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The anti-inflammatory and immunomodulatory abilities of oral selective phosphodiesterase 4 (PDE4) inhibitors enabled the approval of roflumilast and apremilast for use in chronic obstructive pulmonary disease and psoriasis/psoriatic arthritis, respectively. However, the antifibrotic potential of PDE4 inhibitors has not yet been explored clinically. BI 1015550 is a novel PDE4 inhibitor showing a preferential enzymatic inhibition of PDE4B. In vitro, BI 1015550 inhibits lipopolysaccharide (LPS)-induced tumor necrosis factor-α (TNF-α) and phytohemagglutinin-induced interleukin-2 synthesis in human peripheral blood mononuclear cells, as well as LPS-induced TNF-α synthesis in human and rat whole blood. In vivo, oral BI 1015550 shows potent anti-inflammatory activity in mice by inhibiting LPS-induced TNF-α synthesis ex vivo and in Suncus murinus by inhibiting neutrophil influx into bronchoalveolar lavage fluid stimulated by nebulized LPS. In Suncus murinus, PDE4 inhibitors induce emesis, a well-known gastrointestinal side effect limiting the use of PDE4 inhibitors in humans, and the therapeutic ratio of BI 1015550 appeared to be substantially improved compared with roflumilast. Oral BI 1015550 was also tested in two well-known mouse models of lung fibrosis (induced by either bleomycin or silica) under therapeutic conditions, and appeared to be effective by modulating various model-specific parameters. To better understand the antifibrotic potential of BI 1015550 in vivo, its direct effect on human fibroblasts from patients with idiopathic pulmonary fibrosis (IPF) was investigated in vitro. BI 1015550 inhibited transforming growth factor-β-stimulated myofibroblast transformation and the mRNA expression of various extracellular matrix proteins, as well as basic fibroblast growth factor plus interleukin-1β-induced cell proliferation. Nintedanib overall was unremarkable in these assays, but interestingly, the inhibition of proliferation was synergistic when it was combined with BI 1015550, leading to a roughly 10-fold shift of the concentration–response curve to the left. In summary, the unique preferential inhibition of PDE4B by BI 1015550 and its anticipated improved tolerability in humans, plus its anti-inflammatory and antifibrotic potential, suggest BI 1015550 to be a promising oral clinical candidate for the treatment of IPF and other fibro-proliferative diseases.

Background Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease for which diagnosis and management remain challenging. Defining the circulating proteome in IPF may identify targets for biomarker development. We sought to quantify the circulating proteome in IPF, determine differential protein expression between subjects with IPF and controls, and examine relationships between protein expression and markers of disease severity. Methods This study involved 300 patients with IPF from the IPF-PRO Registry and 100 participants without known lung disease. Plasma collected at enrolment was analysed using aptamer-based proteomics (1305 proteins). Linear regression was used to determine differential protein expression between participants with IPF and controls and associations between protein expression and disease severity measures (percent predicted values for forced vital capacity [FVC] and diffusion capacity of the lung for carbon monoxide [DLco]; composite physiologic index [CPI]). Multivariable models were fit to select proteins that best distinguished IPF from controls. Results Five hundred fifty one proteins had significantly different levels between IPF and controls, of which 47 showed a |log2(fold-change)| > 0.585 (i.e. > 1.5-fold difference). Among the proteins with the greatest difference in levels in patients with IPF versus controls were the glycoproteins thrombospondin 1 and von Willebrand factor and immune-related proteins C-C motif chemokine ligand 17 and bactericidal permeability-increasing protein. Multivariable classification modelling identified nine proteins that, when considered together, distinguished IPF versus control status with high accuracy (area under receiver operating curve = 0.99). Among participants with IPF, 14 proteins were significantly associated with FVC % predicted, 23 with DLco % predicted, 14 with CPI. Four proteins (roundabout homolog-2, spondin-1, polymeric immunoglobulin receptor, intercellular adhesion molecule 5) demonstrated the expected relationship across all three disease severity measures. When considered in pathways analyses, proteins associated with the presence or severity of IPF were enriched in pathways involved in platelet and haemostatic responses, vascular or platelet derived growth factor signalling, immune activation, and extracellular matrix organisation. Conclusions Patients with IPF have a distinct circulating proteome and can be distinguished using a nine-protein profile. Several proteins strongly associate with disease severity. The proteins identified may represent biomarker candidates and implicate pathways for further investigation. Trial registration ClinicalTrials.gov (NCT01915511).

