Mechanistic evaluation of chromium bioremediation in Acinetobacter junii strain b2w: A proteomic approach.

Growing industrialization and unchecked release of industrial waste, including heavy metals have resulted in disastrous effects on environment. Considering the problem of heavy metal pollution, the present research was designed to study the bioremediation of chromium, a highly toxic and prominent heavy metal pollutant by Acinetobacter junii strain b2w isolated from the Mithi river, Mumbai, India. The bacterial isolate could grow without affecting its growth kinetics up to a concentration of 200 ppm of chromium and showed resistance towards 400 ppm of chromium. It was able to bioremediate 83.06% of total chromium and reduces 98.24% of Cr6+ to C3+ at a concentration of 10 ppm of chromium. The bacterial isolate could grow well at a wide pH range from 5 to 9, salinity of up to 3.5% and could also tolerate heavy metals such as Cd, Zn, As, Hg, Pb and Cu. Thus, indicating its possible on-ground applicability for bioremediation of chromium. Acinetobacter junii bioaccumulate chromium without disrupting the cell integrity and biosorption. However, chromium alters the functional groups on bacterial cell surface and led to decrease in sulfate-containing molecules. Further, the protein expression study has revealed that Cr significantly up-regulates proteins broadly classified under envelope stress responses, oxidative stress responses, energy metabolism and quorum sensing and growth regulator. The possible mechanisms of Cr detoxification in Acinetobacter junii strain b2w could be reduction, bioaccumulation and efflux along with neutralization of oxidative stress generated by Cr. Thus, based on bacterial bioremediation potential and its molecular response, it can be proposed that the isolated Acinetobacter junii has potential applicability for chromium bioremediation.

Controlled Uptake of an Iridium Complex inside Engineered apo-Ferritin Nanocages: Study of Structure and Catalysis.

The effect of the mutation at the core of the ferritin nanocage (apo-rHLFr) on the uptake of IrCp* has been investigated by structural and spectroscopic methods. Site-specific mutations of two polar residues viz., Asp38 and Arg52 were investigated. The uptake of IrCp* was increased by about 1.5-fold on mutation of Arg52 by His compared to the wild-type variant, while mutation of Asp38 by His had no effect on the uptake. All the variants of the Ir-embedded ferritin cages remained as stable as the wild-type analogue. These hybrid bio-nanocages of ferritin were found to efficiently catalyze transfer hydrogenation of various substituted acetophenones forming the corresponding chiral alcohols with up to 88 % conversion and 70 % enantioselectivity. An electron-withdrawing substituent on the reactant enhanced the Turnover frequency of the reaction. Molecular docking analyses suggested that the substrate binds in different orientations at the active site in different mutants of the nanocage.

Identification of a copper ion recognition peptide sequence in the subunit II of cytochrome c oxidase: a combined theoretical and experimental study.

The role of the pentapeptide, NHSFM, derived from the surface exposed part of the metal ion binding loop of the subunit II of cytochrome c oxidase on the maturation of the binuclear purple CuA center of the enzyme has been investigated using several experimental and computational methods. The copper ion was found to form 1:1 complex of the pentapeptide with a binding constant ~ 104 M-1 to 105 M-1, where a 4 ligand coordination from the peptide in a type 2 copper center was revealed. The pH dependence of the metal-peptide was associated with a [Formula: see text] of ~ 10 suggesting deprotonation of the N-terminal amine. EXAFS studies as well as DFT calculations of the metal-peptide complexes revealed pH dependent changes in the metal-ligand bond distances. Spectroscopic properties of the metal peptides calculated from TDDFT studies agreed with the experimental results. Restrained molecular dynamics (RMD) simulations indicated coordination of a carbonyl oxygen from the asparagine (N) side chain and of water molecules apart from histidine (H), methionine (M) and terminal amine of asparagine (N) in a distorted square planar geometry of Cu-NHSFM. Analyses of the backbone distances as well as B-factors for the metal peptide suggested that the peptide backbone becomes more compact and rigid on binding of the metal ion. This indicated that binding of copper ion to this pentapeptide in the protein possibly cause movement of the protein backbone bringing other coordinating residues closer to the copper ion, and thus helping in sequential uptake of copper ions to the protein.

