Altered AADC Enzyme Activity Found in Boy With Novel Mutation

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by Steve Bryson, PhD |

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A novel gene mutation — which caused a case of aromatic l-amino acid decarboxylase (AADC) deficiency in a young boy — was found to directly impair a crucial step in the activity of the AADC enzyme without affecting its stability or production, a study reported.

The researchers’ investigation helped them determine the structural basis for the severity of the AADC deficiency in this 2-year-old patient — and provided a rationale for precision therapy.

“The obtained results allow … for a tailored therapy in the reported patient, instead of trying different medications looking for the best clinical response (as it is usually practiced in AADC deficiency),” the team wrote.

The study, “The novel P330L pathogenic variant of aromatic amino acid decarboxylase maps on the catalytic flexible loop underlying its crucial role,” was published in the journal Cellular and Molecular Life Sciences.

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Enzymes are proteins that act as catalysts to accelerate biochemical reactions.

AADC deficiency is caused by mutations in the DDC gene, which provides the instructions to make the aromatic l-amino acid decarboxylase (AADC) enzyme. This enzyme plays an essential role in producing several nerve cell signaling molecules, or neurotransmitters, such as serotonin and dopamine.

The AADC enzyme is composed of two identical chains of amino acids, the building blocks of proteins. Attached to the enzyme is pyridoxal phosphate (PLP), the active form of vitamin B6, which participates in the enzyme’s catalytic activity.

The binding of serotonin or dopamine precursor molecules to the enzyme triggers a reaction that removes a chemical structure called a carboxyl group — hence the name decarboxylase — leaving either serotonin or dopamine as a final product. The place on the enzyme where this reaction takes place is called the active site.

To date, more than 90 mutations in the DDC gene have been identified. These mutations change the amino acids, thus altering the structure of the AADC enzyme, leading to reduced enzyme production or activity, abnormally low serotonin or dopamine, and disease symptoms.

Although these mutations are found throughout the enzyme, none have been identified on the catalytic loop — a portion of the AADC enzyme that covers the active site like a lid, which is necessary for the reaction to occur.

Now, for the first time, researchers based at the University of Verona, in Italy, reported the case of an AADC deficiency patient who carried a mutation found on the catalytic loop. This mutation is called Pro330Leu (or P330L), in which a proline amino acid is changed to a leucine amino acid at position 330 of the protein chain.

The boy, now 2.5 years old, was born with a normal body weight. However, since infancy, he has been unable to suck. He also experienced recurrent vomiting with excessive salivation.

After the age of 8 months, he had episodes of anxiety, crying, and increased sweating. He remained stable with pyridoxine, or vitamin B6, a first-line AADC deficiency treatment.

At age 2, however, he acquired a SARS-CoV2 infection, the virus that causes COVID-19, along with the appearance of oculogyric crises — movements of the eyeballs into a fixed, upward position.

While folinate treatment provided some clinical improvements, he experienced decreased muscle tone and a slowing of mental or physical abilities. MRI scans and electroencephalograms (EEG) of brain activity showed mild, non-specific abnormalities.

Urine analysis showed a substantial increase in dopamine precursors, a pattern characteristic of AADC deficiency. Blood tests revealed AADC enzymatic activity was severely impaired, using the dopamine precursor L-dopa and the serotonin precursor L-5HTP. A genetic assessment revealed the P330L mutation, which had not been previously reported in relevant databases.

Modeling studies showed that Pro330 is placed inside the active site when the catalytic loop is closed. Replacing the proline with leucine amino acid was found to be unfavorable, shifting the tendency of the loop, or lid, to be open. “The P330L replacement is expected to affect the positioning of the loop,” the team wrote.

Computer modeling supported the importance of Pro300 in keeping the catalytic loop properly fitted and correctly orienting a tyrosine amino acid that directly participates in the chemical reaction. Light-based spectroscopic analysis confirmed that this mutation does not affect the enzyme structure overall but instead compromises the flexibility of the catalytic loop.

AADC activity analysis showed that the P330L mutation decreased the enzyme’s activity by 674 times when L-dopa was used as a precursor, and by 194 times with L-5HTP.

“Thus, the P330L substitution compromises catalysis rather than overall protein structural features,” the researchers wrote.

Further examination demonstrated that the P330L mutation, which impaired loop closing, led to an altered orientation of PLP, which affected AADC’s ability to produce serotonin or dopamine efficiently.

“The substitution with Leu on one hand destroys the [bonding] network rendering the [catalytic loop] less able to sit in the closed state, on the other it influences [PLP] placing, thus affecting catalysis rather than global protein structure,” the researchers concluded.

“Given the severe catalytic impairment exhibited by this novel AADC protein variant, the therapeutic suggestion is to sustain the ongoing clinical management with a dopamine (and serotonin) [activator], to enhance the extremely low endogenous activity,” the researchers added. “If it appears to be clinically ineffective, gene therapy should be considered.”