Phenotypic and Enzymatic Comparative Analysis of the KPC Variants,KPC-2 and Its Recently Discovered Variant KPC-15

Sixteen different variants (KPC-2 to KPC-17) in the KPC family have been reported, and most current studies are focusing on KPC-2 and KPC-3. The KPC-15 variant, which isolated from Klebsiella pneumoniae in a Chinese hospital, was a recently discovered KPC enzyme. To compare the characteristics of KPC-15 and KPC-2, the variants were determined by susceptibility testing, PCR amplification and sequencing, and study of kinetic parameters. The strain harboring the KPC-15 showed resistance to 18 conventional antimicrobial agents, especially to cabapenem antibiotics, and the strain involving the KPC-2 also indicated resistance to cabapenem antibiotics, but both strains were susceptible to polymyxin B and colistin. The conjugation experiments showed that the changes of MIC values to the antibiotics were due to the transferred plasmids. The differences of amino acids were characterised at sites of 119 leucine and 146 lysine with KPC-15 and KPC-2. The minimum evolution tree indicated the KPC alleles evolution, and showed that the KPC-15 appeared to be homogenous with KPC-4 closely. Steady-state kinetic parameters showed the catalytic efficiency of KPC-15 was higher than that of KPC-2 for all tested antibiotics in this study. The catalytic efficiency of KPC-15 caused resistance to β-lactam antibiotics was higher than that of KPC-2. Meanwhile, an evolutionary transformation changed KPC from an efficient carbapenemase to its variants (KPC-15) with better ceftazidimase catalytic efficiency, and the old antibiotics polymyxin B and colistin might play a role in the therapy for multi-resistant strains.     

Quantitative Comparison of Catalytic Mechanisms and Overall Reactions in Convergently Evolved Enzymes: Implications for Classification of Enzyme Function

Functionally analogous enzymes are those that catalyze similar reactions on similar substrates but do not share common ancestry, providing a window on the different structural strategies nature has used to evolve required catalysts. Identification and use of this information to improve reaction classification and computational annotation of enzymes newly discovered in the genome projects would benefit from systematic determination of reaction similarities. Here, we quantified similarity in bond changes for overall reactions and catalytic mechanisms for 95 pairs of functionally analogous enzymes (non-homologous enzymes with identical first three numbers of their EC codes) from the MACiE database. Similarity of overall reactions was computed by comparing the sets of bond changes in the transformations from substrates to products. For similarity of mechanisms, sets of bond changes occurring in each mechanistic step were compared; these similarities were then used to guide global and local alignments of mechanistic steps. Using this metric, only 44% of pairs of functionally analogous enzymes in the dataset had significantly similar overall reactions. For these enzymes, convergence to the same mechanism occurred in 33% of cases, with most pairs having at least one identical mechanistic step. Using our metric, overall reaction similarity serves as an upper bound for mechanistic similarity in functional analogs. For example, the four carbon-oxygen lyases acting on phosphates (EC 4.2.3) show neither significant overall reaction similarity nor significant mechanistic similarity. By contrast, the three carboxylic-ester hydrolases (EC 3.1.1) catalyze overall reactions with identical bond changes and have converged to almost identical mechanisms. The large proportion of enzyme pairs that do not show significant overall reaction similarity (56%) suggests that at least for the functionally analogous enzymes studied here, more stringent criteria could be used to refine definitions of EC sub-subclasses for improved discrimination in their classification of enzyme reactions. The results also indicate that mechanistic convergence of reaction steps is widespread, suggesting that quantitative measurement of mechanistic similarity can inform approaches for functional annotation.     

