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This curriculum from the BASIL (Biochemistry Authentic Scientific Inquiry Laboratory) biochemistry consortium aims to get students to transition from thinking like students to thinking like scientists. Students will explore proteins with known structure but unknown function using computational analyses and wet-lab techniques. BASIL is designed for undergraduate biochemistry lab courses, but can be adapted to first year (or even high school) settings, as well as upper-level undergraduate or graduate coursework. It is targeted to students in biology, biochemistry, chemistry, or related majors. Further details about the BASIL biochemistry consortium can be found on the BASIL blog. The curriculum is flexible and can be adapted to match the available facilities, the strengths of the instructor and the learning goals of a course and institution. These lessons are often used as part of upper-level laboratory coursework with at least one semester of biochemistry as a pre-requisite or co-requisite. The lab has been designed for classes ranging from 10-24 students (working in teams of two or three) per lab section. This lesson can be adapted to laboratory courses for introductory biology, cell and molecular biology, or advanced biology labs., This work is supported by NSF IUSE 1503811, 1503699, 1502720, 1503676, 1503710, 1503798, 1503734, 1709170, 1709805, 1709592, 1710583, 1709355, 1709278, and 1710051

Thin women have not traditionally been considered ideal candidates for autologous breast reconstruction. The purpose of this study was to examine the use of deep inferior epigastric perforator (DIEP) flap reconstruction in thin women undergoing immediate unilateral breast reconstruction. A retrospective review of 1,040 consecutive patients was performed. In total, 381 patients met the inclusion criteria. To improve clinical interpretability, patients were divided into three groups based on body mass index: “thin” (BMI ≤ 22.99), “traditional” (>23 and ≤29.99), and “obese” (BMI >30) candidates. Flap characteristics were compared to mastectomy weights, and postoperative complications were analyzed. In all groups, flap size was generally more than sufficient to match the mastectomy specimen, as flap weight:mastectomy weight ratio ws greater than 1 in all groups with no significant difference between groups (1.1 in thin patients, 1.0 in traditional patients, and 1.0 in obese patients). Fat necrosis prevalence was lowest in the thin group (12.5%), compared to the traditional (15.9%, P = 0.443) or obese (14.4%, P = 0.698) groups. Prevalence of breast infection were lower in the thin patients (5.2%) versus the traditional (8.7%, P = 0.287) or obese (14.4%, P = 0.033). Abdominal wound healing complications and seroma were also lowest in thin patients. DIEP flap breast reconstruction may be an effective method for unilateral breast reconstruction in thin patients, with sufficient flap weights and lower incidence of complications than in heavier patients. As such, low BMI may not present a barrier in the reconstruction of a breast mound matching native breast size. © 2015 Wiley Periodicals, Inc. Microsurgery, 2015.

This study objective was to develop a method to characterize the diversity of trade-offs between life functions expressed by dairy cows. Trade-offs between life functions involve adaptive responses of dairy cows to suboptimal nutritional environments. Until now, they have been explored mainly by examining unfavorable correlations between two traits. These two-trait approaches are limiting for exploring the diversity of trade-offs among cows. A multi-trait and dynamic method was developed to phenotype trade-offs between life functions involved in cow fitness (lactation, reproduction, and ability to survive) and explore their diversity. Records from 334 lactating cows reared in two experimental INRA (France) units were used to study the dynamics of cow milk yield, body condition changes and reproduction performance. The analysis focused on the first 13 weeks postpartum, when cows are supposed to experience a negative energy balance. Ten variables accounting for the dynamics of responses were calculated and included in a clustering analysis. Four main clusters of trade-offs were obtained. Profile 1 of trade-off (N=53) included cows giving priority to lactation and mobilizing much of their body fat reserves, with poor reproductive performance. Trade-off profile 2 (N=111) identified cows mobilizing much of their body fat reserves, giving priority to reproduction at the expense of high milk yield. Trade-off profile 3 (N=67) consisted of thin cows presenting difficulties in all functions: a large body-reserve mobilization after calving that does not benefit to milk yield and long delays before reproducing and low success rates. Profile 4 of trade-off (N=103) was composed of cows with no trade-off between functions, since they recorded average milk yield, maintained their body condition and had good reproductive performances. Our approach highlighted the relevance of considering the three life functions simultaneously when phenotyping dairy cows for their ability to manage prioritization between life functions and this multi-trait clustering approach represents an operational tool to do so, using readily available farm data. Since classification of cows into clusters is not fully determined by the breed or parity, our study underlined also the utility of better understanding the mechanisms that drive nutrient allocation between life functions. We also believe in the benefit of considering this individual diversity, as a herd management tool for farmers.

