Pseudonocardia Thermophila Research Articles

Multi-method analysis revealed the mechanism of substrate selectivity in NHase: A gatekeeper residue at the activity center

Recognizing the critical need to elucidate the molecular determinants of this selectivity offers a pathway to engineer enzymes with broader and more versatile catalytic capabilities. Through integrated methods including phylogenetic analysis, molecular docking, and structural analysis, we identified a pivotal amino acid residue, αTrp116, linking the substrate binding pocket and the active site of a NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). This residue acts as a crucial determinant of substrate specificity within the NHase enzyme. The mutant αW116R modified the substrate specificity of PtNHase, significantly enhancing its catalytic efficiency towards aromatic substrates. The catalytic activity for aromatic compounds such as 3-Cyanopyridine was 14-fold that of the wild-type, whereas its activity for aliphatic substrates diminished to one-sixth. MD simulations revealed that replacing αTrp116 with Arg allowed aromatic nitrile substrates to achieve more favorable conformations within the active site. Based on the mutant αW116R, we further constructed a combinatorial variant Pt-4, tailored for aromatic substrates, which exhibited an enzyme activity 50 times that of the wild-type. These results highlight the critical influence of amino acid residues in the enzyme's active site on substrate specificity and offer fresh perspectives and approaches for the evolution of enzymes.

Structure-oriented engineering of nitrile hydratase: Reshaping of substrate access tunnel and binding pocket for efficient synthesis of cinnamamide

Cinnamamide and its derivatives are the most common and important building blocks widely present in natural products. Currently, nitrile hydratase (NHase, EC 4.2.1.84) has been widely used in large-scale industrial production of nicotinamide and acrylamide, while its catalytic activity is extremely low or inactive for bulky nitrile substrates such as cinnamonitrile. Therefore, beneficial variant βF37P/L48P/F51N were obtained from PtNHase of Pseudonocardia thermophila JCM3095 by reshaping of substrate access tunnel and binding pocket, which exhibited 14.88-fold improved catalytic efficiency compared to the wild-type PtNHase. Structure analysis, molecular dynamics simulations and dynamical cross-correlation matrix (DCCM) analysis revealed that the introduced mutations enlarged the substrate access tunnel and binding pocket, enhanced overall anti-correlated movements of enzymes, which would promote product release during the dynamic process of catalysis. In a hydration process, the complete conversion of 5 mM cinnamonitrile was achieved by βF37P/L48P/F51N in a 50 mL reaction, with cinnamamide yield of almost 100 % and productivity of 0.736 g L−1 h−1. The study demonstrates the co-evolution of substrate access tunnel and binding pocket is an effective strategy, and provides a valuable reference for future research. Furthermore, NHases have huge potential for catalyzing bulky nitriles to form corresponding amides in large-scale industrial production.

High-Level Expression of Nitrile Hydratase in Escherichia coli for 2-Amino-2,3-Dimethylbutyramide Synthesis

In the synthesis of imidazolinone herbicides, 2-Amino-2,3-dimethylbutyramide (ADBA) is an important intermedium. In this study, the recombinant production of nitrile hydratase (NHase) in Escherichia coli for ADBA synthesis was explored. A local library containing recombinant NHases from various sources was screened using a colorimetric method. NHase from Pseudonocardia thermophila JCM3095 was selected, fused with a His-tag and one-step purified. The enzymatic properties of recombinant NHase were studied and indicated robust thermal stability and inhibition of cyanide ions due to substrate degradation. After systematic optimization of fermentation conditions, the OD600 (optical density at 600 nm), enzyme activity and specific activity of recombinant strain E. coli BL21(DE3)/pET-28a+NHase reached 19.4, 3.72 U/mL and 1.04 U/mg protein at 42 h, representing 5.86-, 26.6- and 4-fold increases, respectively. These results offered an efficient recombinant whole-cell biocatalyst for ADBA synthesis.