Increased Stability and Catalytic Efficiency of Yeast Hexokinase Upon Interaction with Zwitterionic Micelles. Kinetics and Conformational Studies

The effect of ligands (glucose, ATP and Mg2+) and zwitterionic micellesof lysophosphatidylcholine (LPC) or N-hexadecyl-N,N-dimethyl-3-ammoniumpropanesulfonate (HPS) in the yeast hexokinase (HK) stability was studied at35°C. The thermal inactivation kinetics followed one-exponentialdecay. The effect of ligands on protecting the enzyme against inactivationfollowed the order: glucose>glucose/Mg2+>ATP/Mg2+Mg2+bufferonly. Both LPC and HPS micelles increased the enzyme stability only whenthe incubation medium contained glucose or glucose/Mg2+,suggesting that the protein conformation is a key prerequisite for theenzyme-micelle interaction to take place. This enzyme-micelle interactionresulted in an increased catalytic efficiency (with a decrease in Km forATP and increase in Vmax as well as in changes on the tertiary (intrinsicfluorescence) structure of the yeast hexokinase.     

Disease Variants of the Human Mitochondrial DNA Helicase Encoded by C10orf2 Differentially Alter Protein Stability,Nucleotide Hydrolysis,and Helicase Activity

Missense mutations in the human C10orf2 gene, encoding the mitochondrial DNA (mtDNA) helicase, co-segregate with mitochondrial diseases such as adult-onset progressive external ophthalmoplegia, hepatocerebral syndrome with mtDNA depletion syndrome, and infantile-onset spinocerebellar ataxia. To understand the biochemical consequences of C10orf2 mutations, we overproduced wild type and 20 mutant forms of human mtDNA helicase in Escherichia coli and developed novel schemes to purify the recombinant enzymes to near homogeneity. A combination of molecular crowding, non-ionic detergents, Mg²⁺ ions, and elevated ionic strength was required to combat insolubility and intrinsic instability of certain mutant variants. A systematic biochemical assessment of the enzymes included analysis of DNA binding affinity, DNA helicase activity, the kinetics of nucleotide hydrolysis, and estimates of thermal stability. In contrast to other studies, we found that all 20 mutant variants retain helicase function under optimized in vitro conditions despite partial reductions in DNA binding affinity, nucleotide hydrolysis, or thermal stability for some mutants. Such partial defects are consistent with the delayed presentation of mitochondrial diseases associated with mutation of C10orf2.     

Protein Dynamics Control the Progression and Efficiency of the Catalytic Reaction Cycle of the Escherichia coli DNA-Repair Enzyme AlkB

A central goal of enzymology is to understand the physicochemical mechanisms that enable proteins to catalyze complex chemical reactions with high efficiency. Recent methodological advances enable the contribution of protein dynamics to enzyme efficiency to be explored more deeply. Here, we utilize enzymological and biophysical studies, including NMR measurements of conformational dynamics, to develop a quantitative mechanistic scheme for the DNA repair enzyme AlkB. Like other iron/2-oxoglutarate-dependent dioxygenases, AlkB employs a two-step mechanism in which oxidation of 2-oxoglutarate generates a highly reactive enzyme-bound oxyferryl intermediate that, in the case of AlkB, slowly hydroxylates an alkylated nucleobase. Our results demonstrate that a microsecond-to-millisecond time scale conformational transition facilitates the proper sequential order of substrate binding to AlkB. Mutations altering the dynamics of this transition allow generation of the oxyferryl intermediate but promote its premature quenching by solvent, which uncouples 2-oxoglutarate turnover from nucleobase oxidation. Therefore, efficient catalysis by AlkB depends upon the dynamics of a specific conformational transition, establishing another paradigm for the control of enzyme function by protein dynamics.     

