Biotechnological production of reduced and oxidized NAD precursors.

Dysregulation of nicotinamide adenine dinucleotide (NAD) homeostasis by increased activity of NAD consumers or reduced NAD biosynthesis plays an important role in the onset of prevalent, often age-related, diseases, such as diabetes, neuropathies or nephropathies. To counteract such dysregulation, NAD replenishment strategies can be used. Among these, administration of vitamin B derivatives (NAD precursors) has garnered attention in recent years.

14-3-3 Activated Bacterial Exotoxins AexT and ExoT Share Actin and the SH2 Domains of CRK Proteins as Targets for ADP-Ribosylation.

Bacterial exotoxins with ADP-ribosyltransferase activity can be divided into distinct clades based on their domain organization. Exotoxins from several clades are known to modify actin at Arg177; but of the 14-3-3 dependent exotoxins only exoenzyme T (AexT) has been reported to ADP-ribosylate actin. Given the extensive similarity among the 14-3-3 dependent exotoxins, we initiated a structural and biochemical comparison of these proteins.

MacroGreen, a simple tool for detection of ADP-ribosylated proteins.

Enzymes in the PARP family partake in the regulation of vital cellular signaling pathways by ADP-ribosylating their targets. The roles of these signaling pathways in disease development and the de-regulation of several PARP enzymes in cancer cells have motivated the pursuit of PARP inhibitors for therapeutic applications. In this rapidly expanding research area, availability of simple research tools will help assess the functions of ADP-ribosylation in a wider range of contexts.

PARP10 Multi-Site Auto- and Histone MARylation Visualized by Acid-Urea Gel Electrophoresis.

Poly-ADP-ribose polymerase (PARP)-family ADP-ribosyltransferases function in various signaling pathways, predominantly in the nucleus and cytosol. Although PARP inhibitors are in clinical practice for cancer therapy, the enzymatic activities of individual PARP family members are yet insufficiently understood. We studied PARP10, a mono-ADP-ribosyltransferase and potential drug target.

The first comprehensive phylogenetic and biochemical analysis of NADH diphosphatases reveals that the enzyme from Tuber melanosporum is highly active towards NAD.

Nudix (for nucleoside diphosphatases linked to other moieties, X) hydrolases are a diverse family of proteins capable of cleaving an enormous variety of substrates, ranging from nucleotide sugars to NAD-capped RNAs. Although all the members of this superfamily share a common conserved catalytic motif, the Nudix box, their substrate specificity lies in specific sequence traits, which give rise to different subfamilies. Among them, NADH pyrophosphatases or diphosphatases (NADDs) are poorly studied and nothing is known about their distribution.

An uncharacterized FMAG_01619 protein from Fusobacterium mortiferum ATCC 9817 demonstrates that some bacterial macrodomains can also act as poly-ADP-ribosylhydrolases.

Macrodomains constitute a conserved fold widely distributed that is not only able to bind ADP-ribose in its free and protein-linked forms but also can catalyse the hydrolysis of the latter. They are involved in the regulation of important cellular processes, such as signalling, differentiation, proliferation and apoptosis, and in host-virus response, and for this, they are considered as promising therapeutic targets to slow tumour progression and viral pathogenesis. Although extensive work has been carried out with them, including their classification into six distinct phylogenetically clades, little is known on bacterial macrodomains, especially if these latter are able to remove poly(ADP-ribose) polymer (PAR) from PARylated proteins, activity that only has been confirmed in human TARG1 (C6orf130) protein.

Comparative inhibitory profile and distribution of bacterial PARPs, using Clostridioides difficile CD160 PARP as a model.

Poly-ADP-ribose polymerases (PARPs) are involved in the regulation of important cellular processes, such as DNA repair, aging and apoptosis, among others. They have been considered as promising therapeutic targets, since human cancer cells carrying BRCA1 and BRCA2 mutations are highly sensitive to human PARP-1 inhibitors. Although extensive work has been carried out with the latter enzyme, little is known on bacterial PARPs, of which only one has been demonstrated to be active.

Biochemical characterization of a new nicotinamidase from an unclassified bacterium thriving in a geothermal water stream microbial mat community.

Nicotinamidases are amidohydrolases that convert nicotinamide into nicotinic acid, contributing to NAD+ homeostasis in most organisms. In order to increase the number of nicotinamidases described to date, this manuscript characterizes a nicotinamidase obtained from a metagenomic library fosmid clone (JFF054_F02) obtained from a geothermal water stream microbial mat community in a Japanese epithermal mine. The enzyme showed an optimum temperature of 90°C, making it the first hyperthermophilic bacterial nicotinamidase to be characterized, since the phylogenetic analysis of this fosmid clone placed it in a clade of uncultured geothermal bacteria.

Structural and functional analysis of macrodomain reveals a network of waters involved in substrate binding and catalysis.

Macrodomains are ubiquitous conserved domains that bind or transform ADP-ribose (ADPr) metabolites. In humans, they are involved in transcription, X-chromosome inactivation, neurodegeneration and modulating PARP1 signalling, making them potential targets for therapeutic agents. Unfortunately, some aspects related to the substrate binding and catalysis of MacroD-like macrodomains still remain unclear, since mutation of the proposed catalytic aspartate does not completely abolish enzyme activity.

Characterization and mutational analysis of a nicotinamide mononucleotide deamidase from Agrobacterium tumefaciens showing high thermal stability and catalytic efficiency.

NAD+ has emerged as a crucial element in both bioenergetic and signaling pathways since it acts as a key regulator of cellular and organismal homeostasis. Among the enzymes involved in its recycling, nicotinamide mononucleotide (NMN) deamidase is one of the key players in the bacterial pyridine nucleotide cycle, where it catalyzes the conversion of NMN into nicotinic acid mononucleotide (NaMN), which is later converted to NAD+ in the Preiss-Handler pathway. The biochemical characteristics of bacterial NMN deamidases have been poorly studied, although they have been investigated in some firmicutes, gamma-proteobacteria and actinobacteria.