SummaryAn 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 2-ketobutyrate 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.

Background-We recently reported that arterial superoxide (O 2Ϫ ) 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 Ϫ -induced vascular dysfunction. However, the interaction between O 2 Ϫ 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 Ϫ leads to impaired endothelium-dependent relaxation and that gene transfer of human guanosine 5Ј-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 Ϫ 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 Ϫ 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 Ϫ and improved endothelium-dependent relaxation and basal NO release in DOCA-salt rats. Conclusions-These

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.