Idiopathic pulmonary fibrosis (IPF) is a progressive and ultimately fatal disease with a variable clinical course. Biomarkers that predict patient outcomes are needed. We leveraged data from 300 patients in the multicenter IPF-PRO Registry to determine associations between circulating proteins and the composite outcome of respiratory death or lung transplant. Plasma collected at enrollment was analyzed using aptamer-based proteomics (1305 proteins). Over a median follow-up of 30.4 months, there were 76 respiratory deaths and 26 lung transplants. In unadjusted univariable analyses, 61 proteins were significantly associated with the outcome (hazard ratio > 2 or p ≤ 0.05). In multivariable analyses, a set of 4 clinical measures and 47 unique proteins predicted the probability of respiratory death or lung transplant with an optimism-corrected C-index of 0.76. Our results suggest that select circulating proteins strongly associate with the risk of mortality in patients with IPF and confer information independent of clinical measures.

Background: IPF is characterized by parenchymal accumulation of collagen-rich extracellular matrix (ECM). Circulating collagen neoepitopes reflect ECM turnover. Aim: Determine whether serum concentrations of neoepitopes are associated with disease severity and clinically relevant outcomes in patients with IPF. Methods: Serum concentrations of 7 neoepitopes (BGM, C3A, C3M, C5M, C6M, CRPM, VICM) were determined in 300 patients enrolled in the IPF-PRO Registry using ELISA-based assays. Analytes were log2 transformed and tested for associations with composite physiologic index (CPI) and DLco and FVC % predicted at enrollment using generalized linear models, and with outcomes of death, respiratory death, death or transplant, respiratory death or transplant, and respiratory hospitalization using Cox proportional hazards regression. Results: C3M, C6M, CRPM and VICM were associated with CPI and DLco % predicted (p Conclusion: Concentrations of select serum neoepitopes are associated with disease severity and risk of respiratory hospitalization in patients with IPF.

BackgroundMatrix metalloproteinases (MMPs) and tissue inhibitors of MMPs (TIMPs) play important roles in the turnover of extracellular matrix and in the pathogenesis of idiopathic pulmonary fibrosis (IPF). This study aimed to determine the utility of circulating MMPs and TIMPs in distinguishing patients with IPF from controls and to explore associations between MMPs/TIMPs and measures of disease severity in patients with IPF.MethodsThe IPF cohort (n = 300) came from the IPF-PRO Registry, an observational multicenter registry of patients with IPF that was diagnosed or confirmed at the enrolling center in the past 6 months. Controls (n = 100) without known lung disease came from a population-based registry. Generalized linear models were used to compare circulating concentrations of MMPs 1, 2, 3, 7, 8, 9, 12, and 13 and TIMPs 1, 2, and 4 between patients with IPF and controls, and to investigate associations between circulating levels of these proteins and measures of IPF severity. Multivariable models were fit to identify the MMP/TIMPs that best distinguished patients with IPF from controls.ResultsAll the MMP/TIMPs analyzed were present at significantly higher levels in patients with IPF compared with controls except for TIMP2. Multivariable analyses selected MMP8, MMP9 and TIMP1 as top candidates for distinguishing patients with IPF from controls. Higher concentrations of MMP7, MMP12, MMP13 and TIMP4 were significantly associated with lower diffusion capacity of the lung for carbon monoxide (DLCO) % predicted and higher composite physiologic index (worse disease). MMP9 was associated with the composite physiologic index. No MMP/TIMPs were associated with forced vital capacity % predicted.ConclusionsCirculating MMPs and TIMPs were broadly elevated among patients with IPF. Select MMP/TIMPs strongly associated with measures of disease severity. Our results identify potential MMP/TIMP targets for further development as disease-related biomarkers.