Comparison of Hydrolysis Efficiency and Enzyme Adsorption of Three Different Cellulosic Materials in the Presence of Poly(ethylene Glycol)

The addition of non-ionic surfactants has recently been confirmed to positively affect the enzymatic hydrolysis of cellulosic materials. However, the functional mechanisms of these surfactants remain unclear. This work investigated the influence of poly(ethylene glycol) (PEG) on the enzymatic hydrolysis of three cellulosic materials, namely, acid steam-exploded corn straw, pure microcrystalline cellulose (Avicel PH101), and bagasse sulfite pulp (BSP). The results showed that PEG addition led to varied effects on the enzymatic hydrolysis of different cellulosic materials. Addition of PEG was most effective on the enzymatic hydrolysis of PH101 and weakly effective on the hydrolysis of BSP. We further investigated PEG concentrations and enzymatic activities in the supernatant during hydrolysis and found that the positive effects of PEG treatment might contribute to its influence on enzyme desorption from different substrates. We also found that the efficiency of PEG depended on its capacity to bind to different substrates. PEG exhibited stronger affinity to pure cellulose than to the two other lignocellulosic substrates. These findings are helpful in further revealing the mechanism of surfactants and improving the enzymatic hydrolysis process.     

Differential Thermal Stability and Oxidative Vulnerability of the Hemoglobin Variants,HbA2 and HbE

Apart from few early biophysical studies, the relative thermal instability of HbE has been only shown by clinical investigations. We have compared in vitro thermal stability of HbE with HbA₂ and HbA using optical spectroscopy. From absorption measurements in the soret region, synchronous fluorescence spectroscopy and dynamic light scattering experiments, we have found thermal stability of the three hemoglobin variants following the order HbE<HbA<HbA₂ in terms of structural unfolding and aggregation pattern. We have found formation of intermolecular dityrosine fluorophores with characteristic fluorescence signature, at pH >11.0 in all the three variants. Under oxidative stress conditions in presence of hydrogen peroxide, HbE has been found to be more vulnerable to aggregation compared to HbA and HbA₂. Taken together, these studies have shown thermal and oxidative instability of HbE and points towards the role of HbE in the upregulation of redox regulators and chaperone proteins in erythrocyte proteome of patients suffering from HbEbeta thalassemia.     

Selective Pressure Causes an RNA Virus to Trade Reproductive Fitness for Increased Structural and Thermal Stability of a Viral Enzyme

The modulation of fitness by single mutational substitutions during environmental change is the most fundamental consequence of natural selection. The antagonistic tradeoffs of pleiotropic mutations that can be selected under changing environments therefore lie at the foundation of evolutionary biology. However, the molecular basis of fitness tradeoffs is rarely determined in terms of how these pleiotropic mutations affect protein structure. Here we use an interdisciplinary approach to study how antagonistic pleiotropy and protein function dictate a fitness tradeoff. We challenged populations of an RNA virus, bacteriophage Φ6, to evolve in a novel temperature environment where heat shock imposed extreme virus mortality. A single amino acid substitution in the viral lysin protein P5 (V207F) favored improved stability, and hence survival of challenged viruses, despite a concomitant tradeoff that decreased viral reproduction. This mutation increased the thermostability of P5. Crystal structures of wild-type, mutant, and ligand-bound P5 reveal the molecular basis of this thermostabilization—the Phe207 side chain fills a hydrophobic cavity that is unoccupied in the wild-type—and identify P5 as a lytic transglycosylase. The mutation did not reduce the enzymatic activity of P5, suggesting that the reproduction tradeoff stems from other factors such as inefficient capsid assembly or disassembly. Our study demonstrates how combining experimental evolution, biochemistry, and structural biology can identify the mechanisms that drive the antagonistic pleiotropic phenotypes of an individual point mutation in the classic evolutionary tug-of-war between survival and reproduction.     

Structural Modeling of the SARS-CoV-2 Spike/Human ACE2 Complex Interface can Identify High-Affinity Variants Associated with Increased Transmissibility

