into the ORF of chiA resulted in the loss of chitinase activity as well as loss of ChiC proteins in a chitinase-negative mutant. In Escherichia coli, ChiC appeared to be expressed from its own promoter.

The deduced primary sequence (cbbL and cbbS) of form I ribulose 1, 5-bisphosphate carboxylase/oxygenase (rubisco) from Bradyrhizobium japonicum places this enzyme within the Type IC subgroup of red-like rubisco enzymes. In addition, B. japonicum appears to organize most of the structural genes of the Calvin-Benson-Bassham (CBB) pathway in at least one major operon. Functional expression and characterization of the B. japonicum and Xanthobacter flavus enzymes from this group revealed that these molecules exhibit diverse kinetic properties despite their relatively high degree of sequence relatedness. Of prime importance was the fact that these closely related enzymes exhibited CO2 and O2 substrate specificities that varied from relatively low values [tau = (VcKo)/(VoKc) = 45] to values that approximated those obtained for higher plants (tau = 75). These results, combined with the metabolic and genetic versatility of the organisms from which these enzymes were derived, suggest a potential rich resource for future biological selection and structure-function studies aimed at elucidating structural features that govern key enzymological properties of rubisco., (Copyright 1999 Academic Press.)

Epoxide carboxylase from the bacterium Xanthobacter strain Py2 is a multicomponent enzyme system which catalyzes the pyridine nucleotide-dependent carboxylation of aliphatic epoxides to beta-ketoacids as illustrated by the reaction epoxypropane + CO2 + NADPH + NAD+ --> acetoacetate + H+ + NADP+ + NADH. The combination of four distinct proteins, designated components I-IV, are required for the reconstitution of epoxide carboxylase activity with racemic mixtures of short-chain (C3-C5) terminal epoxyalkanes. In this work, components III and IV of the epoxide carboxylase system are shown to confer specificity for epoxyalkane enantiomers. Components I-III supported the carboxylation of (R)-epoxypropane, while components I, II, and IV supported the carboxylation of (S)-epoxypropane. At fixed concentrations of components I and II, the rates of (R)- and (S)-epoxypropane carboxylation saturated with increasing concentrations of component III or IV to give identical maximal rates for the two epoxide substrates. (S)-Epoxypropane was an inactivator of (R)-epoxypropane carboxylation by components I- III, while (R)-epoxypropane was an inactivator of (S)-epoxypropane carboxylation by components I, II, and IV. These inactivating effects were fully reversed upon the addition of the correct complementing dehydrogenase component. Amino acid sequence analysis of components III and IV demonstrates that they belong to the short-chain dehydrogenase/reductase (SDR) family of enzymes. Both components contain highly conserved residues within the coenzyme binding fold and catalytic regions found in SDR enzymes. Components III and IV are proposed to catalyze the NAD+-dependent abstraction of a hydride from a chiral secondary alcohol-like intermediate bound to the active site component of the enzyme system to form the corresponding beta-ketone intermediate. A multicomponent epoxide carboxylase system was purified to homogeneity from Nocardia corallina B276, a bacterium phylogenetically unrelated to Xanthobacter Py2, and found to consist of four proteins with functions identical to those of the Xanthobacter Py2 system. The stereoselective dehydrogenases of the Xanthobacter epoxide carboxylase system were able to substitute for the corresponding components of the N. corallina system when using (R)- and (S)-epoxypropane as substrates, and vice versa. These results provide the first demonstration of the involvement of stereospecific dehydrogenases in aliphatic epoxide metabolism and provide new insights into microbial strategies for the utilization of chiral organic molecules.

Background: . Enzymes from psychrophilic (cold-adapted) microorganisms operate at temperatures close to 0 degreesC, where the activity of their mesophilic and thermophilic counterparts is drastically reduced. It has generally been assumed that thermophily is associated with rigid proteins, whereas psychrophilic enzymes have a tendency to be more flexible., Results: . Insights into the cold adaptation of proteins are gained on the basis of a psychrophilic protein's molecular structure. To this end, we have determined the structure of the recombinant form of a psychrophilic alpha-amylase from Alteromonas haloplanctis at 2.4 A resolution. We have compared this with the structure of the wild-type enzyme, recently solved at 2.0 A resolution, and with available structures of their mesophilic counterparts. These comparative studies have enabled us to identify possible determinants of cold adaptation., Conclusions: . We propose that an increased resilience of the molecular surface and a less rigid protein core, with less interdomain interactions, are determining factors of the conformational flexibility that allows efficient enzyme catalysis in cold environments.