Examination of the Catalytic Role of the Axial Cystine Ligand in the Co-Type Nitrile Hydratase from Pseudonocardia thermophila JCM 3095

The strictly conserved αSer162 residue in the Co-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), which forms a hydrogen bond to the axial αCys108-S atom, was mutated into an Ala residue. The αSer162Ala yielded two different protein species: one was the apoform (αSerA) that exhibited no observable activity, and the second (αSerB) contained its full complement of cobalt ions and was active with a kcat value of 63 ± 3 s−1 towards acrylonitrile at pH 7.5. The X-ray crystal structure of αSerA was determined at 1.85 Å resolution and contained no detectable cobalt per α2β2 heterotetramer. The axial αCys108 ligand itself was also mutated into Ser, Met, and His ligands. All three of these αCys108 mutant enzymes contained only half of the cobalt complement of wild-type PtNHase, but were able to hydrate acrylonitrile with kcat values of 120 ± 6, 29 ± 3, and 14 ± 1 s−1 for the αCys108His, Ser, and Met mutant enzymes, respectively. As all three of these mutant enzymes are catalytically competent, these data provide the first experimental evidence that transient disulfide bond formation is not catalytically essential for NHases.

Engineering of the thermophilic nitrile hydratase from Pseudonocardia thermophila JCM3095 for large-scale nicotinamide production based on sequence-activity relationships

The green biocatalyst nitrile hydratase (NHase) is able to bio-transform 3-cyanopyridine into nicotinamide. As the NHase reaction is exothermic, an enzyme with high activity and stability is needed for nicotinamide production. In this study, we used sequence analysis and site-directed mutagenesis to generate a mutant of thermophilic NHase from Pseudonocardia thermophila JCM3095 with substantially enhanced activity and developed a powerful process for nicotinamide bio-production. The specific activity of αF126Y/αF168Y mutant was successfully increased by 3.98-fold over that of the wild-type enzyme. The half-life of such mutant was longer than 2 h, which was comparable to its parent enzyme. The relative activity of the αF126Y/αF168Y mutant after treatment with 1 M 3-cyanopyridine and 2 M nicotinamide was 73.2% and 63.7%, respectively, showing minor loss of its original stability. Structural analysis demonstrated that hydrogen bonds at the active site and α-β subunit interface of the NHase contribute to the improved activity and the maintenance of stability. Escherichia coli transformant harboring the mutant NHase was used for nicotinamide bio-production, yielding a nicotinamide productivity of 251.1 g/(L·h), which is higher than the productivity obtained using other NHase-containing strains and transformants. The newly established variant is therefore a promising alternative for the industrial production of nicotinamides.

Computational Design of Nitrile Hydratase from Pseudonocardia thermophila JCM3095 for Improved Thermostability

High thermostability and catalytic activity are key properties for nitrile hydratase (NHase, EC 4.2.1.84) as a well-industrialized catalyst. In this study, rational design was applied to tailor the thermostability of NHase from Pseudonocardia thermophila JCM3095 (PtNHase) by combining FireProt server prediction and molecular dynamics (MD) simulation. Site-directed mutagenesis of non-catalytic residues provided by the rational design was subsequentially performed. The positive multiple-point mutant, namely, M10 (αI5P/αT18Y/αQ31L/αD92H/βA20P/βP38L/βF118W/βS130Y/βC189N/βC218V), was obtained and further analyzed. The Melting temperature (Tm) of the M10 mutant showed an increase by 3.2 °C and a substantial increase in residual activity of the enzyme at elevated temperatures was also observed. Moreover, the M10 mutant also showed a 2.1-fold increase in catalytic activity compared with the wild-type PtNHase. Molecular docking and MD simulations demonstrated better substrate affinity and improved thermostability for the mutant.

Elucidating the role of the axial cysteine residue in NHase catalysis and the enzyme maturation