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis; it uses tetrahydrobiopterin and molecular oxygen to convert tyrosine to DOPA. Its amino terminal 150 amino acids comprise a domain whose structure is involved in regulating the enzyme's activity. Modes of regulation include phosphorylation by multiple kinases at four different serine residues, and dephosphorylation by two phosphatases. The enzyme is inhibited in feedback fashion by the catecholamine neurotransmitters. Dopamine binds to TyrH competitively with tetrahydrobiopterin, and interacts with the R domain. TyrH activity is modulated by protein-protein interactions with enzymes in the same pathway or the tetrahydrobiopterin pathway, structural proteins considered to be chaperones that mediate the neuron's oxidative state, and the protein that transfers dopamine into secretory vesicles. TyrH is modified in the presence of NO, resulting in nitration of tyrosine residues and the glutathionylation of cysteine residues.

The physiological role of the liver enzyme phenylalanine hydroxylase (PheH)1 is to catalyze the hydroxylation of excess phenylalanine in the diet to form tyrosine, using tetrahydrobiopterin (BH4) as the source of electrons for this monooxygenation reaction (Figure 1) [1]. The importance of the enzyme is demonstrated by the devastating effects of insufficient PheH activity. The resulting disease, phenylketonuria, results in poor growth and progressive intellectual impairment, with eventual death of the affected patient at a young age in the absence of treatment [2]. Figure 1 The reaction catalyzed by phenylalanine hydroxylase PheH is a member of the family of aromatic amino acid hydroxylases, along with tyrosine hydroxylase (TyrH) and tryptophan hydroxylase (TrpH) [1]. TyrH hydroxylase in the adrenal gland and central nervous system catalyzes the rate-limiting step in the formation of catecholamine neurotransmitters. TrpH in the central nervous system catalyzes the rate-limiting step in the formation of serotonin. All three enzymes are homotetramers, with each monomer containing an N-terminal regulatory domain of 100-160 residues, a homologous catalytic domain of ~300 residues, and a C-terminal tetramerization domain of ~45 residues that contains a C-terminal helix. There is no structure available of an intact eukaryotic aromatic amino acid hydroxylase. In the case of PheH, the most complete structures are of a dimeric rat enzyme that lacks the C-terminal helix [3] and of the tetrameric human enzyme lacking the N-terminal regulatory domain [4]. The structure of the dimeric rat enzyme shows that the N-terminus of the regulatory domain extends across the active site opening. This feature has led to residues 19-29 being designated an auto-inhibitory domain critical for regulation of PheH [5]. Maintaining proper levels of PheH activity is clearly critical, and the enzyme activity is regulated in response to levels of free phenylalanine. The seminal work leading to our present understanding of the regulation of PheH was carried out by Shiman and coworkers [6–9]. PheH exhibits positive cooperativity when the rate of the reaction is determined as a function of phenylalanine concentration and hyperbolic kinetics when the concentration of the tetrahydropterin is varied. In addition, there is a lag in the formation of tyrosine unless the enzyme is first incubated with phenylalanine [9], and the lag is more pronounced if the enzyme is first incubated with a tetrahydropterin. In the model developed to explain these observations, the resting form of the enzyme is inactive. Binding of BH4 stabilizes the inactive form, while binding of phenylalanine at a regulatory site activates the enzyme. The structure of the combined catalytic and regulatory domains of PheH provided a structural rationale for this model, by showing that residues 19-29 of the regulatory domain lie across the active site in the resting enzyme and presumably keep the enzyme from binding substrates [3]. Binding of phenylalanine at a regulatory site would then shift the enzyme to a conformation in which the active site was open. Direct structural evidence for this model is lacking. There is no reported structure of a form of PheH with both a regulatory domain and either phenylalanine or a pterin bound, so that the actual structural change that results in activation has not been established. In the absence of a direct structural support for a regulatory binding site for phenylalanine, the presence of such a site has come under question. A protein containing maltose binding protein fused to the N-terminus of the human PheH regulatory domain has been reported to bind radio-labeled phenylalanine [10]. In contrast, Thorolfsson et al. [11] could not detect any binding to the isolated regulatory domain by differential scanning calorimetry and concluded that activation is due to interactions between the active sites and that the role of the regulatory domain is to allow communication between active sites. Such a model would be consistent with the structure, which shows each regulatory domain interacting with two catalytic domains. To address the existence of a regulatory binding site for phenylalanine in PheH, we have expressed the isolated regulatory domain of rat PheH and analyzed the effects of phenylalanine on its structure. The results establish that there is a binding site for phenylalanine in this domain, consistent with the existence of an allosteric site for phenylalanine in PheH.