Introduction: The circulating metabolome, reflecting underlying cellular processes and biology, has not been characterised in patients with IPF. Aim: To determine differential metabolite expression in patients with IPF vs controls. Methods: Patients with IPF that was diagnosed or confirmed at the enrolling center in the past 6 months (n=300) were drawn from the multicenter IPF-PRO Registry. Controls (n=100) without known lung disease were of similar age and sex distribution. Metabolites (408 in 11 major groups) were quantified using the Absolute IDQ p400HR kit. Of 398 metabolites detected, 303 were carried forward to analysis. Data were log2 transformed and linear regression used to compare metabolite expression in IPF vs control groups. Results: Patients with IPF were predominantly male (74.3%), former smokers (67.3%), with a median age of 70. Median (Q1, Q3) % predicted FVC and DLCO were 69.7 (61.0, 80.2) and 40.6 (31.7, 49.4), respectively. 229 metabolites were differentially expressed in the IPF vs. control group based on p-values corrected to control the false discovery rate at 5%, with 79 having a |log2FC|≥0.585 and 40 having a |log2FC|≥1. The top 40 metabolites that were differentially expressed came from 5 major groups (Figure). Conclusions: IPF associates with a distinct circulating metabolome. Further analysis of the IPF metabolome might aid the development of diagnostic biomarkers or the definition of novel disease endotypes.

Nitric oxide (NO) is a key physiological mediator and disturbed regulation of NO release is associated with the pathophysiology of almost all inflammatory diseases. A multitude of inhibitors of NOSs (nitric oxide synthases) have been developed, initially with low or even no selectivity against the constitutively expressed NOS isoforms, eNOS (endothelial NOS) and nNOS (neuronal NOS). In the meanwhile these efforts yielded potent and highly selective iNOS (inducible NOS) inhibitors. Moreover, iNOS inhibitors have been shown to exert beneficial anti-inflammatory effects in a wide variety of acute and chronic animal models of inflammation. In the present mini-review, we summarize some of our current knowledge of inhibitors of the iNOS isoenzyme, their biochemical properties and efficacy in animal models of pulmonary diseases and in human disease itself. Moreover, the potential benefit of iNOS inhibition in animal models of COPD (chronic obstructive pulmonary disease), such as cigarette smoke-induced pulmonary inflammation, has not been explicitly studied so far. In this context, we demonstrated recently that both a semi-selective iNOS inhibitor {L-NIL [N6-(1-iminoethyl)-L-lysine hydrochloride]} and highly selective iNOS inhibitors (GW274150 and BYK402750) potently diminished inflammation in a cigarette smoke mouse model mimicking certain aspects of human COPD. Therefore, despite the disappointing results from recent asthma trials, iNOS inhibition could still be of therapeutic utility in COPD, a concept which needs to be challenged and validated in human disease.

Imidazopyridine derivates were recently shown to be a novel class of selective and arginine-competitive inhibitors of inducible nitric-oxide synthase (iNOS), and 2-[2-(4-methoxypyridin-2-yl)-ethyl]-3H-imidazo[4,5-b]pyridine (BYK191023) was found to have very high selectivity in enzymatic and cellular models ( Mol Pharmacol 69: 328-337, 2006 ). Here, we show that BYK191023 irreversibly inactivates murine iNOS in an NADPH- and time-dependent manner, whereas it acts only as a reversible l-arginine-competitive inhibitor in the absence of NADPH or during anaerobic preincubation. Time-dependent irreversible inhibition by BYK191023 could also be demonstrated in intact cells using the RAW macrophage or iNOS-overexpressing human embryonic kidney 293 cell lines. The mechanism of BYK191023 inhibition in the presence of NADPH was studied using spectral, kinetic, chromatographic, and radioligand binding methods. BYK191023-bound iNOS was spectrally indistinguishable from l-arginine-bound iNOS, pointing to an interaction of BYK191023 with the catalytic center of the enzyme. [(3)H]BYK191023 was recovered quantitatively from irreversibly inactivated iNOS, and no inhibitor metabolite was detected by high-performance liquid chromatography (HPLC). Size exclusion chromatography revealed only about 20% iNOS dissociation into monomers. Furthermore, HPLC and spectrophotometric analysis showed that the irreversible inhibition was associated with loss of heme from iNOS and a reduced ability to form the distinctive ferrous heme-CO complex (cytochrome P450). Thus, enzyme inactivation is mainly caused by heme loss, and it occurs in the inhibitor-bound enzyme in the presence of electron flux from NADPH.