Nitrile hydratases are metalloenzymes that contain trivalent Fe/or Co ions in their active site, coordinated in an N2S3 ligand environment. The sulfur atoms are post‐translationally modified and have three different oxidation states (αCys, Cys‐SOH and Cys‐SOO−) while backbone amines account for the nitrogen ligands and are deprotonated. Theoretical calculations assume that the αCys ligand is a thiolate, making it a strong π‐donor ligand and a good a nucleophile. A critical outstanding mechanistic question is whether the formation of a transient disulfide bond is essential for catalytic turnover. Such a transient disulfide bond was proposed based on QM/MM calculations between the αCys sulfur and the sulfur of the proposed sulfenic acid cyclic intermediate. However, more recent theoretical studies is self‐inconsistent in its conclusions regarding a transient disulfide formation. To examine the role of the αCys108 ligand in catalysis, it was mutated to A, M, S, and H in the Co‐type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase). ICP‐MS demonstrated reduced metal ion content for each mutant: <5% (αC108A), ~44% (αC108M), ~50% (αC108S) and ~55% ( αC108H) and catalytic activity also decreased to ~0.3%, ~0.8%, ~1.6%, and ~6.7%, respectively, towards acrylonitrile. The X‐ray crystal structure of αC108A confirmed the mutation and the decreased active site metal content. Therefore, the axial thiolate is important to properly tune the electronic properties of the active site metal ion, but is not essential to catalysis. These data provide insight into the critical mechanistic question of whether the axial αCys ligand forms a transient disulfide bond during turnover.Support or Funding InformationThis work was supported by (CHE‐1808711, RCH; CHE‐1308672, BB; CHE‐1532168 BB & RCH), the Todd Wehr Foundation, Bruker Biospin, and the National Institutes of Health/NIBIB National Biomedical EPR Center (P41‐EB001980)

Analyzing the function of the insert region found between the α and β-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis

The functional roles of the (His)17 region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHaseΔ238−257 and MbNHaseΔ219−272, were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α2β2 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)17 motif provides an MbNHase enzyme that is ∼55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseact) but ∼28% more active when expressed in the presence of PtNHaseact. MbNHaseΔ219−272 exhibits ∼55% and ∼89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseact. Proteolytic cleavage of MbNHase provides an α2β2 heterotetramer that is modestly more active compared to WT MbNHase (kcat = 163 ± 4 vs 131 ± 3 s−1). Combination of these data establish that neither the (His)17 nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.

Evidence for the participation of an extra α-helix at β-subunit surface in the thermal stability of Co-type nitrile hydratase.

Nitrile hydratase (NHase) has attracted considerable attention since it can efficiently catalyze the hydration of nitriles to valuable amides. However, the poor stability of NHase is one of the main drawbacks in the industrial application. In this study, we compared the structural difference between Fe-type and Co-type NHase and found that an extra α helix existed at the β-subunit surface of Co-type NHase (defined as the β-6th helix). Then, the effects of the β-6th helix were investigated on the thermal stability and the catalytic kinetics of a Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229). When the β-6th helix was deleted or disrupted, the thermal stability of NHase1229 was reduced to 17.6 and 12.9% of that of wild NHase1229, respectively. Thus, the β-6th helix is important for the thermal stability of Co-type NHase. Based on the structural characteristics of Co-type NHase, the β-6th helix may be interacted with another helix at the α-subunit (defined as the α-2nd helix) by hydrophobic network just as a "magnetic suction buckle" on the enzyme surface to stabilize the binding of α- and β-subunits. The β-6th helix is located at the mouth of the substrate and product tunnel, so it plays crucial roles in catalytic process. Furthermore, the β-6th helix in NHase1229 was swapped with a thermophilic NHase fragment from Pseudonocardia thermophila JCM3095 (NHase1229-Swap). The thermal stability of NHase1229-Swap was significantly improved, and the half-life was approximately 2.4-fold at 40°C than that of the wild NHase1229. The knowledge is useful for improving the stability of NHases by restriction fragment swapping.

AkzoNobel will boost its production of colloidal silica in Sweden

The cobalt-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase) was successfully encapsulated in tetramethyl orthosilicate sol–gel matrices to produce a PtNHase:sol–gel biomaterial. The PtNHase:sol–gel biomaterial catalyzed the conversion of 600 mM acrylonitrile to acrylamide in 60 min at 35 °C with a yields of >90%. Treatment of the biomaterial with proteases confirmed that the catalytic activity is due to the encapsulated enzyme and not surface bound NHase. The biomaterial retained 50% of its activity after being used for a total of 13 consecutive reactions for the conversion of acrylonitrile to acrylamide. The thermostability and long-term storage of the PtNHase:sol–gel are substantially improved compared to the soluble NHase. Additionally, the biomaterial is significantly more stable at high concentrations of methanol (50% and 70%, v/v) as a co-solvent for the hydration of acrylonitrile than native PtNHase. These data indicate that PtNHase:sol–gel biomaterials can be used to develop new synthetic avenues involving nitriles as starting materials given that the conversion of the nitrile moiety to the corresponding amide occurs under mild temperature and pH conditions.

