Structural Consequences of Calmodulin EF Hand Mutations.

Calmodulin (CaM) is a cytosolic Ca2+-binding protein that serves as a control element for many enzymes. It consists of two globular domains, each containing two EF hand pairs capable of binding Ca2+, joined by a flexible central linker region. CaM is able to bind and activate its target proteins in the Ca2+-replete and Ca2+-deplete forms. To study the Ca2+-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca2+-binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. These CaM mutant proteins are deficient in binding Ca2+ in either the N-lobe EF hands (CaM12), C-lobe EF hands (CaM34), or all four EF hands (CaM1234). To investigate potential structural changes these mutations may cause, we performed detailed NMR studies of CaM12, CaM34, and CaM1234 including determining the solution structure of CaM1234. We then investigated if these CaM mutants affected the interaction of CaM with a target protein known to interact with apoCaM by determining the solution structure of CaM34 bound to the iNOS CaM binding domain peptide. The structures provide direct structural evidence of changes that are present in these Ca2+-deficient CaM mutants and show these mutations increase the hydrophobic exposed surface and decrease the electronegative surface potential throughout each lobe of CaM. These Ca2+-deficient CaM mutants may not be a true representation of apoCaM and may not allow for native-like interactions of apoCaM with its target proteins.

Structural Studies of a Complex Between Endothelial Nitric Oxide Synthase and Calmodulin at Physiological Calcium Concentration.

The small acidic protein calmodulin (CaM) serves as a Ca2+ sensor and control element for many enzymes including nitric oxide synthase (NOS) enzymes that play major roles in key physiological and pathological processes. CaM binding causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. In this report, NMR spectroscopy was used to determine the solution structure of the endothelial NOS (eNOS) peptide in complex with CaM at the lowest Ca2+ concentration (225 nM) required for CaM to bind to eNOS and corresponds to a physiological elevated Ca2+ level found in mammalian cells. Under these conditions, the CaM-eNOS complex has a Ca2+-replete C-terminal lobe bound to the eNOS peptide and a Ca2+ free N-terminal lobe loosely associated with the eNOS peptide. With increasing Ca2+ concentration, the binding of Ca2+ by the N-lobe of CaM results in a stronger interaction with the C-terminal region of the eNOS peptide and increased α-helical structure of the peptide that may be part of the mechanism resulting in electron transfer from the FMN to the heme in the oxygenase domain of the enzyme. Surface plasmon resonance studies performed under the same conditions show Ca2+ concentration-dependent binding kinetics were consistent with the NMR structural results. This investigation shows that structural studies performed under more physiological relevant conditions provide information on subtle changes in structure that may not be apparent when experiments are performed in excess Ca2+ concentrations.

An omics approach to rational feed: Enhancing growth in CHO cultures with NMR metabolomics and 2D-DIGE proteomics.

Expression of recombinant proteins exerts stress on cell culture systems, affecting the expression of endogenous proteins, and contributing to the depletion of nutrients and accumulation of waste metabolites. In this work, 2D-DIGE proteomics was employed to analyze differential expression of proteins following stable transfection of a Chinese Hamster Ovary (CHO) cell line to constitutively express a heavy-chain monoclonal antibody. Thirty-four proteins of significant differential expression were identified and cross-referenced with cellular functions and metabolic pathways to identify points of cell stress. Subsequently, 1D-(1)H NMR metabolomics experiments analyzed cultures to observe nutrient depletion and waste metabolite accumulations to further examine these cell stresses and pathways. From among fifty metabolites tracked in time-course, eight were observed to be completely depleted from the production media, including: glucose, glutamine, proline, serine, cystine, asparagine, choline, and hypoxanthine, while twenty-three excreted metabolites were also observed to accumulate. The differentially expressed proteins, as well as the nutrient depletion and accumulation of these metabolites corresponded with upregulated pathways and cell systems related to anaplerotic TCA-replenishment, NADH/NADPH replenishment, tetrahydrofolate cycle C1 cofactor conversions, limitations to lipid synthesis, and redox modulation. A nutrient cocktail was assembled to improve the growth medium and alleviate these cell stresses to achieve a ∼75% improvement to peak cell densities.

Chemical shift assignments of calmodulin constructs with EF hand mutations.

