Chloroplast ferredoxin-NADP⁺reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP⁺ photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2′-phosphoryl group of NADP⁺, replacement of the C-terminal tyrosine (Tyr³⁰⁸ in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, thek _cat value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD⁺ complexes revealed that the nicotinamide ring of NAD⁺ binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with thek _cat values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2′-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP⁺ reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.

D-Serine acts as a co-agonist of N-methyl-d-aspartate receptors (NMDAR) which appear overactivated in AD, while d-aspartate is a modulatory molecule acting on NMDAR as a second agonist. The aim of this work is to clarify whether the levels of these d-amino acids in serum are deregulated in AD, with the final goal to identify novel and precocious biomarkers in AD. Serum levels of l- and d-enantiomers of serine and aspartate were determined by HPLC using a pre-column derivatization procedure and a selective enzymatic degradation. Experimental data obtained from age-matched healthy subjects (HS) and AD patients were statistically evaluated by considering age, gender, and disease progression, and compared. Minor changes were apparent in the serum l- and d-aspartate levels in AD patients compared to HS. A positive correlation for the d-serine level and age was apparent in the AD cohort. Notably, the serum d-serine level and the d-/total serine ratio significantly increased with the progression of the disease. Gender seems to have a minor effect on the levels of all analytes tested. This work proposes that the serum d-serine level and d-/total serine ratio values as novel and valuable biomarkers for the progression of AD: the latter parameter allows to discriminate CDR 2 and CDR 1 patients from healthy (CDR 0) individuals.

D-amino acid oxidase (DAAO) is a well-known flavoenzyme that catalyzes the oxidative FAD-dependent deamination of D-amino acids. As a result of the absolute stereoselectivity and broad substrate specificity, microbial DAAOs have been employed as industrial biocatalysts in the production of semi-synthetic cephalosporins and enantiomerically pure amino acids. Moreover, in mammals, DAAO is present in specific brain areas and degrades D-serine, an endogenous coagonist of the N-methyl-D-aspartate receptors (NMDARs). Dysregulation of D-serine metabolism due to an altered DAAO functionality is related to pathological NMDARs dysfunctions such as in amyotrophic lateral sclerosis and schizophrenia. In this protocol paper, we describe a variety of direct assays based on the determination of molecular oxygen consumption, reduction of alternative electron acceptors, or α-keto acid production, of coupled assays to detect the hydrogen peroxide or the ammonium production, and an indirect assay of the α-keto acid production based on a chemical derivatization. These analytical assays allow the determination of DAAO activity both on recombinant enzyme preparations, in cells, and in tissue samples.