The biosynthesis of norleucine and norvaline by E. coli as byproducts of the branched chain amino acid biosynthesis occurs via the promiscuous enzymes of the (iso)-leucine pathway (Bogosian et al. ; Sycheva et al. ; Soini et al. ). Due to their broad range of keto acid substrate acceptance, the leuABCD-operon enzymes are proposed to facilitate a direct carbon chain elongation from the central intermediate pyruvate to the branched chain amino acid precursor 2-ketobutyrate which is synthesized from threonine under standard conditions. The following extension reactions of 2-ketobutyrate are catalyzed by the leucine biosynthesis enzymes isopropylmalate synthase (IPMS, leuA gene), isopropylmalate isomerase (IPMI, leuCD genes) and isopropylmalate dehydrogenase (IPMD, leuB gene). Enzymatic specificity of IPMS has not been investigated in E. coli yet but is known for a variety of substrates of the highly conserved homolog protein in Salmonella typhimurium (Kohlhaw et al. ) which can utilize, e.g., 2-ketobutyrate and pyruvate, for condensation with acetyl-CoA. Subsequent conversion of the intermediate compounds by IPMI and IPMD via this keto acid elongation pathway forms 2-ketovalerate and 2-ketocaproate. The last step in norleucine and norvaline biosynthesis consists of the transamination of 2-ketocaproate and 2-ketovalerate by aminotransferases IlvE, TyrA and AvtA.
Norvaline belongs to the group of non-usual amino acid analogs that may be formed under certain circumstances as byproducts of the branched-chain amino acid biosynthetic pathway in E. coli and other Gram-negative microorganisms. These amino acids can accumulate and are secreted under certain cultivation conditions.
leading to norvaline and 1-butanol biosynthesis.
AB - Objective. - Arginase is a nitric oxide synthase-alternative pathway for L-arginine breakdown leading to biosynthesis of urea and L-ornithine. Arginase pathway is inducible by inflammatory molecules - such as cytokines and bacterial endotoxin - in macrophages and smooth muscle cells. The presence of an arginase pathway in human endothelial cells and its possible modulation by inflammation are unknown. Methods. - We have: (i) characterised arginase pathway in terms of activity, isoform type and gene expression in a primary human umbilical vein endothelial cells (HUVEC) line; (ii) evaluated arginase functional role in cell proliferation with the aid of L-norvaline, an arginase inhibitor and (iii) determined the effects of tumour necrosis factor-α and endotoxin on arginase pathway. Results. - HUVEC showed a baseline arginase activity and expression of both arginase isoforms (arginase I and II (A-I and A-II, respectively)) which resulted in L-norvaline-inhibitable cellular polyamine synthesis. The baseline arginase activity is important for HUVEC proliferation as cell cycle analysis and nuclear factor Ki-67 immunostaining revealed. Following incubation with inflammatory molecules, arginase activity increased but HUVEC cell cycling decreased. Conclusions. - A-I and A-II are constitutively expressed in HUVEC where they take part to the regulation of cell cycling. Although arginase activity is positively modulated by inflammatory molecules, it is insufficient to counteract the overall cell cycling inhibiting effects of inflammation.