Alpha-synuclein (a-Syn), a protein implicated in Parkinson disease, contributes significantly to dopamine metabolism. a-Syn binding inhibits the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in catecholamine synthesis. Phosphorylation of TH stimulates its activity, an effect that is reversed by protein phosphatase 2A (PP2A). In cells, a-Syn overexpression activates PP2A. Here we demonstrate that a-Syn significantly inhibited TH activity in vitro and in vivo and that phosphorylation of a-Syn serine 129 (Ser-129) modulated this effect. In MN9D cells, a-Syn overexpression reduced TH serine 19 phosphorylation (Ser(P)-19). In dopaminergic tissues from mice overexpressing human a-Syn in catecholamine neurons only, TH-Ser-19 and TH-Ser-40 phosphorylation and activity were also reduced, whereas PP2A was more active. Cerebellum, which lacks excess a-Syn, had PP2A activity identical to controls. Conversely, a-Syn knock-out mice had elevated TH-Ser-19 phosphorylation and activity and less active PP2A in dopaminergic tissues. Using an a-Syn Ser-129 dephosphorylation mimic, with serine mutated to alanine, TH was more inhibited, whereas PP2A was more active in vitro and in vivo. Phosphorylation of a-Syn Ser-129 by Polo-like-kinase 2 in vitro reduced the ability of a-Syn to inhibit TH or activate PP2A, identifying a novel regulatory role for Ser-129 on a-Syn. These findings extend our understanding of normal a-Syn biology and have implications for the dopamine dysfunction of Parkinson disease.

Electrospray mass spectrometry (ES-MS) is a new speciation technique that has the great interest to be able to probe the element, the ligand and the complex in order to reach the speciation. This paper will focus on the use of ES-MS for the speciation of actinides/lanthanides on several systems of interest in various fields such as the interaction between DTPA (decorporant) and europium, HEBP and uranium, BTP (new extracting agent) and lanthanides with comparison with known chemistry as well as whenever possible with other speciation techniques.