The COVID-19 pandemic has triggered concerns about the emergence of more infectious and pathogenic viral strains. As a public health measure, efficient screening methods are needed to determine the functional effects of new sequence variants. Here we show that structural modeling of SARS-CoV-2 Spike protein binding to the human ACE2 receptor, the first step in host-cell entry, predicts many novel variant combinations with enhanced binding affinities. By focusing on natural variants at the Spike-hACE2 interface and assessing over 700 mutant complexes, our analysis reveals that high-affinity Spike mutations (including N440K, S443A, G476S, E484R, G502P) tend to cluster near known human ACE2 recognition sites (K31 and K353). These Spike regions are structurally flexible, allowing certain mutations to optimize interface interaction energies. Although most human ACE2 variants tend to weaken binding affinity, they can interact with Spike mutations to generate high-affinity double mutant complexes, suggesting variation in individual susceptibility to infection. Applying structural analysis to highly transmissible variants, we find that circulating point mutations S477N, E484K and N501Y form high-affinity complexes (~40% more than wild-type). By combining predicted affinities and available antibody escape data, we show that fast-spreading viral variants exploit combinatorial mutations possessing both enhanced affinity and antibody resistance, including S477N/E484K, E484K/N501Y and K417T/E484K/N501Y. Thus, three-dimensional modeling of the Spike/hACE2 complex predicts changes in structure and binding affinity that correlate with transmissibility and therefore can help inform future intervention strategies.     

Comparative Biology of Two Natural Variants of the IncQ-2 Family Plasmids,pRAS3.1 and pRAS3.2

Plasmids pRAS3.1 and pRAS3.2 are two closely related, natural variants of the IncQ-2 plasmid family that have identical plasmid backbones except for two differences. Plasmid pRAS3.1 has five 6-bp repeat sequences in the promoter region of the mobB gene and four 22-bp iterons in its oriV region, whereas pRAS3.2 has only four 6-bp repeats and three 22-bp iterons. Plasmid pRAS3.1 was found to have a higher copy number than pRAS3.2, and we show that the extra 6-bp repeat results in an increase in mobB and downstream mobA/repB expression. Placement of repB (primase) behind an arabinose-inducible promoter in trans resulted in an increase in repB expression and an approximately twofold increase in the copy number of plasmids with identical numbers of 22-bp iterons. The pRAS3 plasmids were shown to have a previously unrecognized toxin-antitoxin plasmid stability module within their replicons. The ability of the pRAS3 plasmids to mobilize the oriT regions of two other plasmids of the IncQ-2 family, pTF-FC2 and pTC-F14, suggested that the mobilization proteins pRAS3 are relaxed and can mobilize oriT regions with substantially different sequences. Plasmids pRAS3.1 and pRAS3.2 were highly incompatible with plasmids pTF-FC2 and pTC-F14, and this incompatibility was removed on inactivation of an open reading frame situated downstream of the mobCDE mobilization genes rather than being due to the 22-bp oriV-associated iterons. We propose that the pRAS3 plasmids represent a third, γ incompatibility group within the IncQ-2 family plasmids.Plasmids of the IncQ family are small (<20 kb), have a broad host range, and are highly promiscuous due to their ability to be mobilized very efficiently by self-transmissible plasmids such as the IncP plasmids. They have been divided into two families, IncQ-1 and IncQ-2, based on the amino acid sequence relatedness of their RepA (helicase), RepB (primase), and RepC (DNA-binding) replication proteins and because the mobilization proteins of the two families are unrelated, consisting of three or five genes, respectively (31). IncQ-1 group plasmids include RSF1010 and the near-identical R1162, pDN1, pIE1107, pIE1115, and pIE1130, while IncQ-2 plasmids include pTF-FC2, pTC-F14, and pRAS3.IncQ-2 plasmids pRAS3.1 and pRAS3.2 were isolated in Norway from the fish pathogens Aeromonas salmonicida subsp. salmonicida and atypical A. salmonicida, respectively, while investigating plasmids that conferred resistance to tetracycline (21). The two plasmids encode identical replication and mobilization proteins, with the most important differences in the plasmid backbone being that pRAS3.1 has four 22-bp iterons in its oriV region and five 6-bp repeat sequences upstream of its mobB gene, whereas pRAS3.2 has only three iterons and four 6-bp repeat sequences. No biological studies were carried out in the initial report of the pRAS3 plasmids. As a contribution to our studies on the evolution of IncQ plasmids, our longer-term aim is to address the question of why two natural versions of the plasmid exist. Here we report on the major differences in the biology of the two plasmids. In addition, we discovered the presence of repC and mobB genes that were not detected when the sequence of pRAS3 plasmids was previously reported. We also discovered a putative toxin-antitoxin (TA) postsegregational system different from that found in other members of the IncQ plasmids and tested it for functionality.The IncQ-1 plasmids are subdivided into incompatibility groups α, β, and γ, (31), whereas the IncQ-2 plasmids are subdivided into two incompatibility groups, α and β (14). In this work we also report on the incompatibility between the pRAS3 plasmids and other members of the IncQ-2 plasmid family as well as the IncQ-1 family plasmids. Furthermore, we compare the functional relatedness of the pRAS3 mobilization system with that of previously studied IncQ-2 plasmids.     

Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.     

Modification of the Monomer Composition of Polyhydroxyalkanoate Synthesized in Saccharomyces cerevisiae Expressing Variants of the β-Oxidation-Associated Multifunctional Enzyme

Expression by Saccharomyces cerevisiae of a polyhydroxyalkanoate (PHA) synthase modified at the carboxy end by the addition of a peroxisome targeting signal derived from the last 34 amino acids of the Brassica napus isocitrate lyase (ICL) and containing the terminal tripeptide Ser-Arg-Met resulted in the synthesis of PHA. The ability of the terminal peptide Ser-Arg-Met and of the 34-amino-acid peptide from the B. napus ICL to target foreign proteins to the peroxisome of S. cerevisiae was demonstrated with green fluorescent protein fusions. PHA synthesis was found to be dependent on the presence of both the enzymes generating the β-oxidation intermediate 3-hydroxyacyl-coenzyme A (3-hydroxyacyl-[CoA]) and the peroxin-encoding PEX5 gene, demonstrating the requirement for a functional peroxisome and a β-oxidation cycle for PHA synthesis. Using a variant of the S. cerevisiae β-oxidation multifunctional enzyme with a mutation inactivating the B domain of the R-3-hydroxyacyl-CoA dehydrogenase, it was possible to modify the PHA monomer composition through an increase in the proportion of the short-chain monomers of five and six carbons.     

Embryoless Wheat Grain: A Natural System for the Study of Gibberellin-induced Enzyme Formation

Yorkstar wheat, grown in New York State, has a high percentage (10-11) of grains without embryos. The embryoless grains have viable aleurone layers and show no sign of injury. These grains are able to support α-amylase synthesis only in the presence of gibberellin A₃ (GA₃). In the absence of GA₃ some protein synthesis occurs in embryoless grains during the early hours of soaking, indicating that such activity occurs prior to and independent of GA₃ induction of α-amylase. The level of β-amylase on a dry weight basis is the same in embryoless and normal grains and decreases with time of soaking. In the presence of GA₃, β-amylase decreases at a slower rate. Isoenzymes of α-amylase from GA₃-treated embryoless and normal grains show quantitative as well as qualitative differences. Cycloheximide (60 μg/ml) completely inhibits the synthesis of α-amylase by embryoless grains. Of the RNA synthesis inhibitors, actinomycin D (60 μg/ml) was ineffective while 6-methylpurine (60 μg/ml) gave 65% inhibition without decreasing the number of isoenzymes.     