Calmodulin (CaM) is a ubiquitous cytosolic Ca(2+)-binding protein able to bind and regulate hundreds of different proteins. It consists of two globular domains joined by a flexible central linker region. Each one of these domains contains two EF hand pairs capable of binding to Ca(2+). Upon Ca(2+) binding CaM undergoes a conformational change exposing hydrophobic patches that interact with its intracellular target proteins. CaM is able to bind to target proteins in the Ca(2+)-replete and Ca(2+)-deplete forms. To study the Ca(2+)-dependent/independent properties of binding and activation of target proteins by CaM, CaM constructs with Ca(2+) binding disrupting mutations of Asp to Ala at position one of each EF hand have been used. One target protein of CaM is nitric oxide synthase, which catalyzes the production of nitric oxide. At elevated Ca(2+) concentrations, CaM binds to neuronal NOS and endothelial NOS, making them the Ca(2+)-dependent NOS enzymes. In contrast, inducible NOS is transcriptionally regulated in vivo and binds to CaM at basal levels of Ca(2+). Here we report the NMR backbone and sidechain resonance assignments of C-lobe Ca(2+)-replete and deplete CaM12, N-lobe Ca(2+)-replete and deplete CaM34, CaM1234 in the absence of Ca(2+) and N-lobe Ca(2+)-replete CaM34 with the iNOS CaM-binding domain peptide.

Dynamics of nitric oxide synthase-calmodulin interactions at physiological calcium concentrations.

The intracellular Ca²âº concentration is an important regulator of many cellular functions. The small acidic protein calmodulin (CaM) serves as a Ca²âº sensor and control element for many enzymes. Nitric oxide synthase (NOS) is one of the proteins that is activated by CaM and plays a major role in a number of key physiological and pathological processes. Previous studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. We have analyzed the structure and dynamics of complexes formed by peptides based on inducible NOS (iNOS) and endothelial NOS (eNOS) with CaM at Ca²âº concentrations that mimic the physiological basal (17 and 100 nM) and elevated levels (225 nM) found in mammalian cells using fluorescence techniques and nuclear magnetic resonance spectroscopy. The results show the CaM-NOS complexes have similar structures at physiological and fully saturated Ca²âº levels; however, their dynamics are remarkably different. At 225 nM Ca²âº, the CaM-NOS complexes show overall an increase in backbone dynamics, when compared to the dynamics of the complexes at saturating Ca²âº concentrations. Specifically, the N-lobe of CaM in the CaM-iNOS complex displays a lower internal mobility (higher S²) and higher exchange protection compared to those of the CaM-eNOS complex. In contrast, the C-lobe of CaM in the CaM-eNOS complex is less dynamic. These results illustrate that structures of CaM-NOS complexes determined at saturated Ca²âº concentrations cannot provide a complete picture because the differences in intramolecular dynamics become visible only at physiological Ca²âº levels.

Chemical shift perturbations induced by residue specific mutations of CaM interacting with NOS peptides.

The regulation of nitric oxide synthase (NOS) by calmodulin (CaM) plays a major role in a number of key physiological and pathological processes. A detailed molecular level picture of how this regulation is achieved is critical for drug development and for our understanding of protein regulation in general. CaM is a small acidic calcium binding protein and is required to fully activate NOS. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the electron transfer between the reductase and oxygenase domains through a process that is thought to be highly dynamic. The interaction of CaM with NOS is modified by a number of post-translation modifications including phosphorylation. Here we present backbone and sidechain (1)H, (15)N NMR assignments of modified CaM interacting with NOS peptides which provides the basis for a detailed study of CaM-NOS interaction dynamics using (15)N relaxation methods.

Pulsed electron paramagnetic resonance study of domain docking in neuronal nitric oxide synthase: the calmodulin and output state perspective.

The binding of calmodulin (CaM) to neuronal nitric oxide synthase (nNOS) enables formation of the output state of nNOS for nitric oxide production. Essential to NOS function is the geometry and dynamics of CaM docking to the NOS oxygenase domain, but little is known about these details. In the present work, the domain docking in a CaM-bound oxygenase/FMN (oxyFMN) construct of nNOS was investigated using the relaxation-induced dipolar modulation enhancement (RIDME) technique, which is a pulsed electron paramagnetic resonance technique sensitive to the magnetic dipole interaction between the electron spins. A cysteine was introduced at position 110 of CaM, after which a nitroxide spin label was attached at the position. The RIDME study of the magnetic dipole interaction between the spin label and the ferric heme centers in the oxygenase domain of nNOS revealed that, with increasing [Ca(2+)], the concentration of nNOS·CaM complexes increases and reaches a maximum at [Ca(2+)]/[CaM] ≥ 4. The RIDME kinetics of CaM-bound nNOS represented monotonous decays without well-defined oscillations. The analysis of these kinetics based on the structural models for the open and docked states has shown that only about 15 ± 3% of the CaM-bound nNOS is in the docked state at any given time, while the remaining 85 ± 3% of the protein is in the open conformations characterized by a wide distribution of distances between the bound CaM and the oxygenase domain. The results of this investigation are consistent with a model that the Ca(2+)-CaM interaction causes CaM docking with the oxygenase domain. The low population of the docked state indicates that the CaM-controlled docking between the FMN and heme domains is highly dynamic.

