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PubMed1997

Crystal structure of nitrile hydratase reveals a novel iron centre in a novel fold.

BACKGROUND: Nitrile hydratases are unusual metalloenzymes that catalyze the hydration of nitriles to their corresponding amides. They are used as biocatalysts in acrylamide production, one of the few commercial scale bioprocesses, as well as in environmental remediation for the removal of nitriles from waste streams. Nitrile hydratases are composed of two subunits, alpha and beta, and they contain one iron atom per alphabeta unit. We have determined the crystal structure of photoactivated iron-containing nitrile hydratase from Rhodococcus sp. R312 to 2.65 A resolution as a first step in the elucidation of its catalytic mechanism. RESULTS: The alpha subunit consists of a long N-terminal arm and a C-terminal domain that forms a novel fold. This fold can be described as a four layered structure, alpha-beta-beta-alpha, with unusual connectivities between the beta strands. The beta subunit also contains a long N-terminal extension, a helical domain, and a C-terminal domain that folds into a beta roll. The two subunits form a tight heterodimer that is the functional unit of the enzyme. The active site is located in a cavity at the subunit-subunit interface. The iron centre is formed by residues from the alpha subunit only-three cysteine thiolates and two mainchain amide nitrogen atoms are ligands. CONCLUSIONS: Nitrile hydratases contain a novel iron centre with a structure not previously observed in proteins; it resembles a hybrid of the iron centres of heme and Fe-S proteins. The low-spin electronic configuration presumably results in part from two Fe-amide nitrogen bonds. The structure is consistent with the metal ion having a role as a Lewis acid in the catalytic reaction.

Binding Sites↗
PubMed2000

Stable isotope incorporation triples the upper mass limit for determination of elemental composition by accurate mass measurement.

By comparing electrospray ionization Fourier-transform ion cyclotron resonance (FT-ICR) mass spectra and collision-induced dissociation (CID) FT-ICR mass spectra of a phospholipid (851 Da) extracted from natural abundance and 99% 13C bacterial growth media, we are able to reduce its number of possible elemental compositions (based on +/-10 ppm externally calibrated mass accuracy and biologically relevant compositional constraints) from 394 to 1. The basic idea is simply that the mass of a molecule containing N carbon atoms increases by N Da when 12C is replaced by 13C. Once the number of carbons is known, the number of possible combinations of other atoms in the molecule is greatly reduced. We demonstrate the method for a stored-waveform inverse Fourier transform-isolated phospholipid from an extract of membrane lipids from Rhodococcus rhodochrous hydrocarbon-degrading bacteria grown on either natural abundance or 99% 13C-enriched mixtures of n-hexadecane and n-octadecane. We project that this method raises the upper mass limit for unique determination of elemental composition from accurate mass measurement by a factor of at least 3, thereby extending "chemical formula" determination to identification and sequencing of larger synthetic and bio-polymers: phospholipids, oligopeptides of more than three to four amino acids, DNA or RNA of more than two nucleotides, oligosaccharides of more than three sugars, etc. The method can also be extended to determination of the number of other atoms for which heavy isotopes are available (e.g., 15N, 34S, 18O, etc.).

Carbon Isotopes↗
PubMed2000

Crystal structure of the haloalkane dehalogenase from Sphingomonas paucimobilis UT26.

The haloalkane dehalogenase from Sphingomonas paucimobilis UT26 (LinB) is the enzyme involved in the degradation of the important environmental pollutant gamma-hexachlorocyclohexane. The enzyme hydrolyzes a broad range of halogenated cyclic and aliphatic compounds. Here, we present the 1.58 A crystal structure of LinB and the 2.0 A structure of LinB with 1,3-propanediol, a product of debromination of 1,3-dibromopropane, in the active site of the enzyme. The enzyme belongs to the alpha/beta hydrolase family and contains a catalytic triad (Asp108, His272, and Glu132) in the lipase-like topological arrangement previously proposed from mutagenesis experiments. The LinB structure was compared with the structures of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 and from Rhodococcus sp. and the structural features involved in the adaptation toward xenobiotic substrates were identified. The arrangement and composition of the alpha-helices in the cap domain results in the differences in the size and shape of the active-site cavity and the entrance tunnel. This is the major determinant of the substrate specificity of this haloalkane dehalogenase.