Identification and functional analysis of the activator gene involved in the biosynthesis of Co-type nitrile hydratase from Aurantimonas manganoxydans

Nitrile hydratase (NHase) has attracted considerable attention to synthesize valuable amides. Here, the gene cluster of Co-type NHase from Aurantimonas manganoxydans ATCC BAA-1229 (NHase1229) was identified through Edman degradation and recombinant expression, including α-subunit, β-subunit, and activator. The activator gene contained 408 bases, which was essential for the biosynthesis of NHase1229 in Escherichia coli. The activator gene can be substituted by Co-type activator from Pseudonocardia thermophila JCM3095 and Pseudomonas putida 5B to assist in the functional expression of NHase1229, not by that of Fe-type NHase from Pseudomonas putida F1. Multi-sequence alignment showed that the activator contained conserved amino acid residues (Trp34, Trp53, Phe 85, Gln35, Glu39, and Glu90), but these residues had no significant effect on activator function. Histidine residues (His112, His120, and His134) at the C-terminus only partially participated in the activator function. Furthermore, the successive α-helices in domain TIGR03889 were identified to be critical for the activator function. The integration efficiency of cobalt into the active site was decreased with the interruption of α-helices. Therefore, Co-type and Fe-type activators have specific functions for the integration of different metal ions, and four conserved α-helices in Co-type activator affect the integration of cobalt. These knowledge is useful for understanding the biosynthesis of Co-type NHase.

The Stability Enhancement of Nitrile Hydratase from Bordetella petrii by Swapping the C-terminal Domain of β subunit.

The thermal stability of most nitrile hydratases (NHase) is poor, which has been enhanced to some extent by molecular modifications in several specific regions of the C-terminal domain (C-domain) of β subunit of NHase. Since the C-domain could be present as a naturally separate domain in a few NHases, the whole C-domain is proposed to be related to the NHase stability. The chimeric NHase (SBpNHase) from the thermal-sensitive BpNHase (NHase from Bordetella petrii) and the relatively thermal-stable PtNHase (NHase from Pseudonocardia thermophila) was constructed by swapping the corresponding C-domains. After 30min incubation at 50°C, the original BpNHase nearly lost its activity, while the SBpNHase retained 50% residual activity, compared with the melting temperature (Tm) (50°C) of the original BpNHase, that of the SBpNHase was 55°C. The SBpNHase with higher thermal stability would be useful for the thermal stability enhancement of NHase and for the understanding of the relationship between the stability of NHase and its structure.

Improvement of stability of nitrile hydratase via protein fragment swapping

Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, is the key enzyme for the production of amides in industries. However, the poor stability of this enzyme under the reaction conditions is a drawback of its industrial application. In this study, we aimed to improve the stability of NHase (PpNHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy. One thermophilic NHase fragment from Comamonas testosteroni 5-MGAM-4D and two fragments from Pseudonocardia thermophila JCM3095 were selected to swap the corresponding fragments of PpNHase. Seven chimeric NHases were designed using STAR (site targeted amino recombination) software and molecular dynamics to determine the crossover sites for fragment recombination. All constructed chimeric NHases showed 1.4- to 3.5-fold enhancement in thermostability and six of them become more tolerant to high-concentration product. Notably, one of these NHases, 3AB, exhibited a 1.4±0.05-fold increase in activity compared to the wild-type PpNHase. Circular dichroism spectrum analysis and homology modeling revealed that the 3AB slightly differed in secondary structure from wild-type PpNHase. The 3AB constructed in this study is useful for further industrial application, and the method for designing the chimeric protein using homologous protein fragment swapping without a decrease in activity may be a strategy to improve the stability of other enzymes.

The active site sulfenic acid ligand in nitrile hydratases can function as a nucleophile.