Covalent conjugation of single-walled carbon nanotube with CYP101 mutant for direct electrocatalysis.

Covalent linkage between the single-walled carbon nanotube (SWCNT) and CYP101 through a specific site of the enzyme can provide a novel method of designing efficient enzyme electrodes using this prototype cytochrome P450 enzyme. We have chemically modified the SWCNT with linker 4-carboxy phenyl maleimide (CPMI) containing maleimide functional groups. The enzyme was covalently attached on to the SWCNT through the maleimide group of the linker (CPMI) to the thiolate group of the surface exposed Cys 58 or Cys 136 of the CYP101 forming a covalently immobilized protein on the nanotube. Thin film of the modified SWCNT-CPMI-CYP101conjugate was made on a glassy carbon (GC) electrode. Direct electrochemistry of the substrate (camphor)-bound enzyme was studied using this immobilized enzyme electrode system and the redox potential was found to be -320mV vs Ag/AgCl (3 M KCl), which agrees with the redox potential of the substrate bound enzyme reported earlier. The electrochemically driven enzymatic mono-oxygenation of camphor by this immobilized enzyme electrode system was studied by measurement of the catalytic current at different concentrations of camphor. The catalytic current was found to increase with increasing concentration of camphor in presence of oxygen. The product formed during the catalysis was identified by mass-spectrometry as hydroxy-camphor.

Kinesin associated protein, DmKAP, binding harnesses the C-terminal ends of the Drosophila kinesin-2 stalk heterodimer.

The heterotrimeric kinesin-2 consists of two distinct motor subunits and an accessory protein, KAP, which binds to the coiled-coil stalk domains and one of the tail domains of the motor subunits. Genetic studies revealed that KAP is essential for the kinesin-2 functions in cilia, flagella, and axon. However, the structural significance of the KAP binding on kinesin-2 assembly and stability is not known. Here, using the Fluorescence Lifetime assay, we show that DmKAP binding selectively reduces the distance between the C-terminal ends of Drosophila kinesin-2 stalk heterodimer. Insertion of a missense mutation (E551K) in the Drosophila kinesin-2α stalk fragment, which was shown to reduce the structural dynamics of the stalk heterodimer earlier, also reduced the distances at both the N- and C-terminal ends of the stalk heterodimer independent of DmKAP. The zipping effect, particularly at the N-terminal end of the mutant stalk heterodimer, is further enhanced in the presence of DmKAP. Together, these results suggest that the KAP binding could alter the structural dynamics of kinesin-2 stalk heterodimer at the C-terminal end and stabilize the association between the stalk domains.

The structural dynamics of the kinesin-2 stalk heterodimer and its biological relevance.

Association between two motor subunits through the rod/stalk domain enables molecular motors to walk processively on protein filaments. Previous studies suggested that structural flexibility in the coiled-coil stalk of kinesins is essential for processive runs. The stalk of heterotrimeric kinesin-2, a comparatively less processive motor, is unstable at ambient temperature. How this structural instability impacts the motor function is unclear. Here, using the Förster Resonance Energy Transfer based assays, we show that the Drosophila kinesin-2α/ß stalk heterodimer is dynamic at physiological conditions. We further show that insertion of a missense mutation (Glu551-Lys) at the C-terminal half of kinesin-2α stalk reduces the dynamics of the heterodimeric stalk in vitro. The mutation, isolated as a recessive lethal allele in a forward genetic screen, is reported to disrupt the motor function in axonal transport and cilia development. Together these two results suggest that the dynamic instability of the kinesin-2 stalk could play a crucial role in maintaining its biological function.