Construction and Application of Variants of the Pseudomonas fluorescens EBC191 Arylacetonitrilase for Increased Production of Acids or Amides

The arylacetonitrilase from Pseudomonas fluorescens EBC191 differs from previously studied arylacetonitrilases by its low enantiospecificity during the turnover of mandelonitrile and by the large amounts of amides that are formed in the course of this reaction. In the sequence of the nitrilase from P. fluorescens, a cysteine residue (Cys163) is present in direct neighborhood (toward the amino terminus) to the catalytic active cysteine residue, which is rather unique among bacterial nitrilases. Therefore, this cysteine residue was exchanged in the nitrilase from P. fluorescens EBC191 for various amino acid residues which are present in other nitrilases at the homologous position. The influence of these mutations on the reaction specificity and enantiospecificity was analyzed with (R,S)-mandelonitrile and (R,S)-2-phenylpropionitrile as substrates. The mutants obtained demonstrated significant differences in their amide-forming capacities. The exchange of Cys163 for asparagine or glutamine residues resulted in significantly increased amounts of amides formed. In contrast, a substitution for alanine or serine residues decreased the amounts of amides formed. The newly discovered mutation was combined with previously identified mutations which also resulted in increased amide formation. Thus, variants which possessed in addition to the mutation Cys163Asn also a deletion at the C terminus of the enzyme and/or the modification Ala165Arg were constructed. These constructs demonstrated increased amide formation capacity in comparison to the mutants carrying only single mutations. The recombinant plasmids that encoded enzyme variants which formed large amounts of mandeloamide or that formed almost stoichiometric amounts of mandelic acid from mandelonitrile were used to transform Escherichia coli strains that expressed a plant-derived (S)-hydroxynitrile lyase. The whole-cell biocatalysts obtained in this way converted benzaldehyde plus cyanide either to (S)-mandeloamide or (S)-mandelic acid with high yields and enantiopurities.Nitrilases (EC 3.5.5.1) are hydrolytic enzymes found in many bacteria, fungi, and plants which convert nitriles to the corresponding carboxylic acids and ammonia. They are members of the CN hydrolase (or nitrilase) superfamily of enzymes, which also encompasses other enzymes which attack C-N bonds, such as aliphatic amidases, carbamoylases, and N-acyltransferases (1). Nitrilases possess a catalytic triad which is composed of a cysteine, a glutamate, and a lysine residue and form during the catalytic cycle a covalent adduct between the cysteine residue and the carbon atom of the nitrile group (11, 12, 29). Nitriles are important intermediates in chemical industry, and several processes which utilize the chemo-, regio-, or enantioselectivity of nitrilases for the production of commercially interesting products have been investigated (13, 16, 17, 18, 22, 26, 27, 33, 34). There is also growing biotechnological interest in nitrilases because they form (as other members of the so-called nitrilase superfamily) spiral quaternary structures which can be studied by electron microscopy and which might be useful as templates in nanotechnology (30, 31).We are currently investigating a nitrilase from Pseudomonas fluorescens EBC191 which converts various substituted phenylacetonitriles [e.g., 2-phenylpropionitrile (2-PPN), mandelonitrile (2-hydroxyphenylacetonitrile), or phenylglycinonitrile (2-aminophenylacetonitrile)] and also aliphatic 2-acetoxynitriles with moderate enantioselectivities into the corresponding α-substituted carboxylic acids. This enzyme forms with certain nitriles also significant amounts of the corresponding amides as side products (3, 5, 8, 15, 19, 24). The enzyme has recently been studied intensively in order to analyze the molecular basis for the substrate specificity, reaction specificity, and enantiospecificity of nitrilases (9, 10). In the course of these investigations, the effects of various carboxy-terminal mutations and mutations in close proximity to the catalytic active cysteine residue were analyzed. These experiments demonstrated that deletions of 47 to 67 amino acids (aa) from the carboxy terminus of the nitrilase resulted in variant forms that demonstrated increased amide formation and an increased formation of the (R)-acids (9). In addition, it was demonstrated that the size of the amino acid residue in direct proximity to the catalytic active cysteine residue (toward the C terminus) is determinative of the enantioselectivity of acid formation. Thus, it was found that only enzyme variants with large amino acid residues at this position showed a high degree of enantioselectivity for the formation of (R)-mandelic acid from racemic mandelonitrile (10). In the present study, we investigated a set of enzyme variants that carried mutations located in the amino-terminal part of the enzyme (in relation to the catalytic active cysteine residue). Thus, several mutations that resulted in changes in the enantioselectivity of the reactions and increased formation of amides were identified.     