Nitrile hydratase (NHase) catalyzes the hydration of nitriles to their corresponding commercially valuable amides at ambient temperatures and physiological pH. Several reaction mechanisms have been proposed for NHase enzymes; however, the source of the nucleophile remains a mystery. Boronic acids have been shown to be potent inhibitors of numerous hydrolytic enzymes due to the open shell of boron, which allows it to expand from a trigonal planar (sp2) form to a tetrahedral form (sp3). Therefore, we examined the inhibition of the Co-type NHase from Pseudonocardia thermophila JCM 3095 (PtNHase) by boronic acids via kinetics and X-ray crystallography. Both 1-butaneboronic acid (BuBA) and phenylboronic acid (PBA) function as potent competitive inhibitors of PtNHase. X-ray crystal structures for BuBA and PBA complexed to PtNHase were solved and refined at 1.5, 1.6, and 1.2 Å resolution. The resulting PtNHase–boronic acid complexes represent a “snapshot” of reaction intermediates and implicate the cysteine-sulfenic acid ligand as the catalytic nucleophile, a heretofore unknown role for the αCys113–OH sulfenic acid ligand. Based on these data, a new mechanism of action for the hydration of nitriles by NHase is presented.

Acrylamide production using encapsulated nitrile hydratase from Pseudonocardia thermophila in a sol–gel matrix

Abstract The cobalt-type nitrile hydratase from Pseudonocardia thermophila JCM 3095 ( Pt NHase) was successfully encapsulated in tetramethyl orthosilicate sol–gel matrices to produce a Pt NHase:sol–gel biomaterial. The Pt NHase:sol–gel biomaterial catalyzed the conversion of 600 mM acrylonitrile to acrylamide in 60 min at 35 °C with a yields of >90%. Treatment of the biomaterial with proteases confirmed that the catalytic activity is due to the encapsulated enzyme and not surface bound NHase. The biomaterial retained 50% of its activity after being used for a total of 13 consecutive reactions for the conversion of acrylonitrile to acrylamide. The thermostability and long-term storage of the Pt NHase:sol–gel are substantially improved compared to the soluble NHase. Additionally, the biomaterial is significantly more stable at high concentrations of methanol (50% and 70%, v/v) as a co-solvent for the hydration of acrylonitrile than native Pt NHase. These data indicate that Pt NHase:sol–gel biomaterials can be used to develop new synthetic avenues involving nitriles as starting materials given that the conversion of the nitrile moiety to the corresponding amide occurs under mild temperature and pH conditions.

Enzyme–Substrate Binding Landscapes in the Process of Nitrile Biodegradation Mediated by Nitrile Hydratase and Amidase

The continuing discharge of nitriles in various industrial processes has caused serious environmental consequences of nitrile pollution. Microorganisms possess several nitrile-degrading pathways by direct interactions of nitriles with nitrile-degrading enzymes. However, these interactions are largely unknown and difficult to experimentally determine but important for interpretation of nitrile metabolisms and design of nitrile-degrading enzymes with better nitrile-converting activity. Here, we undertook a molecular modeling study of enzyme-substrate binding modes in the bi-enzyme pathway for degradation of nitrile to acid. Docking results showed that the top substrates having favorable interactions with nitrile hydratase from Rhodococcus erythropolis AJ270 (ReNHase), nitrile hydratase from Pseudonocardia thermophila JCM 3095 (PtNHase), and amidase from Rhodococcus sp. N-771 (RhAmidase) were benzonitrile, 3-cyanopyridine, and L-methioninamide, respectively. We further analyzed the interactional profiles of these top poses with corresponding enzymes, showing that specific residues within the enzyme's binding pockets formed diverse contacts with substrates. This information on binding landscapes and interactional profiles is of great importance for the design of nitrile-degrading enzyme mutants with better oxidation activity toward nitriles or amides in the process of pollutant treatments.

Improving stability of nitrile hydratase by bridging the salt-bridges in specific thermal-sensitive regions

The regions and types suitable mutations for bridging salt-bridges to intensify enzyme stability are identified in this study. Using nitrile hydratase (NHase) as the model enzyme, three deformation-prone thermal-sensitive regions (A1, A2 and A3 in β-subunit), identified by RMSF calculations of the thermophilic NHase 1V29 from Bacillus SC-105-1 and 1UGQ from Pseudonocardia thermophila JCM3095, were determined and the stabilized salt-bridge interactions were transferred into the corresponding region of industrialized mesophilic NHase-TH from Rhodococcus ruber TH. Three types of salt bridges—active-center-adjacent (in A1), internal neighboring-residue-bridged (in A2) and C-terminal-residue-bridged (A3)—were constructed in NHase-TH. The engineered NHase-TH-A1 showed reduced expression of β-subunit, reduced activity and irregular stability. NHase-TH-A2 exhibited a enhanced expression of β-subunit but complete loss of activity; while NHase-TH-A3 exhibited not only a slightly enhanced expression of β-subunit and enzyme activity, but also a 160% increase in thermal stability, a 7% enhanced product tolerance and a 75% enhanced resistance to cell-disruption by ultrasonication. The molecular dynamic (MD) simulation revealed that NHase-TH-A3, with a moderate RMSD value, generates 10 new salt bridges in both internal-subunit and interfacial-subunit, confirming that a C-terminal salt-bridge strategy is powerful for enzyme stability intensification through triggering global changes of the salt bridge networks.