Binding Sites↗
PubMed2001

Mössbauer and EPR studies of the photoactivation of nitrile hydratase.

The alphabeta dimer of active nitrile hydratase from Rhodococcus sp. R312 contains one low-spin ferric ion that is coordinated by three Cys residues, two N-amide groups from the protein backbone, and one OH(-). The enzyme isolated from bacteria grown in the dark is inactive and contains the iron site as a six-coordinate diamagnetic Fe-nitrosyl complex, called NH(dark). The active state can be obtained from the dark state by photolysis of the Fe-NO bond at room temperature. Activation is accompanied by the conversion of NH(dark) to a low-spin ferric complex, NH(light), exhibiting an S = (1)/(2) EPR signal with g values of 2.27, 2.13, and 1.97. We have characterized both NH(dark) and NH(light) with Mössbauer spectroscopy. The z-axis of the 57Fe magnetic hyperfine tensor, A, of NH(light) was found to be rotated by approximately 45 degrees relative to the z-axis of the g tensor (g(z) = 1.97). Comparison of the A tensor of NH(light) with the A tensors of low-spin ferric hemes indicates a substantially larger degree of covalency for nitrile hydratase. We have also performed photolysis experiments between 2 and 20 K and characterized the photolyzed products by EPR and Mössbauer spectroscopy. Photolysis at 4.2 K in the Mössbauer spectrometer yielded a five-coordinate low-spin ferric species, NH(A), which converted back into NH(dark) when the sample was briefly warmed to 77 K. We also describe preliminary EPR photolysis studies that have yielded new intermediates.

Darkness↗
PubMed2003

Protonation structures of Cys-sulfinic and Cys-sulfenic acids in the photosensitive nitrile hydratase revealed by Fourier transform infrared spectroscopy.

Nitrile hydratase (NHase) from Rhodococcus N-771, which catalyzes hydration of nitriles to the corresponding amides, exhibits novel photosensitivity; in the dark, it is in the inactive form that binds an endogenous nitric oxide (NO) molecule at the non-heme iron center, and photodissociation of the NO activates the enzyme. NHase is also known to have a unique active site structure. Two cysteine ligands to the iron center, alphaCys112 and alphaCys114, are post-translationally modified to sulfinic acid (Cys-SO(2)H) and sulfenic acid (Cys-SOH), respectively, which are thought to play a crucial role in the catalytic reaction. Here, we have determined the protonation structures of these Cys-SO(2)H and Cys-SOH groups using Fourier transform infrared (FTIR) spectroscopy in combination with density functional theory (DFT) calculations. The light-induced FTIR difference spectrum of NHase between the dark inactive and light active forms exhibited two prominent signals at (1154-1148)/1126 and (1040-1034)/1019 cm(-1), which downshifted to 1141/1114 and 1026/1012 cm(-1), respectively, in the uniformly (34)S-labeled NHase. In addition, a minor signal at 915/908 cm(-1) also showed a considerable downshift upon (34)S labeling. These (34)S-sensitive signals were basically conserved in D(2)O buffer with only slight shifts. Vibrational frequencies of methanesulfenic acid (CH(3)SOH) and methanesulfinic acid (CH(3)SO(2)H), simple model compounds of Cys-SOH and Cys-SO(2)H, respectively, were calculated using the DFT method in both the protonated and deprotonated forms and in metal complexes. Comparison of the calculated frequencies and isotope shifts with the observed ones provided the assignment of the two major signals around 1140 and 1030 cm(-1) to the asymmetric and symmetric SO(2) stretching vibrations, respectively, of the S-bonded Cys-SO(2)(-) complex, and the assignment of the minor signal around 910 cm(-1) most likely to the SO stretch of the S-bonded Cys-SO(-) complex. These assignments and the small frequency shifts upon deuteration are consistent with the view that the deprotonated alphaCys112-SO(2)(-) and alphaCys114-SO(-) are hydrogen-bonded with the protons from betaArg56 and/or betaArg141, forming a reactive cavity at the interface of the alpha and beta subunits. There is further speculation that either of these groups is hydrogen bonded to a reactant water molecule, increasing its basicity to facilitate the nucleophilic attack on the nitrile substrate bound to the iron center.