Solution structure of calmodulin bound to the target peptide of endothelial nitric oxide synthase phosphorylated at Thr495.

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes, and it is important to understand how this enzyme is regulated. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood at this time. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic and at least in part controlled by several possible phosphorylation sites. We have determined the solution structure of CaM bound to a peptide that contains a phosphorylated threonine corresponding to Thr495 in full size endothelial NOS (eNOS) to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. Our biophysical studies show that phosphorylation of Thr495 introduces electrostatic repulsions between the target sequence and CaM as well as a diminished propensity for the peptide to form an α-helix. The calcium affinity of the CaM-target peptide complex is reduced because of phosphorylation, and this leads to weaker binding at low physiological calcium concentrations. This study provides an explanation for the reduced level of NO production by eNOS carrying a phosphorylated Thr495 residue.

Quantum Hall effect in hydrogenated graphene.

The quantum Hall effect is observed in a two-dimensional electron gas formed in millimeter-scale hydrogenated graphene, with a mobility less than 10 cm2/V·s and corresponding Ioffe-Regel disorder parameter (k(F)λ)(-1) ≫ 1. In a zero magnetic field and low temperatures, the hydrogenated graphene is insulating with a two-point resistance of the order of 250h/e2. The application of a strong magnetic field generates a negative colossal magnetoresistance, with the two-point resistance saturating within 0.5% of h/2e2 at 45 T. Our observations are consistent with the opening of an impurity-induced gap in the density of states of graphene. The interplay between electron localization by defect scattering and magnetic confinement in two-dimensional atomic crystals is discussed.

The calmodulin-binding, short linear motif, NSCaTE is conserved in L-type channel ancestors of vertebrate Cav1.2 and Cav1.3 channels.

NSCaTE is a short linear motif of (xWxxx(I or L)xxxx), composed of residues with a high helix-forming propensity within a mostly disordered N-terminus that is conserved in L-type calcium channels from protostome invertebrates to humans. NSCaTE is an optional, lower affinity and calcium-sensitive binding site for calmodulin (CaM) which competes for CaM binding with a more ancient, C-terminal IQ domain on L-type channels. CaM bound to N- and C- terminal tails serve as dual detectors to changing intracellular Ca(2+) concentrations, promoting calcium-dependent inactivation of L-type calcium channels. NSCaTE is absent in some arthropod species, and is also lacking in vertebrate L-type isoforms, Cav1.1 and Cav1.4 channels. The pervasiveness of a methionine just downstream from NSCaTE suggests that L-type channels could generate alternative N-termini lacking NSCaTE through the choice of translational start sites. Long N-terminus with an NSCaTE motif in L-type calcium channel homolog LCav1 from pond snail Lymnaea stagnalis has a faster calcium-dependent inactivation than a shortened N-termini lacking NSCaTE. NSCaTE effects are present in low concentrations of internal buffer (0.5 mM EGTA), but disappears in high buffer conditions (10 mM EGTA). Snail and mammalian NSCaTE have an alpha-helical propensity upon binding Ca(2+)-CaM and can saturate both CaM N-terminal and C-terminal domains in the absence of a competing IQ motif. NSCaTE evolved in ancestors of the first animals with internal organs for promoting a more rapid, calcium-sensitive inactivation of L-type channels.

Pulsed ENDOR determination of the arginine location in the ferrous-NO form of neuronal NOS.