Tyrosine hydroxylase is phosphorylated at four serine residues in its amino-terminus by multiple kinases. Phosphorylation of serine 40 by cAMP-dependent protein kinase results in alleviation of dopamine inhibition [J. Biol. Chem. 267 (1992) 12639]. The other serines are at positions 8, 19, and 31. The effect of phosphorylation at these serines has been investigated using mutated forms of tyrosine hydroxylase containing glutamates at the positions of the serines. The S8E, S19E, and S31E tyrosine hydroxylase variants have similar steady-state kinetic parameters and similar binding affinity for catecholamines to wild-type enzyme. The S8E, S19E, S31E, and S40E variants differ in stability at elevated temperatures. The S40E variant is the least stable, while the others are all more stable than wild-type enzyme. The increased stability of S8E, S19E, and S31E tyrosine hydroxylases may be one of the physiological effects of phosphorylation. It may also have implications for the interpretation of activities of heterogeneous mixtures of tyrosine hydroxylase which have been phosphorylated.

Cyclin-dependent kinase 5 (Cdk5) is emerging as a neuronal protein kinase involved in multiple aspects of neurotransmission in both post- and presynaptic compartments. Within the reward/motor circuitry of the basal ganglia, Cdk5 regulates dopamine neurotransmission via phosphorylation of the postsynaptic signal transduction pathway integrator, DARPP-32 (dopamine- and cyclic AMP-regulated phosphoprotein, M(r) 32,000). Cdk5 has also been implicated in regulating various steps in the presynaptic vesicle cycle. Here we report that Cdk5 phosphorylates tyrosine hydroxylase (TH), the key enzyme for synthesis of dopamine. Using phosphopeptide mapping, site-directed mutagenesis, and phosphorylation state-specific antibodies, the site was identified as Ser31, a previously defined extracellular signal-regulated kinases 1/2 (ERK1/2) site. The phosphorylation of Ser31 by Cdk5 versus ERK1/2 was investigated in intact mouse striatal tissue using a pharmacological approach. The results indicated that Cdk5 phosphorylates TH directly and also regulates ERK1/2-dependent phosphorylation of TH through the phosphorylation of mitogen-activated protein kinase kinase 1 (MEK1). Finally, phospho-Ser31 TH levels were increased in dopaminergic neurons of rats trained to chronically self-administer cocaine. These results demonstrate direct and indirect regulation of the phosphorylation state of a Cdk5/ERK1/2 site on TH and suggest a role for these pathways in the neuroadaptive changes associated with chronic cocaine exposure.

Tyrosine hydroxylase (TyrH) catalyzes the conversion of tyrosine to dihydroxyphenylalanine (DOPA), the rate-limiting step in the biosynthesis of dopamine. Four mutations in the TyrH gene have recently been described in cases of autosomal recessive DOPA-responsive dystonia (Swaans et al., Ann Hum Genet 2000;64:25-31). All four are predicted to result in changes in single amino acid residues in the catalytic domain of the protein: T245P, T283M, R306H, and T463M. To determine the effects of these mutations on the molecular properties of the enzyme, mutant proteins containing the individual single amino acid changes have been expressed in bacteria and purified. Only the T283M mutation results in a decrease in the enzyme k(cat) value, while the T245P enzyme has a slightly higher value than the wild-type enzyme. The only case in which a K(m) value for either tyrosine or tetrahydrobiopterin is perturbed is the T245P enzyme, for which the K(m) value for tyrosine has increased about 50%. In contrast to the minor effects of the mutations on enzyme activity, the stability is decreased significantly by the mutations. The R306H and T283M enzymes are the least stable, losing activity 30- and 50-fold more rapidly than the wild-type enzyme. The apparent T(m) value for unfolding was decreased by 3.9, 8.2, and 7.2 degrees for the T245P, R306H, and T463M enzymes, while the T283M enzyme was too unstable for measurement of a T(m) value. The results establish that the physiological effects of the mutations are primarily due to the decreased stability of the mutant proteins rather than decreases in their intrinsic activities.