The protein inhibitor of nNOS (PIN/DLC1/LC8) binding does not inhibit the NADPH-dependent heme reduction in nNOS, a key step in NO synthesis.

The neuronal nitric oxide synthase (nNOS) is an essential enzyme involved in the synthesis of nitric oxide (NO), a potent neurotransmitter. Although previous studies have indicated that the dynein light chain 1 (DLC1) binding to nNOS could inhibit the NO synthesis, the claim is challenged by contradicting reports. Thus, the mechanism of nNOS regulation remained unclear. nNOS has a heme-bearing, Cytochrome P450 core, and the functional enzyme is a dimer. The electron flow from NADPH to Flavin, and finally to the heme of the paired nNOS subunit within a dimer, is facilitated upon calmodulin (CaM) binding. Here, we show that DLC1 binding to nNOS-CaM complex does not affect the electron transport from the reductase to the oxygenase domain. Therefore, it cannot inhibit the rate of NADPH-dependent heme reduction in nNOS, which results in l-Arginine oxidation. Also, the NO release activity does not decrease with increasing DLC1 concentration in the reaction mix, which further confirmed that DLC1 does not inhibit nNOS activity. These findings suggest that the DLC1 binding may have other implications for the nNOS function in the cell.

Role of substituents on the reactivity and product selectivity in reactions of naphthalene derivatives catalyzed by the orphan thermostable cytochrome P450, CYP175A1.

The thermostable nature of CYP175A1 enzyme is of potential interest for the biocatalysis at ambient temperature or at elevated temperature under environmentally benign conditions. Although little is known about the substrate selectivity of this enzyme, the biocatalytic activities of CYP175A1 on different substituted naphthalenes have been studied in oxidative pathway, and the effect of the substituent on the reaction has been determined. The enzyme first acts as a peroxygenase to convert these substituted naphthalenes to the corresponding naphthols, which subsequently undergo in-situ oxidative dimerization to form dyes of different colors possibly by the peroxidase-type activity of CYP175A1. The product analyses and kinetic measurements suggested that the presence of electron releasing substituent (ERS) in the substrate enhanced the substrate conversion, whereas the presence of electron withdrawing substituent (EWS) in the substrate drastically reduced the substrate conversion. The position of the ERS in the substrate was also found to play an important role in the transformation of the substrate. The results further demonstrate that mutation of the Leu80 residue to Phe enhances the reactivity of the enzyme by favoring the substrate association in the active site. The observed rates of the enzymatic oxygenation reaction of the substituted naphthalenes followed the Hammett correlation of substituent effect, supporting aromatic electrophilic substitution mechanism catalyzed by the cytochrome P450 enzyme.

Mechanism of copper incorporation in subunit II of cytochrome C oxidase from Thermus thermophilus: identification of intermediate species.

Detailed spectroscopic and kinetic studies of incorporation of copper ion in the wild type (WT) and the D111AA (AA = K, N, or E) mutants of the metal ion binding site of the soluble fragment of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) showed the formation of at least two distinct intermediates. The global analyses of the multiwavelength kinetic results suggested a four-step reaction scheme involving two distinct intermediates in the pathway of incorporation of copper ions into the apoprotein forming the purple dinuclear CuA. An early intermediate similar to the red copper binding proteins was detected in the WT as well as in all the mutants. The second intermediate was a green copper species in the case of WT TtCuA. Mutation of Asp111, however, formed a second intermediate that is distinctly different from that formed in the case of the WT protein, suggesting that mutants follow pathways of copper ion incorporation different from that in the WT protein. The electrostatic interaction between Asp111 and the coordinating His114 possibly plays a subtle role in the mechanism of incorporation of metal ion into the protein. The overall Kd for WT TtCuA was found to be ~8 nM, which changed with mutation of the Asp111 residue. The activation and thermodynamic parameters were also determined from the temperature- and pH-dependent multiwavelength kinetics, and the results are discussed to unravel the role of Asp111 in the mechanism of formation of the dinuclear CuA center in cytochrome c oxidase.