Pseudonocardia mongoliensis sp. nov. and Pseudonocardia khuvsgulensis sp. nov., isolated from soil

Two actinomycetes, designated MN08-A0270(T) and MN08-A0297(T), were isolated from soil from the area around Khuvsgul Lake, Khuvsgul province, Mongolia, and subjected to phenotypic and genotypic characterization. They produced well-developed, branched substrate hyphae and, similar to closely related species of the genus Pseudonocardia, produced zigzag-shaped aerial hyphae by acropetal budding and blastospores. A comparative analysis of 16S rRNA gene sequences indicated that strains MN08-A0270(T) and MN08-A0297(T) formed two distinct clades within the genus Pseudonocardia and were respectively most closely related to Pseudonocardia yunnanensis NBRC 15681(T) (97.3 % similarity) and Pseudonocardia thermophila IMSNU 20112(T) (97.1 %). Chemotaxonomic characteristics, including cell-wall diaminopimelic acid, whole-cell sugars, fatty acid components and major menaquinones, suggested that the two organisms belonged to the genus Pseudonocardia. Strains MN08-A0270(T) and MN08-A0297(T) could be differentiated from each other and from closely related species of the genus Pseudonocardia by physiological and biochemical characteristics, predominant fatty acids, menaquinones and whole-cell sugar components. Combined with the results of a broad range of phenotypic tests and DNA-DNA hybridization data and phylogenetic analysis, these results support the conclusion that these strains represent two novel species of the genus Pseudonocardia, for which we propose the names Pseudonocardia mongoliensis sp. nov. (type strain MN08-A0270(T) = NBRC 105885(T) = VTCC D9-25(T)) and Pseudonocardia khuvsgulensis sp. nov. (type strain MN08-A0297(T) = NBRC 105886(T) = VTCC D9-26(T)).

Pseudonocardia rhizophila sp. nov., a novel actinomycete isolated from a rhizosphere soil

During the course of our research on new actinobacterial sources, a novel actinomycete strain YIM 67013(T) was isolated from rhizosphere soil of Tripterygium wilfordii Hook. f. collected from Yunnan province, south-west China. The strain formed well-differentiated aerial and substrate mycelia, grew in the presence of up to 15% NaCl (optimum, 0-3% NaCl). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain YIM 67013(T) belongs to the genus Pseudonocardia, with highest similarity to Pseudonocardia thermophila DSM 43832(T) (97.6%). Sequence similarities between strain YIM 67013(T) and the other Pseudonocardia species ranged from 96.6 to 93.2%. Key morphological and physiological characteristics as well as chemotaxonomic features of strain YIM 67013(T) were congruent with the description of the genus Pseudonocardia. The G+C content of the genomic DNA was 69.7 mol%. Based on comparative analysis of physiological, biochemical and chemotaxonomic data, including low DNA-DNA hybridization results, it is proposed that strain YIM 67013(T) represents a novel species of the genus Pseudonocardia, named Pseudonocardia rhizophila sp. nov. The type strain is YIM 67013(T) (= CCTCC AA 209043(T) = DSM 45381(T)).

Mechanical aspects of nitrile hydratase enzymatic activity. Steered molecular dynamics simulations of Pseudonocardia thermophila JCM 3095

Nitrile hydratase (NHase), an important biotechnological enzyme, has been investigated using a steered molecular dynamics computer modelling for the first time. An external force applied to the docked ligands was used to determine transport paths for acrylonitrile (substrate) and acrylamide (product). The average drag force of 120 pN within the enzyme channel is 50% higher than that in model water. The major hindrance of 500 pN is generated by βPhe37 residue. This region may be responsible for the stereoselectivity of NHases.