Mammalian nitric oxide synthases (NOSs) are enzymes responsible for oxidation of L-arginine (L-Arg) to nitric oxide (NO). Mechanisms of reactions at the catalytic heme site are not well understood, and it is of current interest to study structures of the heme species that activates O(2) and transforms the substrate. The NOS ferrous-NO complex is a close mimic of the obligatory ferric (hydro)peroxo intermediate in NOS catalysis. In this work, pulsed electron-nuclear double resonance (ENDOR) was used to probe the position of the l-Arg substrate at the NO(•)-coordinated ferrous heme center(s) in the oxygenase domain of rat neuronal NOS (nNOS). The analysis of (2)H and (15)N ENDOR spectra of samples containing d(7)- or guanidino-(15)N(2) labeled L-Arg has resulted in distance estimates for the nearby guanidino nitrogen and the nearby proton (deuteron) at C(δ). The L-Arg position was found to be noticeably different from that in the X-ray crystal structure of nNOS ferrous-NO complex [Li et al. J. Biol. Inorg. Chem.2006, 11, 753-768], with the nearby guanidino nitrogen being ~0.5 Šcloser to, and the nearby H(δ) about 1 Šfurther from, the NO ligand than in the X-ray structure. The difference might be related to the structural constraints imposed on the protein by the crystal. Importantly, in spite of its closer position, the guanidino nitrogen does not form a hydrogen bond with the NO ligand, as evidenced by the absence of significant isotropic hfi constant for N(g1). This is consistent with the previous reports that it is not the L-Arg substrate itself that would most likely serve as a direct proton donor to the diatomic ligands (NO and O(2)) bound to the heme.

Development of metal-chelating inhibitors for the Class II fructose 1,6-bisphosphate (FBP) aldolase.

It has long been suggested that the essential and ubiquitous enzyme fructose 1,6-bisphosphate (FBP) aldolase could be a good drug target against bacteria and fungi, since lower organisms possess a metal-dependant (Class II) FBP aldolase, as opposed to higher organisms which possess a Schiff-base forming (Class I) FBP aldolase. We have tested the capacity of derivatives of the metal-chelating compound dipicolinic acid (DPA), as well a thiol-containing compound, to inhibit purified recombinant Class II FBP aldolases from Mycobacterium tuberculosis, Pseudomonas aeruginosa, Bacillus cereus, Bacillus anthracis, and from the Rice Blast causative agent Magnaporthe grisea. The aldolase from M. tuberculosis was the most sensitive to the metal-chelating inhibitors, with an IC(50) of 5.2 µM with 2,3-dimercaptopropanesulfonate (DMPS) and 28 µM with DPA. DMPS and the synthesized inhibitor 6-(phosphonomethyl)picolinic acid inhibited the enzyme in a time-dependent, competitive fashion, with second order rate constants of 273 and 270 M(-1) s(-1) respectively for the binding of these compounds to the M. tuberculosis aldolase's active site in the presence of the substrate FBP (K(M) 27.9 µM). The most potent first generation inhibitors were modeled into the active site of the M. tuberculosis aldolase structure, with results indicating that the metal chelators tested cannot bind the catalytic zinc in a bidentate fashion while it remains in its catalytic location, and that most enzyme-ligand interactions involve the phosphate binding pocket residues.

Structure and dynamics of calmodulin (CaM) bound to nitric oxide synthase peptides: effects of a phosphomimetic CaM mutation.

Nitric oxide synthase (NOS) plays a major role in a number of key physiological and pathological processes. Knowledge of how this is regulated is important. The small acidic calcium binding protein, calmodulin (CaM), is required to fully activate the enzyme. The exact mechanism of how CaM activates NOS is not fully understood. Studies have shown CaM to act like a switch that causes a conformational change in NOS to allow for the transfer of an electron between the reductase and oxygenase domains through a process that is thought to be highly dynamic. To investigate the dynamic properties of CaM-NOS interactions, we determined the solution structure of CaM bound to the inducible NOS (iNOS) and endothelial NOS (eNOS) CaM binding region peptides. In addition, we investigated the effect of CaM phosphorylation. Tyrosine 99 (Y99) of CaM is reported to be phosphorylated in vivo. We have produced a phosphomimetic Y99E CaM to investigate the structural and functional effects that the phosphorylation of this residue may have on nitric oxide production. All three mammalian NOS isoforms were included in the investigation. Our results show that a phosphomimetic Y99E CaM significantly reduces the maximal synthase activity of eNOS by 40% while having little effect on nNOS or iNOS activity. A comparative nuclear magnetic resonance study between phosphomimetic Y99E CaM and wild-type CaM bound to the eNOS CaM binding region peptide was performed. This investigation provides important insights into how the increased electronegativity of a phosphorylated CaM protein affects the binding, dynamics, and activation of the NOS enzymes.

Role of an isoform-specific serine residue in FMN-heme electron transfer in inducible nitric oxide synthase.