Short-term regulation of catecholamine biosynthesis involves reversible phosphorylation of several serine residues in the N-terminal regulatory domain of tyrosine hydroxylase. The MAP kinases ERK1/2 have been identified as responsible for phosphorylation of Ser31. As an initial step in elucidating the effects of phosphorylation of Ser31 on the structure and activity of tyrosine hydroxylase, the kinetics of phosphorylation of the rat enzyme by recombinant rat ERK2 have been characterized. Complete phosphorylation results in incorporation of 2 mol of phosphate into each subunit of tyrosine hydroxylase. The S8A and S31A enzymes only incorporate a single phosphate, while the S19A and S40A enzymes incorporate two. Phosphorylation of S8A tyrosine hydroxylase is nine times as rapid as phosphorylation of the S31A enzyme, consistent with a ninefold preference of ERK2 for Ser31 over Ser8.

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of nitroalkanes to the respective aldehydes with production of nitrite and hydrogen peroxide. The sequences of several peptides from the fungal enzyme were used to design oligonucleotides for the isolation of a portion of the NAO gene from an F. oxysporum genomic DNA preparation. This sequence was used to clone the cDNA for NAO from an F. oxysporum cDNA library. The sequence of the cloned cDNA showed that NOA is a member of the acyl-CoA dehydrogenase (ACAD) superfamily. The members of this family share with NAO a mechanism that is initiated by proton removal from carbon, suggesting a common chemical reaction for this superfamily. NAO was expressed in Escherichia coli and the recombinant enzyme was characterized. Recombinant NAO has identical kinetic parameters to enzyme isolated from F. oxysporum but is isolated with oxidized FAD rather than the nitrobutyl-FAD found in the fungal enzyme. NAO purified from E. coli or from F. oxysporum has no detectable ACAD activity on short- or medium-chain acyl CoAs, and medium-chain acyl-CoA dehydrogenase and short-chain acyl-CoA dehydrogenase are unable to catalyze oxidation of nitroalkanes.

Ser395 and Ser396 in the active site of rat tyrosine hydroxylase are conserved in all three members of the family of pterin-dependent hydroxylases, phenylalanine hydroxylase, tyrosine hydroxylase, and tryptophan hydroxylase. Ser395 is appropriately positioned to form a hydrogen bond to the imidazole nitrogen of His331, an axial ligand to the active site iron, while Ser396 is located on the wall of the active site cleft. Site-directed mutagenesis has been used to analyze the roles of these two residues in catalysis. The specific activities for formation of dihydroxyphenylalanine by the S395A, S395T, and S396A enzymes are 1.3, 26, and 69% of the wild-type values, respectively. Both the S395A and S396A enzymes bind a stoichiometric amount of iron and exhibit wild-type spectra when complexed with dopamine. The K(M) values for tyrosine, 6-methyltetrahydropterin, and tetrahydrobiopterin are unaffected by replacement of either residue with alanine. Although the V(max) value for tyrosine hydroxylation by the S395A enzyme is decreased by 2 orders of magnitude, the V(max) value for tetrahydropterin oxidation by either the S395A or the S396A enzyme is unchanged from the wild-type value. With both mutant enzymes, there is quantitative formation of 4a-hydroxypterin from 6-methyltetrahydropterin. These results establish that Ser395 is required for amino acid hydroxylation but not for cleavage of the oxygen-oxygen bond, while Ser396 is not essential. These results also establish that cleavage of the oxygen-oxygen bond occurs in a separate step from amino acid hydroxylation.

The aromatic amino acid hydroxylases tyrosine and phenylalanine hydroxylase both contain non-heme iron, utilize oxygen and tetrahydrobiopterin, and are tetramers of identical subunits. The catalytic domains of these enzymes are homologous, and recent X-ray crystallographic analyses show the active sites of the two enzymes are very similar. The hydroxyl oxygens of tyrosine 371 in tyrosine hydroxylase and of tyrosine 325 of phenylalanine hydroxylase are 5 and 4.5 A, respectively, away from the active site iron in the enzymes. To determine whether this residue has a role in the catalytic mechanism as previously suggested [Erlandsen, H., et al. (1997) Nat. Struct. Biol. 4, 995-1000], tyrosine 371 of tyrosine hydroxylase was altered to phenylalanine by site-directed mutagenesis. The Y371F protein was fully active in tyrosine hydroxylation, eliminating an essential mechanistic role for this residue. There was no change in the product distribution seen with phenylalanine or 4-methylphenylalanine as a substrate, suggesting that the reactivity of the hydroxylating intermediate was unaffected. However, the KM value for phenylalanine was decreased 10-fold in the mutant protein. These results are interpreted as an indication of greater conformational flexibility in the active site of the mutant protein.

