Botulinum Neurotoxin Types A,B, and E: Fragmentations by Autoproteolysis and Other Mechanisms Including by <Emphasis Type="Italic">O</Emphasis>-Phenanthroline–Dithiothreitol,and Association of the Dinucleotides NAD<Superscript>+</Superscript>/NADH with the Heavy Chain of the Three Neurotoxins

The first evidence of autoproteolytic activity of the ~50-kDa light chain of the clostridial neurotoxins (NT) is traceable to the observations that the light chains of botulinum NT serotypes A and E, separated from their ~100-kDa heavy chain conjugate, were found cleaved at the amino side of Tyr250 and Arg244, respectively [DasGupta and Foley (1989). Biochimie 71: 1183–1200]. Specific cleavages of the recombinant light chain of NT type A, including at Tyr249–Tyr250, firmly established that the cleavages reported earlier were due to autoproteolysis [Ahmed et al. (2001). J. Protein Chem. 20: 221–231; Ahmed et al. (2003). Biochemistry 42:12539–12549] and not by contaminating proteases or non-enzymatic. We now report many cleavages in the NT types A, B and E and also in their separated light and heavy chains, and identification of several of the peptide bonds cleaved. None of the identified cleaved bonds (–P₁–P₁′ –) in one serotype (except Asp–Pro) was found common in other serotypes or cleaved within itself at a second site. After separation from the heavy chain self-cleavages of the light chains of type A, B and E at Tyr249–Tyr250, Gln258–Ser259 and Ile243–Arg244, respectively indicate an intriguing feature (in the aligned sequences these bonds of type A and B are 2 and type A and E are 4 peptide bonds apart) that may have some role in the NT’s structure–function relationship yet to be understood. We point out that autoproteolysis of a single peptide bond (Phe418–Thr419 or Phe422–Glu423) in NT type A reported by Ahmed et al. (2001) can potentially generate proteolytically active light chain freed of the heavy chain; this is an efficient pathway, that by-passes nicking by a trypsin-like protease(s) inside the intrachain disulfide bridge and its reductive cleavage. We offer probable explanations for the observed cleavages such as acid- and metal-mediated (non-catalytic and non-stoichiometric) reactions in addition to autoproteolysis but cannot predict which mechanism(s) of cleavage occur or prevail following NT’s entry in the body as poison or therapeutic agent. The metal chelator O-phenanthroline (above critical miceller concentration) in the presence of dithiothreitol cleaved type E NT at limited sites generating discrete 114-, 87-, 49-, 42-, and 31-kDa fragments but degraded NTs type A and B extensively. The limited cleavage of type E NT was dependent on the presence of metal ion(s) bound to the protein and its native (urea sensitive) conformation. The self-cleavage of the NTs at specific sites prompted us to search for specific binding sites on the NTs analogous to SNARE-motifs—the 9-residuelong motifs present on the NT’s natural substrates (SNAP-25, syntaxin, VAMP/synaptobrevin); such putative binding motifs (sites) noted on all clostridial NTs are reported here. Their relationship to the observed autoproteolysis remains to be determined experimentally. The dinucleotide NAD⁺/NADH associated with the NTs type A, B and E (2–3 NADH per protein molecule) via their H-chains, and a portion of the H-chain (toward the C-terminus) appears to exhibit limited amino acid sequence homology with lactate dehydrogenase—a representative NAD⁺/NADH binding protein.     

Expression of a novel P275L variant of NADH:cytochrome b5 reductase gives functional insight into the conserved motif important for pyridine nucleotide binding

The clinical disorder of recessive congenital methemoglobinemia (RCM, OMIN 250800) is associated with mutations in NADH:cytochrome b5 reductase (cb5r) and manifests as cyanosis from birth. Screening a cyanotic infant indicated elevated methemoglobin levels and decreased cb5r activity suggesting RCM. Sequencing the DIA1 gene encoding cb5r revealed a novel mutation, C27161T (NCBI accession number: NT_011520), resulting in replacement of proline at amino acid 275 with leucine (P275L). To understand how this mutation would affect cb5r's function, the P275L variant was expressed in a heterologous expression system and spectroscopic, thermodynamic, and thermostability studies were performed. The leucine substitution at residue 275 was found to significantly decrease the affinity towards the physiological reducing substrate, NADH, without affecting the activity of the P275L variant. From the rat model, residue 275 is predicted to be part of a conserved "CGPPPM" motif important for the binding and correct positioning of the NADH reducing substrate. Thus P275 influences the interaction with NADH which was confirmed by the change in affinity towards the physiological reducing substrate.     

Structural evidence for direct hydride transfer from NADH to cytochrome P450nor

Nitric oxide reductase cytochrome P450nor catalyzes an unusual reaction, direct electron transfer from NAD(P)H to bound heme. Here, we succeeded in determining the crystal structure of P450nor in a complex with an NADH analogue, nicotinic acid adenine dinucleotide, which provides conclusive evidence for the mechanism of the unprecedented electron transfer. Comparison of the structure with those of dinucleotide-free forms revealed a global conformational change accompanied by intriguing local movements caused by the binding of the pyridine nucleotide. Arg64 and Arg174 fix the pyrophosphate moiety upon the dinucleotide binding. Stereo-selective hydride transfer from NADH to NO-bound heme was suggested from the structure, the nicotinic acid ring being fixed near the heme by the conserved Thr residue in the I-helix and the upward-shifted propionate side-chain of the heme. A proton channel near the NADH channel is formed upon the dinucleotide binding, which should direct continuous transfer of the hydride and proton. A salt-bridge network (Glu71-Arg64-Asp88) was shown to be crucial for a high catalytic turnover.