In the crystal structure of a calmodulin (CaM)-bound FMN domain of human inducible nitric oxide synthase (NOS), the CaM-binding region together with CaM forms a hinge, and pivots on an R536(NOS)/E47(CaM) pair (Xia et al. J Biol Chem 284:30708-30717, 2009). Notably, isoform-specific human inducible NOS S562 and C563 residues form hydrogen bonds with the R536 residue through their backbone oxygens. In this study, we investigated the roles of the S562 and C563 residues in the NOS FMN-heme interdomain electron transfer (IET), the rates of which can be used to probe the interdomain FMN/heme alignment. Human inducible NOS S562K and C563R mutants of an oxygenase/FMN (oxyFMN) construct were made by introducing charged residues at these sites as found in human neuronal NOS and endothelial NOS isoforms, respectively. The IET rate constant of the S562K mutant is notably decreased by one third, and its flavin fluorescence intensity per micromole per liter is diminished by approximately 24 %. These results suggest that a positive charge at position 562 destabilizes the hydrogen-bond-mediated NOS/CaM alignment, resulting in slower FMN-heme IET in the mutant. On the other hand, the IET rate constant of the C563R mutant is similar to that of the wild-type, indicating that the mutational effect is site-specific. Moreover, the human inducible NOS oxyFMN R536E mutant was constructed to disrupt the bridging CaM/NOS interaction, and its FMN-heme IET rate was decreased by 96 %. These results demonstrated a new role of the isoform-specific serine residue of the key CaM/FMN(NOS) bridging site in regulating the FMN-heme IET (possibly by tuning the alignment of the FMN and heme domains).

Pulsed ENDOR determination of relative orientation of g-frame and molecular frame of imidazole-coordinated heme center of iNOS.

Mammalian nitric oxide synthase (NOS) is a flavo-hemoprotein that catalyzes the oxidation of L-arginine to nitric oxide. Information about the relative alignment of the heme and FMN domains of NOS is important for understanding the electron transfer between the heme and FMN centers, but no crystal structure data for NOS holoenzyme are available. In our previous work [Astashkin, A. V.; Elmore, B. O.; Fan, W.; Guillemette, J. G.; Feng, C. J. Am. Chem. Soc. 2010, 132, 12059-12067], the distance between the imidazole-coordinated low-spin Fe(III) heme and FMN semiquinone in a human inducible NOS (iNOS) oxygenase/FMN construct has been determined by pulsed electron paramagnetic resonance (EPR). The orientation of the Fe-FMN radius vector, R(Fe-FMN), with respect to the heme g-frame was also determined. In the present study, pulsed electron-nuclear double resonance (ENDOR) investigation of the deuterons at carbons C2 and C5 in the deuterated coordinated imidazole was used to determine the relative orientation of the heme g-frame and molecular frame, from which R(Fe-FMN) can be referenced to the heme molecular frame. Numerical simulations of the ENDOR spectra showed that the g-factor axis corresponding to the low-field EPR turning point is perpendicular to the heme plane, whereas the axis corresponding to the high-field turning point is in the heme plane and makes an angle of about 80° with the coordinated imidazole plane. The FMN-heme domain docking model obtained in the previous work was found to be in qualitative agreement with the combined experimental results of the two pulsed EPR works.

Intraprotein electron transfer between the FMN and heme domains in endothelial nitric oxide synthase holoenzyme.

Intraprotein electron transfer (IET) from flavin mononucleotide (FMN) to heme is an essential step in nitric oxide (NO) synthesis by NO synthase (NOS). The IET kinetics in neuronal and inducible NOS (nNOS and iNOS) holoenzymes have been previously determined in our laboratories by laser flash photolysis [reviewed in: C.J. Feng, G. Tollin, Dalton Trans., (2009) 6692-6700]. Here we report the kinetics of the IET in a bovine endothelial NOS (eNOS) holoenzyme in the presence and absence of added calmodulin (CaM). The IET rate constant in the presence of CaM is estimated to be ~4.3s(-1). No IET was observed in the absence of CaM, indicating that CaM is the primary factor in controlling the FMN-heme IET in the eNOS enzyme. The IET rate constant value for the eNOS holoenzyme is approximately 10 times smaller than those obtained for the iNOS and CaM-bound nNOS holoenzymes. Possible mechanisms underlying the difference in IET kinetics among the NOS isoforms are discussed. Because the rate-limiting step in the IET process in these enzymes is the conformational change from input state to output state, a slower conformational change (than in the other isoforms) is most likely to cause the slower IET in eNOS.

