L-Citrulline production

FIGURE 22.4 Mechanism of nitric oxide and L-citrulline production by NO synthases through the intermediate formation of iV-hydroxyl-L-arginine. 

Over the past 5 years, diverse lines of evidence have converged to suggest the presence of a mammalian-type NOS in plants. Evidence for the presence of NOS activity was first described in the leguminous plant Lupinus Albus [187]. A L-arginine-dependent L-citrulline production, possibly induced by Rhizobium lipopolysaccharides, was measured in roots and nodules using the [14C]arginine/citrulline procedure. L-citrulline production was inhibited by the mammalian NOS antagonist N(G)-... 

Kwon, N.S., Nathan, C.F., Gilker, C., Griffith, O.W., Matthews, D.E., and Stuehr, D.J. (1990). L-citrulline production from L-arginine by macrophage nitric oxide synthase. The ureido oxygen derives from dioxygen. J. Biol. Chem. 265, 13442-13445. 

C. Nitric oxide is an important compound that acts as a biological messenger in many physiological responses. L-Citrulline is a product of the oxidation of L-argenine in the formation of nitric oxide. Bradykinin is formed from a precursor kininogen. 

There are cytosolic and membrane-bound isoforms of NO synthase. Certain soluble and particulate isoforms are constitutive and other soluble isoforms are inducible. The constitutive enzyme is, by definition, present in the catalytically active form and needs only to be stimulated by an appropriate chemical species, following which there is immediate formation of NO plus L-citrulline. This form of NO synthase requires calcium, and for the most part calmodulin, for stimulation of enzymatic activity. It is likely that an increase in intracellular free calcium in the presence of calmodulin is the signal for stimulation of NO synthase, and therefore, the production of NO. This view is consistent with the general understanding that, in vascular tissue, all endothelium-dependent vaso-... 

Schematic illustration of the interrelationships between glutamate and NO in synaptic function in the cetebellum. The presynaptic nerve terminal synthesizes, stores, and releases glutamate (G) as the neurotransmitter by exocytosis as illustrated. The glutamate diffu.ses across the synaptic cleft and interacts with postsynaptic NMDA recepti>rs ( ) that are coupled to calcium (Ca ) channels. Ca influx occurs and the free intracellular Ca complexes with calmtxlulin and activates NO synthase. NADPH is also required hir conversion, and the products of the reaction are NO plus L-citrulline. NO diffuses out of the piistsynaptic cell to interact with nearby target cells, one of which is the presynaptic neuron that released the glutamate in the first place. NO stimulates cytosolic guanylate cyclase and cyclic GMP (cGMP) formation presynaptically, hut the consequence of this pre.synaptic modification is unknown.

Nitric oxide synthase is an unusual enzyme in that substrates with even numbers of electrons (L-arginine, O2, NADPH) are transformed into an odd-electron product (NO) in addition to even-electron products (L-citrulline, NADP,... 

As reviewed in Chapter 3 of this book, nitric oxide is the product of the enzymatic oxidation of one of the guanidino nitrogen groups of L-arginine to the free radical nitric oxide and L-citrulline (Marietta etal., 1988). At present there appears to be at least three distinct isoforms of nitric oxide synthase that are primarily differentiated at the level of gene expression (Schmidt, 1992 Nathan,... 

NO synthases are oxygenases that carry out a two-step oxidation of L-arginine to L-citrulline with production of NO. In the first step, a normal monooxygenase reaction, i -N -hydroxyarginine is formed (Eq. 18-65, step a). In the second step (Eq. 18-65, step b) NO is formed in a three-electron oxidation. In this equation the symbols and + indicate positions of incorporation of labeled 02 atoms in the intermediate and final products. 

NOS is an important signaling enzyme that synthesizes L-citrulline and nitric oxide (NO) from L-arginine and O2 via two turnovers in a P450-like catalytic cycle (Scheme 2). NOS participates in physiological processes such as neurotransmission, vasodilation, and immune response [54,55]. Improper regulation of NO production can lead to diseases such as septic shock, heart disease, arthritis, and diabetes. 

In vivo NO" is generated from the essential amino acid L-arginine by a family of enzymes called nitric oxide synthases (NOS) [1], Three isoforms have been described and cloned - endothelial NOS (eNOS), brain or neuronal NOS (nNOS), and inducible macrophage-type NOS (iNOS). All isoforms of NOS make the same products, NO and L-citrulline, by incorporating an O-atom of dioxygen into L-arginine. Only the amount of NO, the conditions under which it is made, and the location where it is synthesized differ among the three isoforms. 

Figure 16 Overiay of human DDAH-1 structures bound to the product L-citrulline (tan) and A/ -(2-methoxyethyl)-L-arginine (22) (biue). Protein residues are shown as stick models and ligands as ball-and-stick models. Rearrangements of active-site Leu, His, and Arg residues accommodate the inhibitor, and hold the guanidinium in a nonproductive orientation. The figure was constructed using coordinates from PDB accession codes 2JAJ and 2JAI, respectively.

The NOSs are best characterized as cytochrome P-450-like hemeprot-eins (Bredt et al., 1991 Stuehr and Ikeda, 1992 White and Marietta, 1992). They can be broadly divided into a reductase domain at the COOH terminus and an oxidative domain at the NH2 terminus (Fig. 1). The primary amino acid sequences of NOS isoforms share common consensus sequence binding sites for calmodulin, NADPH, flavin-adenine dinucleotide (FAD), and flavin mononucleotide (FMN) (Bredt et al., 1991 Marsden et al., 1992 Sessa et al., 1992 Xie et al., 1992 Lyons et al., 1992 Lowenstein et al., 1992). Each enzyme functions as a dimeric protein in catalyzing the NADPH-dependent five-electron oxidation of L-arginine to generate NO. L-Citrulline is a by-product (Back et al., 1993 Abu and Stuehr, 1993). Electrons are supplied by NADPH, transferred along the flavins and calmodulin, and presented to the catalytic heme center (Stuehr and Ikeda, 1992 White and Marietta, 1992). The NOS apoenzyme requires tetrahydrobiopterin, prosthetic heme (ferroprotoporphyrin IX), calmodulin, FMN, and FAD as cofactors for monomer assembly and/or catalytic activity (Abu and Stuehr, 1993 Mayer and Werner, 1994 Kwon etal., 1989 Stuehr and Ikeda, 1992 Stuehr and Griffith, 1992 White and Marietta, 1992 McMillan etal., 1992 Klatt... 

The cytotoxicity of activated macrophages depends of the presence of L-arginine biotransformed to L-citrulline and NO2 (Hibbs etal. 1987). N -monomethyl-L-arginine prevented the synthesis of both these products as well as the expression of cytotoxicity. 

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