Cysteine↗
PubMed1996

Resonance Raman spectroscopy of nitrile hydratase, a novel iron-sulfur enzyme.

Resonance Raman spectra of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase, a novel non-heme iron enzyme, have a large number of peaks in the 300-500 cm-1 region; observation of shifts in these peaks after labeling with 34S shows that they arise from cysteine coordinated to the ferric ion in the protein. The rich Raman spectra result from coupling of the Fe-S stretch with cysteine side chain deformation modes; the observation of 15N isotope shifts in most of these peaks suggests participation of N-donor metal ligands and peptide backbone amide nitrogens in these modes as well. The aggregate 34S isotope shift is too large to result from a single cysteine ligand, consistent with the analysis of EXAFS data that shows two or three S-donor ligands [Scarrow et al. (1996) Biochemistry 35, 10078-10088]. Widespread 2H isotope shifts seen after exchange of the protein into 2H2O suggest the presence of hydrogen bonds to the coordinated cysteine sulfurs. Comparison of the resonance Raman spectra of nitrile hydratase prepared at pH 7.3 and 9.0 shows a shift of intensity into the higher-energy peaks in the spectra of the latter sample. This is interpreted as resulting from an increase in Fe-S bond strength at the higher pH and is supported by observation of a small decrease in Fe-S bond length in the EXAFS analysis [Scarrow et al. (1996) Biochemistry 35, 10078-10088]. Such a decrease in Fe-S bond length is also consistent with pH dependent changes in EPR spectra and could reflect the loss of one or more hydrogen bonds to sulfur ligands.

Brevibacterium↗
PubMed1996

X-ray spectroscopy of nitrile hydratase at pH 7 and 9.

The iron K-edge X-ray absorption spectrum of Rhodococcus sp. R312 (formerly Brevibacterium sp. R312) nitrile hydratase in frozen solutions at pH 7 and 9 has been analyzed to determine details of the iron coordination. EXAFS analysis implies two or three sulfur ligands per iron and overall six coordination; together with previous EPR and ENDOR results, this implies an N3S2O ligation sphere. The bond lengths from EXAFS analysis [rav(Fe-S) = 2.21 A at pH 7.3; rav(Fe-N/O) = 1.99 A] support cis coordination of two cysteine ligands and conclusively rule out nitric oxide coordination to the iron, a possibility proposed on the basis of an FTIR difference experiment [Noguchi, T., Honda, J., Nagamune, T., Sasabe, H., Inoue, Y., & Endo, I. (1995) FEBS Lett. 358, 9-12]. The higher-frequency EXAFS can be simulated well by inclusion of multiple scattering from two or three imidazole ligands; the fit to the data is improved if first-sphere multiple scattering pathways are also included. A slight shortening (by 0.02 +/- 0.01 A) of one or both Fe-S bonds when the pH is raised from 7.3 to 9.0 is consistent with shifts observed in the Raman spectrum [Brennan et al. (1996) Biochemistry 35, 10068-10077].

Absorptiometry, Photon↗
PubMedDate not supplied

Continuous desulfurization of dibenzothiophene with Rhodococcusrhodochrous IGTS8 (ATCC 53968).