Wild type rabbit tryptophan hydroxylase (TRH) and two truncated mutant proteins have been expressed in Escherichia coli. The wild type protein was only expressed at low levels, whereas the mutant protein lacking the 101 amino-terminal regulatory domain was predominantly found in inclusion bodies. The protein that also lacked the carboxyl-terminal 28 amino acids, TRH102-416, was expressed as 30% of total cell protein. Analytical ultracentrifugation showed that TRH102-416 was predominantly a monomer in solution. The enzyme exhibited an absolute requirement for iron (ferrous or ferric) for activity and did not turn over in the presence of cobalt or copper. With either phenylalanine or tryptophan as substrate, stoichiometric formation of the 4a-hydroxypterin was found. Steady state kinetic parameters were determined with both of these amino acids using both tetrahydrobiopterin and 6-methyltetrahydropterin.

The aromatic amino acid hydroxylases tyrosine hydroxylase (TyrH) and phenylalanine hydroxylase (PheH) have essentially identical active sites; however, PheH is nearly incapable of hydroxylating tyrosine, while TyrH can readily hydroxylate both tyrosine and phenylalanine. Previous studies have indicated that Asp425 of TyrH is important in determining the substrate specificity of that enzyme [Daubner, S. C., Melendez, J., and Fitzpatrick, P. F. (2000) Biochemistry 39, 9652-9661]. Alanine-scanning mutagenesis of amino acids 423-427, a mobile loop containing Asp425, shows that only mutagenesis of Asp425 alters the activity of the enzyme significantly. Saturation mutagenesis of Asp425 results in large (up to 10(4)) decreases in the V(max) and V(max)/K(tyr) values for tyrosine hydroxylation, but only small decreases or even increases in the V(max) and V(max)/K(phe) values for phenylalanine hydroxylation. The decrease in the tyrosine hydroxylation activity of the mutant proteins is due to an uncoupling of tetrahydropterin oxidation from amino acid hydroxylation with tyrosine as the amino acid substrate. In contrast, with the exception of the D425W mutant, the extent of coupling of tetrahydropterin oxidation and amino acid hydroxylation is unaffected or increases with phenylalanine as the amino acid substrate. The decrease in the V(max) value with tyrosine as the substrate shows a negative correlation with the hydrophobicity of the amino acid residue at position 425. The results are consistent with a critical role of Asp425 being to prevent a hydrophobic interaction that results in a restricted active site in which hydroxylation of tyrosine does not occur.

Sampson's menstrual reflux theory is commonly used to explain the origin of endometriotic lesions in women. However, their pathogenesis remains largely unknown. Validated research models need to be developed with the aim of studying mechanisms by which endometrial cells from menstrual reflux implant and grow, and investigating if potential new markers or molecules could be envisaged as diagnostic or therapeutic tools. The present review looks at the different existing murine and non-human primate models of endometriosis, and evaluates their principal uses.