Excessive release of nitric oxide from inducible nitric-oxide synthase (iNOS) has been postulated to contribute to pathology in a number of inflammatory diseases. We recently identified imidazopyridine derivatives as a novel class of potent nitricoxide synthase inhibitors with high selectivity for the inducible isoform. In the present study, we tested the in vivo potency of BYK191023 [2-[2-(4-methoxy-pyridin-2-yl)-ethyl]-3H-imidazo-[4,5-b]pyridine], a selected member of this inhibitor class, in three different rat models of lipopolysaccharide-induced systemic inflammation. Delayed administration of BYK191023 dose-dependently suppressed the lipopolysaccharide-induced increase in plasma nitrate/nitrite (NO(x)) levels with an ED(50) of 14.9 micromol/kg/h. In a model of systemic hypotension following high-dose lipopolysaccharide challenge, curative administration of BYK191023 at a dose that inhibited 83% of the NO(x) increase completely prevented the gradual decrease in mean arterial blood pressure observed in vehicle-treated control animals. The vasopressor effect was specific for endotoxemic animals since BYK191023 did not affect blood pressure in saline-challenged controls. In addition, in a model of lipopolysaccharide-induced vascular hyporesponsiveness, BYK191023 infusion partially restored normal blood pressure responses to norepinephrine and sodium nitroprusside via an l-arginine competitive mechanism. Taken together, BYK191023 is a member of a novel class of highly isoform-selective iNOS inhibitors with promising in vivo activity suitable for mechanistic studies on the role of selective iNOS inhibition as well as clinical development.

Glial cell line-derived neurotrophic factor (GDNF) protects dopaminergic neurones against toxic and physical damage. In addition, GDNF promotes differentiation and structural integrity of dopaminergic neurones. Here we show that GDNF can support the function of primary dopaminergic neurones by triggering activation of GTP-cyclohydrolase I (GTPCH I), a key enzyme in catecholamine biosynthesis. GDNF stimulation of primary dopaminergic neurones expressing both tyrosine 3-monooxygenase and GTPCH I resulted in a dose-dependent doubling of GTPCH I activity, and a concomitant increase in tetrahydrobiopterin levels whereas tyrosine 3-monooxygenase activity was not altered. Actinomycin D, as an inhibitor of de novo biosynthesis, abolished any GDNF-mediated up-regulation of GTPCH I activity. However, GTPCH I mRNA levels in primary dopaminergic neurones were not altered by GDNF treatment, suggesting that the mode of action for that up-regulation is not directly connected to the regulation of GTPCH I transcription. We conclude that

Mast cells (MC) are biologically potent, ubiquitously distributed immune cells with fundamental roles in host integrity and disease. MC diversity and function is regulated by exogenous nitric oxide; however, the production and function of endogenously produced NO in MC is enigmatic. We used rat peritoneal MC (PMC) as an in vivo model to examine intracellular NO production. Live cell confocal analysis of PMC using the NO-sensitive probe diaminofluorescein showed distinct patterns of intracellular NO formation with either antigen (Ag)/IgE (short term) or interferon-gamma (IFN-gamma) (long term). Ag/IgE-induced NO production is preceded by increased intracellular Ca2+, implying constitutive nitric-oxide synthase (NOS) activity. NO formation inhibits MC degranulation. NOS has obligate requirements for tetrahydrobiopterin (BH4), a product of GTP-cyclohydrolase I (CHI), IFN-gamma-stimulated PMC increased CHI mRNA, protein, and enzymatic activity, while decreasing CHI feedback regulatory protein mRNA, causing sustained NO production. Treatment with the CHI inhibitor, 2,4-diamino-6-hydroxypyrimidine, inhibited NO in both IFN-gamma and Ag/IgE systems, increasing MC degranulation. Reconstitution with the exogenous BH4 substrate, sepiapterin, restored NO formation and inhibited exocytosis. Thus, Ag/IgE and IFN-gamma induced intracellular NO plays a key role in MC mediator release, and alterations in NOS activity via BH4 availability may be critical to the heterogeneous responsiveness of MC.

