Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions.

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation

The genetic basis of hemolytic anemia caused by pyrimidine 5' nucleotidase deficiency.

Pyrimidine 5' nucleotidase (P5'N-1) deficiency is an autosomal recessive condition causing hemolytic anemia characterized by marked basophilic stippling and the accumulation of high concentrations of pyrimidine nucleotides within the erythrocyte. It is implicated in the anemia of lead poisoning and is possibly associated with learning difficulties. Recently, a protein with P5'N-1 activity was analyzed and a provisional complementary DNA (cDNA) sequence published. This sequence was used to study 3 families with P5'N-1 deficiency. This approach generated a genomic DNA sequence that was used to search GenBank and identify the gene for P5'N-1. It is found on chromosome 7, consists of 10 exons with alternative splicing of exon 2, and produces proteins 286 and 297 amino acids long. Three homozygous mutations were identified in this gene in 4 subjects with P5'N-1 deficiency: codon 98 GAT-->GTT, Asp-->Val (linked to a silent polymorphism codon 92, TAC-->TAT), codon 177, CAA-->TAA, Gln-->termination, and IVS9-1, G-->T. The latter mutation results in the loss of exon 9 (201 bp) from the cDNA. None of these mutations was found in 100 normal controls. The DNA analysis was complicated by P5'N-1 pseudogenes found on chromosomes 4 and 7. This study is the first description of the structure and location of the P5'N-1 gene, and 3 mutations have been identified in affected patients from separate kindreds. (Blood. 2001;97:3327-3332

Metabolic Profiling of Alternative NAD Biosynthetic Routes in Mouse Tissues

NAD plays essential redox and non-redox roles in cell biology. In mammals, its de novo and recycling biosynthetic pathways encompass two independent branches, the "amidated" and "deamidated" routes. Here we focused on the indispensable enzymes gating these two routes, i.e. nicotinamide mononucleotide adenylyltransferase (NMNAT), which in mammals comprises three distinct isozymes, and NAD synthetase (NADS). First, we measured the in vitro activity of the enzymes, and the levels of all their substrates and products in a number of tissues from the C57BL/6 mouse. Second, from these data, we derived in vivo estimates of enzymes'rates and quantitative contributions to NAD homeostasis. The NMNAT activity, mainly represented by nuclear NMNAT1, appears to be high and nonrate-limiting in all examined tissues, except in blood. The NADS activity, however, appears rate-limiting in lung and skeletal muscle, where its undetectable levels parallel a relative accumulation of the enzyme's substrate NaAD (nicotinic acid adenine dinucleotide). In all tissues, the amidated NAD route was predominant, displaying highest rates in liver and kidney, and lowest in blood. In contrast, the minor deamidated route showed higher relative proportions in blood and small intestine, and higher absolute values in liver and small intestine. Such results provide the first comprehensive picture of the balance of the two alternative NAD biosynthetic routes in different mammalian tissues under physiological conditions. This fills a gap in the current knowledge of NAD biosynthesis, and provides a crucial information for the study of NAD metabolism and its role in disease

Highly stable atropisomers by electrophilic amination of a chiral γ-lactam within the synthesis of an elusive conformationally restricted analogue of α-methylhomoserine

Starting from chiral-protected 4-hydroxymethyl pyrrolidin-2-ones, the otherwise elusive 3,4-trans-3,3,4-trisubstituted isosteres of α-methyl homoserine, tethered on a γ-lactam ring, were prepared exploiting stereoselective electrophilic aminations. These reactions led to the isolation and characterization of a novel type of atropisomers, exceedingly stable at room temperature, that were directly converted to the desired products by a novel non-reductive N–N bond cleavage reaction