Functional Approach to the Catalytic Site of the Sarcoplasmic Reticulum Ca2+-ATPase: Binding and Hydrolysis of ATP in the Absence of Ca2+

Isolated sarcoplasmic reticulum vesicles in the presence of Mg(2+) and absence of Ca(2+) retain significant ATP hydrolytic activity that can be attributed to the Ca(2+)-ATPase protein. At neutral pH and the presence of 5 mM Mg(2+), the dependence of the hydrolysis rate on a linear ATP concentration scale can be fitted by a single hyperbolic function. MgATP hydrolysis is inhibited by either free Mg(2+) or free ATP. The rate of ATP hydrolysis is not perturbed by vanadate, whereas the rate of p-nitrophenyl phosphate hydrolysis is not altered by a nonhydrolyzable ATP analog. ATP binding affinity at neutral pH and in a Ca(2+)-free medium is increased by Mg(2+) but decreased by vanadate when Mg(2+) is present. It is suggested that MgATP hydrolysis in the absence of Ca(2+) requires some optimal adjustment of the enzyme cytoplasmic domains. The Ca(2+)-independent activity is operative at basal levels of cytoplasmic Ca(2+) or when the Ca(2+) binding transition is impeded.     

Formate Oxidase,an Enzyme of the Glucose-Methanol-Choline Oxidoreductase Family,Has a His-Arg Pair and 8-Formyl-FAD at the Catalytic Site

Formate oxidase of Aspergillus oryzae RIB40 contains an 8-replaced FAD with molecular mass of 799 as cofactor. The ¹H-NMR spectrum of the cofactor fraction obtained from the enzyme indicated that the 8-replaced FAD in the fraction was 8-formyl-FAD, present in open form and hemiacetal form. The oxidation-reduction potentials of the open and hemiacetal forms were estimated by cyclic voltammetry to be ?47 and ?177 mV vs. Normal Hydrogen Electrode respectively. The structure of the enzyme was constructed using diffraction data to 2.24 Å resolution collected from a crystal of the enzyme. His₅₁₁ and Arg₅₅₄ were situated close to the pyrimidine part of the isoalloxazine ring of 8-formyl-FAD in open form. The enzyme had 8-formyl-FAD, the oxidation potential of which was approximately 160 mV more positive than that of FAD, and the His-Arg pair at the catalytic site, unlike the other enzymes belonging to the glucose-methanol-choline oxidoreductase family.     

Hydrothermal Synthesis of Monolithic Co3Se4 Nanowire Electrodes for Oxygen Evolution and Overall Water Splitting with High Efficiency and Extraordinary Catalytic Stability

Cobalt selenide has been proposed to be an effective low‐cost electrocatalyst toward the oxygen evolution reaction (OER) due to its well‐suited electronic configuration. However, pure cobalt selenide has by far still exhibited catalytic activity far below what is expected. Herein, this paper for the first time reports the synthesis of new monoclinic Co₃Se₄ thin nanowires on cobalt foam (CF) via a facile one‐pot hydrothermal process using selenourea. When used to catalyze the OER in basic solution, the conditioned monolithic self‐supported Co₃Se₄/CF electrode shows an exceptionally high catalytic current of 397 mA cm^?2 at a low overpotential (η) of 320 mV, a small Tafel slope of 44 mV dec^?1, a turnover frequency of 6.44 × 10^?2 s^?1 at η = 320 mV, and excellent electrocatalytic stability at various current densities. Furthermore, an electrolyzer is assembled using two symmetrical Co₃Se₄/CF electrodes as anode and cathode, which can deliver 10 and 20 mA cm^?2 at low cell voltages of 1.59 and 1.63 V, respectively. More significantly, the electrolyzer can operate at 10 mA cm^?2 over 3500 h and at 100 mA cm^?2 for at least 2000 h without noticeable degradation, showing extraordinary operational stability.