Phosphorylation of Ser40 in the regulatory domain of tyrosine hydroxylase activates the enzyme by increasing the rate constant for dissociation of inhibitory catecholamines from the active site by three orders of magnitude. To probe the changes in the structure of the N-terminal domain upon phosphorylation, individual phenylalanine residues at positions 14, 34, and 74 were replaced with tryptophan in a form of the protein in which the endogenous tryptophans had all been mutated to phenylalanine (W3F TyrH). The steady-state fluorescence anisotropy of F74W W3F TyrH was unaffected by phosphorylation, but the anisotropies of both F14W and F34W W3F TyrH increased significantly upon phosphorylation. The fluorescence of the single tryptophan residue at position 74 was less readily quenched by acrylamide than those at the other two positions; fluorescence increased the rate constant for quenching of the residues at positions 14 and 34, but did not affect that for the residue at position 74. Frequency domain analyses were consistent with phosphorylation having no effect on the amplitude of the rotational motion of the indole ring at position 74, resulting in a small increase in the rotational motion of the residue at position 14, and resulting in a larger increase in the rotational motion of the residue at position 34. These results are consistent with the local environment at position 74 being unaffected by phosphorylation, that at position 34 becoming much more flexible upon phosphorylation, and that at position 14 becoming slightly more flexible upon phosphorylation. The results support a model in which phosphorylation at Ser40 at the N-terminus of the regulatory domain causes a conformational change to a more open conformation in which the N-terminus of the protein no longer inhibits dissociation of a bound catecholamine from the active site.

The flavoprotein nitroalkane oxidase (NAO) from Fusarium oxysporum catalyzes the oxidation of primary and secondary nitroalkanes to their respective aldehydes and ketones. Structurally, the enzyme is a member of the acyl-CoA dehydrogenase superfamily. To date no enzymes other than that from F. oxysporum have been annotated as NAOs. To identify additional potential NAOs, the available database was searched for enzymes in which the active site residues Asp402, Arg409, and Ser276 were conserved. Of the several fungal enzymes identified in this fashion, PODANSg2158 from Podospora anserina was selected for expression and characterization. The recombinant enzyme is a flavoprotein with activity on nitroalkanes comparable to the F. oxysporum NAO, although the substrate specificity is somewhat different. Asp399, Arg406, and Ser273 in PODANSg2158 correspond to the active site triad in F. oxysporum NAO. The k(cat)/K(M)-pH profile with nitroethane shows a pK(a) of 5.9 that is assigned to Asp399 as the active site base. Mutation of Asp399 to asparagine decreases the k(cat)/K(M) value for nitroethane over 2 orders of magnitude. The R406K and S373A mutations decrease this kinetic parameter by 64- and 3-fold, respectively. The structure of PODANSg2158 has been determined at a resolution of 2.0 A, confirming its identification as an NAO.

The role of a polypeptide loop in tyrosine hydroxylase (TyrH) whose homolog in phenylalanine hydroxylase (PheH) takes on a different conformation when substrates are bound has been studied using site-directed mutagenesis. The loop spans positions 177 to 191; alanine was introduced into those positions, introducing one alanine substitution per TyrH variant. Mutagenesis of residues in the center of the loop resulted in alterations in the KM values for substrates, the Vmax value for dihydroxyphenylalanine (DOPA) synthesis, and the coupling of tetrahydropterin oxidation to tyrosine hydroxylation. The variant with the most altered KM value for 6-methyltetrahydropterin was TyrH F184A. The variants with the most affected K(tyr) values were those with substitutions in the center of the loop, TyrH K183A, F184A, D185A, P186A and D187A. These five variants also had the most reduced Vmax values for DOPA synthesis. Alanine substitution in positions 182-186 resulted in lowered ratios of tyrosine hydroxylation to tetrahydropterin oxidation. TyrH F184Y and PheH Y138F, variants with the residue at the center of the loop substituted with the residue present at the homologous position in the other hydroxylase, were also studied. The V/K(tyr) to V/K(phe) ratios for these variants were altered significantly, but the results did not suggest that F184 of TyrH or Y138 of PheH plays a dominant role in determining amino acid substrate specificity.

The French Commissariat a l’Energie Atomique (CEA) has, since mid-2001, performed a preliminary evaluation of different methods to produce hydrogen from nuclear energy. The objective is to compare the hydrogen production costs via high temperature electrolysis or via thermochemical cycles, which are nowadays the two main routes for the long term production of hydrogen without greenhouse effect, both from the technical and economical points of view...