Background— We recently reported that arterial superoxide (O 2 − ) is augmented by increased endothelin-1 (ET-1) in deoxycorticosterone acetate (DOCA)-salt hypertension, a model of low renin hypertension. Tetrahydrobiopterin (BH 4 ), a potent reducing molecule with antioxidant properties and an essential cofactor for endothelial nitric oxide synthase, protects against O 2 − –induced vascular dysfunction. However, the interaction between O 2 − and BH 4 on endothelial function and the underlying mechanisms are unknown. Methods and Results— The present study tested the hypothesis that BH 4 deficiency due to ET-1–induced O 2 − leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5′-triphosphate (GTP) cyclohydrolase I (GTPCH I), the first and rate-limiting enzyme for BH 4 biosynthesis, reverses such deficiency and endothelial dysfunction in carotid arteries of DOCA-salt rats. There were significantly increased arterial O 2 − levels and decreased GTPCH I activity and BH 4 levels in DOCA-salt compared with sham rats. Treatment of arteries of DOCA-salt rats with the selective ET A receptor antagonist ABT-627, NADPH oxidase inhibitor apocynin, or superoxide dismutase (SOD) mimetic tempol abolished O 2 − and restored BH 4 levels. Basal arterial NO release and endothelium-dependent relaxations were impaired in DOCA-salt rats, conditions that were improved by apocynin or tempol treatment. Gene transfer of GTPCH I restored arterial GTPCH I activity and BH 4 levels, resulting in reduced O 2 − and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions— These results indicate that a BH 4 deficiency resulting from ET-1–induced O 2 − via an ET A /NADPH oxidase pathway leads to endothelial dysfunction, and gene transfer of GTPCH I reverses the BH 4 deficiency and endothelial dysfunction by reducing O 2 − in low renin mineralocorticoid hypertension.

Complex-I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkinson’s disease and the toxicity of MPTP and its metabolite MPP + . Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones and protects against the toxic consequences of glutathione depletion. Here we studied the effects of manipulating tetrahydrobiopterin levels on MPP + toxicity in organotypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 lM MPP + for 2 days, followed by 8 days ‘recovery’ in control medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylaseimmunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4-diamino-6-hydroxypyrimidine had no significant synergistic effect on MPP + toxicity. In contrast, the tetrahydrobiopterin precursor L-sepiapterin attenuated the MPP + -induced dopamine depletion and loss of tyrosine hydroxylase-positive cells in a dose-dependent manner with 40 l ML -sepiapterin providing maximal protection. Accordingly, increasing intracellular tetrahydrobiopterin levels may protect

GTP cyclohydrolase I controls the de novo pathway for the synthesis of tetrahydrobiopterin, which is the essential cofactor for tryptophan 5-monooxygenase and thus, for serotonin production. In mouse bone marrow-derived mast cells, the kit ligand selectively up-regulates GTP cyclohydrolase I activity (Ziegler, I., Hultner, L., Egger, D., Kempkes, B., Mailhammer, R., Gillis, S., and Rodl, W. (1993) J. Biol. Chem. 268, 12544–12551). Immunoblot analysis now confirms that this long term enhancement is caused by increased expression of the enzyme. Furthermore we show that GTP cyclohydrolase I is subject to modification at the post-translational level. In vivolabeling with [32P]orthophosphate demonstrates that in primary mast cells and in transfected RBL-2H3 cells overexpressing GTP cyclohydrolase I, the enzyme exists in a phosphorylated form. Antigen binding to the high affinity receptor for IgE triggers an additional and transient phosphorylation of GTP cyclohydrolase I with a concomitant rise in its activity, and in consequence, cellular tetrahydrobiopterin levels increase. These events culminate 8 min after stimulation and can be mimicked by phorbol ester. The hyperphosphorylation is greatly reduced by the protein kinase C inhibitor Ro-31-8220. In vitro phosphorylation studies indicate that GTP cyclohydrolase I is a substrate for both casein kinase II and protein kinase C.

