Characterization of the PLP-dependent decarboxylases GADL1 and CSAD

Abstract

Pyridoxal 5’-phosphate (PLP) -dependent enzymes constitute an essential superfamily of enzymes for all living organisms. Due to the chemistry of their cofactor, PLP, these enzymes catalyze a wide variety of reactions. PLP-dependent decarboxylases (PLPDCs) play key roles in amino acid metabolism and are considered important biocatalysts for the pharmaceutical and chemical industries. In this project, we focused on the recently discovered enzyme glutamate decarboxylase-like 1 (GADL1) and its homolog, cysteine sulfinic acid (CSA) decarboxylase (CSAD). We investigated the expression pattern as well as the structural and biochemical features of both GADL1 and CSAD. Furthermore, we studied the physiological role of GADL1 by developing the first knockout mouse model for this enzyme. In addition, we reported crystal structures of mouse GADL1 (MmGADL1) and CSAD (MmCSAD).

We found that GADL1 decarboxylates aspartate (Asp) to β-alanine, and consequently, it plays a role in the production of carnosine. Both carnosine and β-alanine are pH buffers, metal chelators, and have antioxidants effects. In addition to β-alanine, we observed that GADL1 decarboxylases CSA to hypotaurine, which leads to the production of taurine, one of the most abundant free amino acids in mammals. Taurine also functions as an antioxidant, membrane stabilizer, and neurotransmitter in the central nervous system (CNS). Similarly, CSAD decarboxylates CSA to hypotaurine and has a higher affinity to CSA than GADL1. The tissue distribution of the two enzymes was investigated, and it was found that in humans, both GADL1 and CSAD are expressed in the brain, whereas only CSAD is found in the liver. In mice, both enzymes are expressed in the brain, olfactory bulb (OB), and skeletal muscle (SKM). In the kidney, GADL1 has lower expression than CSAD and in the liver, only the expression of CSAD was found.

To find the physiological role of GADL1 we generated Gadl1−/− mice. These mice were deficient in β-alanine, carnosine, and anserine, particularly in the OB, brain, and SKM, indicating a role for GADL1 in carnosine dipeptide production. Furthermore, Gadl1−/− mice had increased levels of oxidative stress markers, indicating the importance of carnosine as a cellular antioxidant. In addition, in different behavioral examinations, Gadl1−/−, mice showed a slightly decreased level of anxiety. Human genetic studies show a strong association of the GADL1 locus with plasma levels of carnosine, subjective well-being, kidney function, and muscle strength. In addition, investigating the GADL1 active site indicates that the enzyme may have multiple physiological substrates in vivo, including Asp and CSA.

Structural studies on MmGADL1 and MmCSAD in this project showed that the overall fold and the conformation of the bound PLP are similar to other PLP-DCs. Both GADL1 and CSAD showed a more loose conformation in solution than in the crystal state, with open/close motions. In addition, in the MmCSAD structure, phenylalanine94 plays a critical role in substrate binding, and its mutation to serine changed MmCSAD affinity towards both CSA and Asp and also affected enzyme stability. The structural studies of MmGADL1 and MmCSAD provided details on substrate recognition in the PLP-DC family. These results are useful for future studies in structure-based inhibitor design and drug discovery.

In conclusion, in this project, the biochemical and physiological roles of CSAD and a novel PLP-DC, GADL1, were investigated. This study also introduces Gadl1 knockout mice as a multi-aspect model to investigate carnosine biology. Using a structural approach, new information was revealed about the structure and features of both MmGADL1 and MmCSAD.