Thermodynamic effects of the alteration of the axial ligand on the unfolding of thermostable cytochrome C.

The role the axial methionine plays in the conformational properties and thermostability of the heme active site has been investigated with the help of site-specific mutations at the axial Met69 position with His (M69H) and Ala (M69A) in thermostable cytochrome c(552) from Thermus thermophilus. Detailed circular dichroism, direct electrochemistry, and other spectroscopic studies have been employed to investigate the thermally induced and GdnHCl-induced unfolding properties of the heme active site of the wild type and the mutants of cytochrome c(552). We observed an unusually high thermodynamic and thermal stability of the M69A mutant compared to that of wild-type cytochrome c(552). However, the M69H mutant exhibited a slightly lower unfolding free energy compared to that of the wild-type protein. The high conformational stability of the M69A mutant was attributed to the presence of residual structure in the unfolded state as well as to the altered conformation in the folded state of this mutant of cytochrome c(552). This study thus supports the view that apart from the folded state, the unfolded state of a protein may also make a significant contribution to the stability of a protein.

Computationally guided bioengineering of the active site, substrate access pathway, and water channels of thermostable cytochrome P450, CYP175A1, for catalyzing the alkane hydroxylation reaction.

Understanding structure-function relationships in proteins is pivotal in their development as industrial biocatalysts. In this regard, rational engineering of protein active site access pathways and various tunnels and channels plays a central role in designing competent enzymes with high stability and enhanced efficiency. Here, we report the rational evolution of a thermostable cytochrome P450, CYP175A1, to catalyze the C-H activation reaction of longer-chain alkanes. A strategy combining computational tools with experiments has shown that the substrate scope and enzymatic activity can be enhanced by rational engineering of certain important channels such as the substrate entry and water channels along with the active site of the enzyme. The evolved enzymes showed an improved catalytic rate for hexadecane hydroxylation with high regioselectivity. The Q67L/Y68F mutation showed binding of the substrate in the active site, water channel mutation L80F/V220T showed improved catalytic activity through the peroxide shunt pathway and substrate entry channel mutation W269F/I270A showed better substrate accessibility to the active pocket. All-atom MD simulations provided the rationale for the inactivity of the wild-type CYP175A1 for hexadecane hydroxylation and predicted the above hot-spot residues to enhance the activity. The reaction mechanism was studied by QM/MM calculations for enzyme-substrate complexes and reaction intermediates. Detailed thermal and thermodynamic stability of all the mutants were analyzed and the results showed that the evolved enzymes were thermally stable. The present strategy showed promising results, and insights gained from this work can be applied to the general enzymatic system to expand substrate scope and improve catalytic activity.

Antibacterial Effects of ZnO Nanodisks: Shape Effect of the Nanostructure on the Lethality in Escherichia coli.

The role of the shape of the nanostructure on the antibacterial effects of ZnO nanodisks has been investigated by detailed mass spectrometry-based proteomics along with other spectroscopic and microscopic studies on E. coli. The primary interaction study of the E. coli cells in the presence of ZnO nanodisks showed rigorous cell surface damage disrupting the cell wall/membrane components detected by microscopic and ATR-FTIR studies. Protein profiling of whole-cell extracts in the presence and absence of ZnO nanodisks identified several proteins that are upregulated and downregulated under the stress of the nanodisks. This suggests that the bacterial response to the primary stress leads to a secondary impact of ZnO nanodisk toxicity via regulation of the expression of specific proteins. Results showed that the ZnO nanodisks lead to the over-expression of peptidyl-dipeptidase Dcp, Transketolase-1, etc., which are important to maintaining the osmotic balance in the cell. The abrupt change in osmotic pressure leads to mechanical injury to the membrane, and nutritional starvation conditions, which is revealed from the expression of the key proteins involved in membrane-protein assembly, maintaining membrane integrity, cell division processes, etc. Thus, indicating a deleterious effect of ZnO nanodisk on the protective layer of E. coli. ZnO nanodisks seem to primarily affect the protective membrane layer, inducing cell death via the development of osmotic shock conditions, as one of the possible reasons for cell death. These results unravel a unique behavior of the disk-shaped ZnO nanostructure in executing lethality in E. coli, which has not been reported for other known shapes or morphologies of ZnO nanoforms.