Desulfurization of a model fuel system consisting of hexadecane and dibenzothiophene (DBT) by Rhodococcus rhodochrous IGTS8 was demonstrated in a 2-L continuous stirred tank reactor (CSTR). The reactor was operated in a semicontinuous and continuous mode with and without recycling of the model fuel. A constant volumetric desulfurization activity A(t), (in mg HBP L(-1) h(-1)) was maintained in the reactor with a feeding strategy of fresh cell suspension based on a first-order decay of the biocatalyst. Maximum desulfurization rates, as measured by specific desulfurization activity, of 1.9 mg HBP/g DCW h were attained. Rates of biocatalyst decay were on the order of 0.072 h(-1). Theoretical predictions of a respiratory quotient (RQ) associated with this biotransformation reaction agree well with experimental data from off-gas analysis. In addition, the ratio of the specific desulfurization activity a(t), (in mg HBP/g DCW h) of recycled and fresh biocatalyst was determined and evaluated.

Bioreactors↗
PubMed2003

Biosynthesis of CaCO3 crystals of complex morphology using a fungus and an actinomycete.

The biosynthesis of CaCO3 by reaction of aqueous Ca2+ ions with a fungus, Fusarium sp., and an actinomycete, Rhodococcus sp. (both plant organisms), is described. In the case of the fungus, cruciform-shaped calcite crystals are obtained (SEM picture A) while the actinomycete yielded the unstable polymorph of CaCO3, vaterite (SEM picture B). Specific proteins secreted by the microorganisms are responsible for the morphology and crystallography control observed. A highlight of this approach is that the microorganisms also provide CO2 for reaction with the Ca2+ ions, making the crystals completely biogenic.

Calcium Carbonate↗
PubMed2002

Practical and convenient enzymatic synthesis of enantiopure alpha-amino acids and amides.

Catalyzed by the nitrile hydratase and the amidease in Rhodococcus sp. AJ270 cells under very mild conditions, a number of alpha-aryl- and alpha-alkyl-substituted DL-glycine nitriles 1 rapidly underwent a highly enantioselective hydrolysis to afford D-(-)-alpha-amino acid amides 2 and L-(+)-alpha-amino acids 3 in high yields with excellent enantiomeric excesses in most cases. The overall enantioselectivity of the biotransformations of nitriles originated from the combined effects of a high L-enantioselective amidase and a low enantioselective nitrile hydratase. The influence of the substrates on both reaction efficiency and enantioselectivity was also discussed in terms of steric and electronic effects. Coupled with chemical hydrolysis of D-(-)-alpha-phenylglycine amide, biotransformation of DL-phenylglycine nitrile was applied in practical scale to produce both D- and L-phenylglycines in high optical purity.

Amides↗
PubMed2003

Nitrile biotransformation for highly enantioselective synthesis of 3-substituted 2,2-dimethylcyclopropanecarboxylic acids and amides.

Biotransformations of differently configured 2,2-dimethyl-3-substitued-cyclopropanecarbonitriles were studied using a nitrile hydratase/amidase-containing Rhodococcus sp. AJ270 whole-cell catalyst under very mild conditions. Although all of the cis-3-aryl-2,2-dimethylcyclopropanecarbonitriles appeared inert toward the biocatalyst, a number of racemic trans-isomers efficiently underwent a highly enantioselective hydrolysis to produce (+)-(1R,3R)-3-aryl-2,2-dimethylcyclopropanecarboxylic acids and (-)-(1S,3S)-3-aryl-2,2-dimethylcyclopropanecarboxamides in high yields with excellent enantiomeric excesses in most cases. The overall enantioselectivity of the biotransformations of nitriles originated from the combined effects of 1R-enantioselective nitrile hydratase and amidase, with the later being a dominant factor. The influence of the substrates on both reaction efficiency and enantioselectivity was discussed in terms of steric and electronic effects. Coupled with chemical transformations, biotransformations of nitriles provided convenient syntheses of optically pure geminally dimethyl-substituted cyclopropanecarboxylic acids and amides, including chrysanthemic acids, in both enantiomeric forms.