Mutation in the flavin mononucleotide domain modulates magnetic circular dichroism spectra of the iNOS ferric cyano complex in a substrate-specific manner.

We have obtained low-temperature magnetic circular dichroism (MCD) spectra for ferric cyano complexes of the wild type and E546N mutant of a human inducible nitric oxide synthase (iNOS) oxygenase/flavin mononucleotide (oxyFMN) construct. The mutation at the FMN domain has previously been shown to modulate the MCD spectra of the l-arginine-bound ferric iNOS heme (Sempombe, J.; et al. J. Am. Chem. Soc. 2009, 131, 6940-6941). The addition of l-arginine to the wild-type protein causes notable changes in the CN(-)-adduct MCD spectrum, while the E546N mutant spectrum is not perturbed. Moreover, the MCD spectral perturbation observed with l-arginine is absent in the CN(-) complexes incubated with N-hydroxy-L-arginine, which is the substrate for the second step of NOS catalysis. These results indicate that interdomain FMN-heme interactions exert a long-range effect on key heme axial ligand-substrate interactions that determine substrate oxidation pathways of NOS.

Evaluation of four microbial Class II fructose 1,6-bisphosphate aldolase enzymes for use as biocatalysts.

Fructose 1,6-bisphosphate (FBP) aldolase has been used as biocatalyst in the synthesis of several pharmaceutical compounds such as monosaccharides and analogs. Is has been suggested that microbial metal-dependant Class II aldolases could be better industrial catalysts than mammalian Class I enzyme because of their greater stability. The Class II aldolases from four microbes were subcloned into the Escherichia coli vector pT7-7, expressed and purified to near homogeneity. The kinetic parameters, temperature stability, pH profile, and tolerance to organic solvents of the Class II enzymes were determined, and compared with the properties of the Class I aldolase from rabbit muscle. Contrary to results obtained previously with the E. coli Class II aldolase, which was reported to be more stable than the mammalian enzyme, other recombinant Class II aldolases were found to be generally less stable than the Class I enzyme, especially in the presence of organic solvents. Class II aldolase from Bacillus cereus showed higher temperature stability than the other enzymes tested, but only the Mycobacterium tuberculosis Class II aldolase had a stability comparable to the Class I mammalian enzyme under assay conditions. The turnover number of the recombinant M. tuberculosis and Magnaporthe grisea Class II type A aldolases was comparable or higher than that of the Class I enzyme. The recombinant B. cereus and Pseudomonas aeruginosa Class II type B aldolases had very low turnover numbers and low metal content, indicating that the E. coli overexpression system may not be suitable for the Class II type B aldolases from these microorganisms.

Cloning, Expression, and Purification of a Nitric Oxide Synthase-Like Protein from Bacillus cereus.

The nitric oxide synthase-like protein from Bacillus cereus (bcNOS) has been cloned, expressed, and characterized. This small hemeprotein (356 amino acids in length) has a mass of 43 kDa and forms a dimer. The recombinant protein showed similar spectral shifts to the mammalian NOS proteins and could bind the substrates L-arginine and N(G)-hydroxy-L-arginine as well as the ligand imidazole. Low levels of activity were recorded for the hydrogen peroxide-dependent oxidation of N(G)-hydroxy-L-arginine and L-arginine by bcNOS, while a reconstituted system with the rat neuronal NOS reductase domain showed no activity. The recombinant bcNOS protein adds to the complement of bacterial NOS-like proteins that are used for the investigation of the mechanism and function of NO in microorganisms.

Electron transfer in a human inducible nitric oxide synthase oxygenase/FMN construct co-expressed with the N-terminal globular domain of calmodulin.

The FMN-heme intraprotein electron transfer (IET) kinetics in a human inducible NOS (iNOS) oxygenase/FMN (oxyFMN) construct co-expressed with NCaM, a truncated calmodulin (CaM) construct that includes only its N-terminal globular domain consisting of residues 1-75, were determined by laser flash photolysis. The IET rate constant is significantly decreased by nearly fourfold (compared to the iNOS oxyFMN co-expressed with full length CaM). This supports an important role of full length CaM in proper interdomain FMN/heme alignment in iNOS. The IET process was not observed with added excess EDTA, suggesting that Ca(2+) depletion results in the FMN domain moving away from the heme domain. The results indicate that a Ca(2+)-dependent reorganization of the truncated CaM construct could cause a major modification of the NCaM/iNOS association resulting in a loss of the IET.