Role of the surface-exposed leucine 155 in the metal ion binding loop of the CuA domain of cytochrome c oxidase from Thermus thermophilus on the function and stability of the protein.

The role of Leu155 in the metal ion binding loop in the soluble CuA binding domain of subunit II of cytochrome c oxidase from Thermus thermophilus (TtCuA) was investigated by site-specific mutations of this residue to arginine (L155R) and glutamic acid (L155E). The UV-visible absorption and electron paramagnetic resonance spectra suggested that the Cu(2)S(2) core of TtCuA was almost unchanged by the mutations. The redox potential of the metal center in the L155R mutant was ~20 mV higher than that in the WT protein, while that of the L155E mutant was almost the same as that of the wild type (WT-TtCuA). The rate of transfer of an electron from cytochrome c(552) to the L155E mutant was much lower than that of transfer to the WT protein, while that for transfer to the L155R mutant was similar to that of WT-TtCuA. The total reorganization energy was increased for both the mutant proteins compared to WT-TtCuA. The results suggest that the presence of a negatively charged residue at the site of Leu155 in TtCuA possibly disfavors the protein-protein interaction between the two redox partners. The mutation also affected the equilibrium pH dependence of the protein. The thermal and thermodynamic stability of TtCuA was drastically decreased upon the mutation, which is most prominent in the L155R mutant. These studies indicate that the hydrophobic patch at the surface of TtCuA consisting of Leu155 is important for the transfer of an electron between cytochrome c(552) and TtCuA.

Oxygenation of monoenoic fatty acids by CYP175A1, an orphan cytochrome P450 from Thermus thermophilus HB27.

The catalytic activity of CYP175A1 toward monooxygenation of saturated and monounsaturated fatty acids of various chain lengths (C16-C24) has been investigated to assess the enzymatic properties of this orphan thermostable cytochrome P450 enzyme. The results showed that the enzyme could catalyze the reaction of monounsaturated fatty acids but not of saturated fatty acids. The product analyses using ESI-MS and GC-MS revealed an important regioselectivity in the CYP175A1 catalyzed monooxygenation of the monoenoic fatty acids depending on the ethylenic double bond (C═C) configuration. When the double bond was in cis-configuration, an epoxy fatty acid was found to be the major product and two allyl-hydroxy fatty acids were found to be the minor products. But when the double bond was in trans-configuration the product distribution was reversed. The oxygenation efficiency was found to be the highest for palmitoleic acid (chain length C16), but there was no direct correlation of the activity with the chain length or the position of unsaturation of the fatty acid. Molecular docking calculations showed that the "U"-type conformations of the monoenoic fatty acids are particularly responsible for their binding in the enzyme pocket, and that is also consistent with the observed regioselectivity in the oxygenation reaction. The present results provide evidence that CYP175A1 can catalyze the regioselective oxygenation reaction of several monoenoic fatty acids though it cannot catalyze the oxygenation of the corresponding saturated analogues. These studies may provide critical information on the nature of the enzyme pocket and of the possible natural substrate of this orphan enzyme.

Sequence specific association of tryptic peptides with multiwalled carbon nanotubes: effect of localization of hydrophobic residues.