Amides↗
PubMed2003

Nitrile biotransformations for highly efficient and enantioselective syntheses of electrophilic oxiranecarboxamides.

Catalyzed by a nitrile hydratase/amidase-containing microbial Rhodococcus sp. AJ270 whole-cell catalyst, a number of racemic trans-2,3-epoxy-3-arylpropanenitriles 1 underwent rapid and efficient hydrolysis under very mild conditions to afford 2R,3S-2-arylglycidamides 2 in excellent yield with enantiomeric excess higher than 99.5%. The overall enantioselectivity of the biotransformations originated from the combined effects of a dominantly high 2S-enantioselective amidase and low 2S-enantioselective nitrile hydratase involved in the cell. The influence of the substrates on both reaction efficiency and enantioselectivity was also discussed in terms of steric and electronic effects.

Amides↗
PubMed2005

Nitrile biotransformations for highly enantioselective synthesis of oxiranecarboxamides with tertiary and quaternary stereocenters; efficient chemoenzymatic approaches to enantiopure alpha-methylated serine and isoserine derivatives.

[reaction: see text] Biotransformations of a number of differently substituted and configured oxiranecarbonitriles using Rhodococcus sp. AJ270, a microbial whole-cell catalyst that contains nitrile hydratase/amidase, were studied. While almost all trans-configured 3-aryl-2-methyloxiranecarbonitriles and 2,3-dimethyl-3-phenyloxiranecarbonitrile were efficiently hydrated by the action of the less enantioselective nitrile hydratase, the amidase exhibited excellent 2S,3R-enantioselectivity against 2-methyl-3-(para-substituted-phenyl)oxiranecarboxamides. Under very mild conditions, biotransformations of nitriles provided an efficient and practical synthesis of 2R,3S-(-)-3-aryl-2-methyloxiranecarboxamides, electrophilic epoxides with tertiary and quaternary stereocenters, in excellent yield with enantiomeric excess greater than 99.5%. The synthetic applications of the resulting enantiomerically pure epoxides were demonstrated by convenient and straightforward syntheses of polyfunctionalized chiral molecules possessing a quaternary stereocenter such as R-(+)-2-hydroxy-2-methyl-3-phenylpropionic acid, 2R,3R-(-)-3-amino-2-hydroxy-2-methyl-3-phenylpropionic acid, and 2S,3S-(+)-2-amino-3-hydroxy-2-methyl-3-phenylpropionic acid, employing the regio- and stereospecific epoxide ring opening reactions of 2R,3S-(-)-2-methyl-3-phenyloxiranecarboxamide as the key steps.

Amides↗
PubMed2005

Effect of a metal alloy fuel catalyst on bacterial growth.

Many microorganisms have been demonstrated to utilize petroleum fuel products to fulfill their nutritional requirement for carbon. As a result, the ability of these microbes to degrade fuel has both a deleterious affect as well as beneficial applications. This study focused on the undesired ability of bacteria to grow on fuel and the potential for some metal alloys to inhibit this biodegradation. The objective of this study was to review the pattern of growth of two reference strains of petroleum-degrading bacteria, Pseudomonas oleovorans and Rhodococcus rhodocrous, in a specific hydrocarbon environment in the presence of a commercially available alloy. The alloy formulated and supplied by Advanced Power Systems International Inc. (APSI) is sold for fuel reformulation and other purposes. The components of the alloy used in the study were antimony, tin, lead, and mercury formulated as pellets. Surface characterization also showed the presence of tin oxide and lead amalgam phases. Hydrocarbon used for the study was primarily 87-octane gasoline. The growth of the bacteria in the water and mineral-supplemented gasoline mixture over 6-8 weeks was monitored by the viable plate count method. While an initial increase in bacteria occurred in the first week, overall bacterial growth was found to be suppressed in the presence of the alloy. Results also indicate that the alloy surface characteristics that convey the catalytic activity may also contribute to the observed antibacterial activity.

