A Novel, Easy Assay Method for Human Cysteine Sulfinic Acid Decarboxylase

Cysteine sulfinic acid decarboxylase catalyzes the last step of taurine biosynthesis in mammals, and belongs to the fold type I superfamily of pyridoxal-5′-phosphate (PLP)-dependent enzymes. Taurine (2-aminoethanesulfonic acid) is the most abundant free amino acid in animal tissues; it is highly present in liver, kidney, muscle, and brain, and plays numerous biological and physiological roles. Despite the importance of taurine in human health, human cysteine sulfinic acid decarboxylase has been poorly characterized at the biochemical level, although its three-dimensional structure has been solved. In the present work, we have recombinantly expressed and purified human cysteine sulfinic acid decarboxylase, and applied a simple spectroscopic direct method based on circular dichroism to measure its enzymatic activity. This method gives a significant advantage in terms of simplicity and reduction of execution time with respect to previously used assays, and will facilitate future studies on the catalytic mechanism of the enzyme. We determined the kinetic constants using L-cysteine sulfinic acid as substrate, and also showed that human cysteine sulfinic acid decarboxylase is capable to catalyze the decarboxylation—besides its natural substrates L-cysteine sulfinic acid and L-cysteic acid—of L-aspartate and L-glutamate, although with much lower efficiency.

GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

Carnosine and related β-alanine–containing peptides are believed to be important antioxidants, pH buffers, and neuromodulators. However, their biosynthetic routes and therapeutic potential are still being debated. This study describes the first animal model lacking the enzyme glutamic acid decarboxylase–like 1 (GADL1). We show that Gadl1−/− mice are deficient in β-alanine, carnosine, and anserine, particularly in the olfactory bulb, cerebral cortex, and skeletal muscle. Gadl1−/− mice also exhibited decreased anxiety, increased levels of oxidative stress markers, alterations in energy and lipid metabolism, and age-related changes. Examination of the GADL1 active site indicated that the enzyme may have multiple physiological substrates, including aspartate and cysteine sulfinic acid. Human genetic studies show strong associations of the GADL1 locus with plasma levels of carnosine, subjective well-being, and muscle strength. Together, this shows the multifaceted and organ-specific roles of carnosine peptides and establishes Gadl1 knockout mice as a versatile model to explore carnosine biology and its therapeutic potential.

GADL1 is a multifunctional decarboxylase with tissue-specific roles in β-alanine and carnosine production

ABSTRACTCarnosine and related β-alanine-containing peptides are believed to be important antioxidants, pH-buffers and neuromodulators. However, their biosynthetic routes and therapeutic potential are still being debated. This study describes the first animal model lacking the enzyme glutamic acid decarboxylase-like 1 (GADL1). We show that Gadl1-/-mice are deficient in β-alanine, carnosine and anserine, particularly in the olfactory bulb, cerebral cortex, and skeletal muscle. Gadl1-/-mice also exhibited decreased anxiety, increased levels of oxidative stress markers, alterations in energy and lipid metabolism, and age-related changes. Examination of the GADL1 active site indicated that the enzyme may have multiple physiological substrates, including aspartate and cysteine sulfinic acid, compatible with organ-specific functions. Human genetic studies show strong associations of the GADL1 locus with plasma levels of carnosine, subjective well-being, and muscle strength, also indicating a role for β-alanine and its peptide derivatives in these traits. Together, this shows the multifaceted and organ specific roles of carnosine peptides and establishes Gadl1 knockout mice as a versatile model to explore carnosine biology and its therapeutic potential.

Quantum Mechanical/Molecular Mechanical investigation of the reduction mechanism of Cysteine Sulfinic acid of Peroxiredoxin via Sulfiredoxin

<div><div><div><p>The formation of the overoxidized cysteine sulfinic acid in proteins has been connected to be associated with various diseases including cancer and age-related diseases. This post-transitional modification of proteins under oxi- dative stress has been known to be irreversible. However, in eukaryotic, the overoxidation of typical 2-Cys perxoiredoxins (Prxs) to sulfinic acid is reversible via a repair enzyme known as sulfiredoxin (Srx) leading to the regulation of both per- oxide signaling and Prxs chaperon activity. In this study, the molecular modeling techniques including molecular dynam- ics simulations (MD) and the hybrid quantum mechanical/molecular mechanical (QM/MM) approach were used to eluci- date the atomistic details of this unique reaction in sulfur chemistry. Our results support the previous experimentally pro- posed mechanism in which the sulfinic acid oxygen perform an in line direct nucleophilic attack on the γ-phosphate of ATP forming sulfinic acid phosphoryl ester intermediate and ADP, via a low barrier of 16.3 kJ mol-1. Subsequently, the formed intermediate is directly reduced via an SN2 mechanism by the Srx-Cys99 forming thiosulfinate. Our results suggest that the rate-limiting step of the reduction mechanism is associated with the reduction step of the thiosulfinate intermedi- ate. This work significantly improves the current knowledge of this unique reaction, which could contribute to the discov- ery of new groups of antioxidants capable of reducing this irreversible overoxidized state in other proteins.</p></div></div></div>

Quantum Mechanical/Molecular Mechanical investigation of the reduction mechanism of Cysteine Sulfinic acid of Peroxiredoxin via Sulfiredoxin

<div><div><div><p>The formation of the overoxidized cysteine sulfinic acid in proteins has been connected to be associated with various diseases including cancer and age-related diseases. This post-transitional modification of proteins under oxi- dative stress has been known to be irreversible. However, in eukaryotic, the overoxidation of typical 2-Cys perxoiredoxins (Prxs) to sulfinic acid is reversible via a repair enzyme known as sulfiredoxin (Srx) leading to the regulation of both per- oxide signaling and Prxs chaperon activity. In this study, the molecular modeling techniques including molecular dynam- ics simulations (MD) and the hybrid quantum mechanical/molecular mechanical (QM/MM) approach were used to eluci- date the atomistic details of this unique reaction in sulfur chemistry. Our results support the previous experimentally pro- posed mechanism in which the sulfinic acid oxygen perform an in line direct nucleophilic attack on the γ-phosphate of ATP forming sulfinic acid phosphoryl ester intermediate and ADP, via a low barrier of 16.3 kJ mol-1. Subsequently, the formed intermediate is directly reduced via an SN2 mechanism by the Srx-Cys99 forming thiosulfinate. Our results suggest that the rate-limiting step of the reduction mechanism is associated with the reduction step of the thiosulfinate intermedi- ate. This work significantly improves the current knowledge of this unique reaction, which could contribute to the discov- ery of new groups of antioxidants capable of reducing this irreversible overoxidized state in other proteins.</p></div></div></div>