A model-free approach has been used to study the association of peptides onto multiwalled carbon nanotubes (MWCNT) in aqueous solution at ambient pH to understand the molecular basis of interaction of the peptides with MWCNT. The peptides obtained by tryptic digestion of cytochrome P450cam from P. putida were allowed to interact with MWCNT, and several peptides were found to bind to the nanotube leading to formation of stable homogeneous dispersion of the bionano conjugates of MWCNT. The peptides bound to the MWCNT were separated from the unbound peptides and sequence analyses by tandem MS/MS technique identified the strongly bound peptides as well as the unbound and the weakly bound peptides. The peptide-MWCNT conjugate was further characterized by TEM as well as Raman, FTIR, vis-NIR absorption, and circular dichroism spectroscopy. A model based on the hydrophobicity of residues in the peptides suggested that the amphiphilic peptides with localized hydrophobic residues at the center or at one end of the sequence form stable dispersions of the peptide-MWCNT conjugates.

Enhanced Substrate Specificity of Thermostable Cytochrome P450 CYP175A1 by Site Saturation Mutation on Tyrosine 68.

Thermostable cytochrome P450 (CYP175A1) cloned from Thermus thermophilus shows mid-point unfolding temperature (Tm) of 88 °C (361 K) along with high thermodynamic stability making it a potential industrially viable biocatalyst. Molecular docking analyses, and structural superposition with steroidogenic and fatty acid metabolizing cytochrome P450 s suggested that the tyrosine 68 may have important role in binding as well as metabolism of substrates by the enzyme. Site-saturation mutation of the tyrosine 68 residue was carried out and several unique mutations were obtained that were properly folded and showed high thermostability. We investigated the effects of variation of the single residue, Tyr68 at the substrate binding pocket of the enzyme on the substrate specificity of CYP175A1. Screening of the mutant colonies of CYP175A1 obtained after saturation mutagenesis of Tyr68 using saturated fatty acid, myristic acid and poly unsaturated fatty acids showed that the Y68K had notable binding and catalytic activity for mono-oxygenation of the saturated fatty acid (myristic acid), which had no major detectable binding affinity towards the WT enzyme. The Y68R mutant of CYP175A1, on the other hand was found to selectively bind and catalyse reaction of cholesterol. The wild type as well as both the mutants of the enzyme however bind poly unsaturated fatty acids. The results thus show that saturation mutation of a single amino acid at the substrate binding pocket of the thermostable cytochrome P450 could induce sufficient changes in the substrate binding pocket of the enzyme that can efficiently change substrate specificity of the enzyme.

Structural design of the active site for covalent attachment of the heme to the protein matrix: studies on a thermostable cytochrome P450.

The molecular basis of the post-translational modification involving covalent attachment of the heme with a glutamic acid observed in some enzymes of the CYP4 family of heme monooxygenases has been investigated using site-directed mutagenesis of CYP175A1 from Thermus thermophilus. Earlier studies of CYP4 as well as the G248E mutant of CYP101A1 showed covalent linkage of the heme to a conserved glutamic acid of helix I. We have introduced Glu/Asp at the Leu80 position in the ß-turn of CYP175A1, on the basis of molecular modeling studies, to assess whether formation of such a covalent linkage is limited only to helix I or whether such modification may also take place with the residue that is spatially located at a position appropriate for activation by the heme peroxidase reaction. Tandem mass spectrometry analyses of the tryptic digest of the wild type and mutants of CYP175A1 were conducted to identify any heme-bound peptide. Tryptic digestion of the L80E mutant of CYP175A1 preincubated with H(2)O(2) showed formation of GLE(-heme)TDWGESWKEARK supporting covalent linkage of Glu80 with the heme in the mutant enzyme. No such heme-bound peptides were found if the sample was not preincubated in H(2)O(2), indicating no activation of the Glu by the heme peroxidase reaction, as proposed earlier. The wild type or L80D mutant of the enzyme did not give any heme-bound peptide. Thus, the results support the idea that covalent attachment of the heme to an amino acid in the protein matrix depends on the structural design of the active site.