Catalysis↗
PubMed2000

Synthesis and antifungal activity of rhodopeptin analogues. 1. Modification of the east and south amino acid moieties.

Structure-activity relationships of the east and south amino acid modified analogues of rhodopeptins, novel antifungal cyclic tetrapeptides isolated from Rhodococcus species Mer-N1033, have been investigated. It was observed that a basic amino acid moiety (lysine or ornithine) as the east amino acid and a hydrophobic and bulky neutral amino acid (i.e., gamma-methylleucine) as the south amino acid were indispensable structure motifs for antifungal activity of rhodopeptin analogues.

Amino Acids↗
PubMed2006

Dramatic enhancement of enantioselectivity of biotransformations of beta-hydroxy nitriles using a simple O-benzyl protection/docking group.

[Structure: see text] Catalyzed by the Rhodococcus erythropolis AJ270 whole cell catalyst, the O-benzylated beta-hydroxy alkanenitriles underwent remarkably high enantioselective biotransformations, whereas the biotransformations of free beta-hydroxy alkanenitriles gave very low enantioselectivity. The easy manipulations of O-protection and O-deprotection, excellent chemical and enantiomeric yields of biotransformations, along with the scalability render this enzymatic transformation attractive and practical for the synthesis of highly enantiopure beta-hydroxy alkanoic acids and their amide derivatives.

Amides↗
PubMed2006

Proteome changes after metabolic engineering to enhance aerobic mineralization of cis-1,2-dichloroethylene.

Metabolically engineered Escherichia coli has previously been used to degrade cis-1,2-dichloroethylene (cis-DCE). The strains express the six genes of an evolved toluene ortho-monooxygenase from Burkholderia cepacia G4 (TOM-Green, which formed a reactive epoxide) with either (1) gamma-glutamylcysteine synthetase (GSHI, which forms glutathione) and the glutathione S-transferase IsoILR1 from Rhodococcus AD45 (which adds glutathione to the reactive cis-DCE epoxide) or (2) with an evolved epoxide hydrolase from Agrobacterium radiobacter AD1 (EchA F108L/I219L/C248I which converts the reactive cis-DCE epoxide to a diol). Here, the impact of this metabolic engineering for bioremediation was assessed by investigating the changes in the proteome through a quantitative shotgun proteomics technique (iTRAQ) by tracking the changes due to the sequential addition of TOM-Green, IsoILR1, and GSHI and due to adding the evolved EchA versus the wild-type enzyme to TOM-Green. For the TOM-Green/EchA system, 8 proteins out of 268 identified proteins were differentially expressed in the strain expressing EchA F108L/I219L/C248I relative to wild-type EchA (e.g., EchA, protein chain elongation factor EF-Ts, 50S ribosomal subunits L7/L12/L32/L29, cysteine synthase A, glycerophosphodiester phosphodiesterase, iron superoxide dismutase). For the TOM-Green/IsoILR1/GSHI system, the expression level of 49 proteins was changed out of 364 identified proteins. The induced proteins due to the addition of TOM-Green, IsoILR1, and GSHI were involved in the oxidative defense mechanism, pyruvate metabolism, and glutathione synthesis (e.g., 30S ribosomal subunit proteins S3 and S16, 50S ribosomal subunit protein L20, alkyl hydroperoxide reductase, lactate dehydrogenase, acetate kinase, cysteine synthase A). Enzymes involved in indole synthesis, fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle were repressed (e.g., tryptophanase, acetyl-CoA carboxylase, phosphoenolpyruvate carboxykinase, malate dehydrogenase). Hence, the metabolic engineering that leads to enhanced aerobic degradation of 1 mM cis-DCE (2.4-4-fold more chloride ions released) and reduced toxicity from cis-DCE epoxide results in enhanced synthesis of glutathione coupled with an induced stress response as well as repression of fatty acid synthesis, gluconeogenesis, and the tricarboxylic acid cycle.

Aerobiosis↗