Mutants of Escherichia coli unable to synthesize a functional pyruvate formate-lyase (PFL) are severely impaired in their capacity to grow by glucose fermentation. In a functional complementation assay designed to isolate the pfl gene from Clostridium butyricum , we fortuitously identified a gene that did not encode a PFL but nonetheless was able to complement the phenotypic defects caused by an E. coli pfl mutation. The clostridial gene encoded a basic 14.5-kDa protein (TcbC) which, based on amino acid similarity and analysis of immediately adjacent DNA sequences, was part of a transposase exhibiting extensive similarity to the product of the site-specific transposon Tn 554 from Staphylococcus aureus . Our studies revealed that the clostridial TcbC protein activated the transcription of the E. coli tdcABCDEFG operon, which encodes an anaerobic l -threonine-degradative pathway. Normally, anaerobic synthesis of the pathway is optimal when E. coli grows in the absence of catabolite-repressing sugars and in the presence of l -threonine. Although anaerobic control of pathway synthesis was maintained, TcbC alleviated glucose repression. One of the products encoded by the tdc operon, TdcE, has recently been shown to be a 2-keto acid formate-lyase (C. Heßlinger, S. A. Fairhurst, and G. Sawers, Mol. Microbiol. 27:477–492, 1998) that can accept pyruvate as an enzyme substrate. Here we show that TdcE is directly responsible for the restoration of fermentative growth to pfl mutants.

Unlike other Escherichia coli K-12 strains, W3110 contains multiple copies of the insertion sequence IS5. Some of these IS5 elements have been involved in tandem duplication of a portion of the chromosome which includes, amonst others, the tdcABC-DEFG operon genes. The nucleotide sequence and insertion site of one of these elements, IS5P, was determined. It was shown that IS5P has inserted within the coding sequence of the tdcA gene and is flanked, not by the remaining portion of the tdcA gene, but by the extreme 3' end of the tdcD gene. In other E. coli K-12 strains the tdcD gene and three other genes, tdcE, tdcF and tdcG, all form part of the tdc operon. Our results demonstrate that during the duplication event the tdcABCgenes have been amplified and separated from the remaining genes tdcE, tdcF and tdcG of the operon, which are each present in single copy.

Inducible arginine oxidation and subsequent NO production by correspondent synthase (iNOS) are important cellular answers to proinflammatory signals. Prolonged NO production has been proved in higher organisms to cause stroke or septic shock. Several classes of potent NOS inhibitors have been reported, most of them targeting the arginine binding site of the oxygenase domain. Here we disclose the SAR and the rational design of potent and selective iNOS inhibitors which may be useful as anti-inflammatory drugs.

Tetrahydrobiopterin (BH4) is an essential cofactor for all three isoforms of NO synthases and serves as electron donor for phenylalanine hydroxylase, tyrosine hydroxylase and tryptophan hydroxylase (1). The enzymes of its de novo biosynthesis pathway, GTP cyclohydrolase I (GTPCH), 6-pyruvoyltetrahydropterin synthase (PTPS) and sepiapterin reductase (SR) have been shown to be phosphorylated by various kinases, including PKC (2, 3, 4). Furthermore, the phosphorylation of GTPCH which catalyses the first and rate limiting step in BH4 synthesis (1) is modulated by potassium ions in PC 12 cells (5), by activation of angiotensin II and PDGF receptors in mesangial cells (6) and by stimulation of the high affinity receptor for IgE, FceRI in mast cells (2).

Estrogens are well known for their protective effects on the cardiovascular system (1,2). Activation of estrogen receptors which have been identified in endothelial and vascular smooth muscle cells leads to changes in gene expression and alterations in cellular function (3,4). Moreover, estrogens potentiate vasodilation mediated by NO production from endothelial NO synthase both after long-term and short-term stimulation (2,4,5). Tetrahydrobiopterin (BH4) is an essential cofactor for all three isoforms of NO synthases and regulates, at least in part, NO production (6,7). Furthermore, there is upcoming evidence that BH4 plays a pivotal role in switching eNOS from a superoxide to a nitric oxide producing state in vivo and in vitro (8,9,10). We hypothesised that estrogen-mediated enhancement of NO generation via eNOS would also be accompanied by activation of BH4 production and that the effect might again be mediated by an increase in the activity of GTP cyclohydrolase I (GTPCH) which catalyses the first and rate limiting step in BH4 synthesis (7).

During the development of vertebrates, the multipotent neural crest cells segregate into neurons and glia of the peripheral nervous system and into pigment cells. The unrestricted precursor cells separate from the dorsal neural tube and undergo fate restriction after their lateral and rostral to caudal migration (Reviewed by Ref. 1). The zebrafish has become a widely used model organism for analyzing the specification of the neural crest-derived cells. Numerous genetic and developmental studies address neuronal defects, changes in pigment cell pattern, or both, in this system.

Summary An immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, crossreacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1 H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.