Biochemical Studies of Aromatic Amino Acid Decarboxylases and Acetaldehyde Synthases
| dc.contributor.author | Liang, Jing | en |
| dc.contributor.committeechair | Li, Jianyong | en |
| dc.contributor.committeemember | Zhu, Jinsong | en |
| dc.contributor.committeemember | Bevan, David R. | en |
| dc.contributor.committeemember | Larson, Timothy J. | en |
| dc.contributor.department | Biochemistry | en |
| dc.date.accessioned | 2020-01-01T07:00:47Z | en |
| dc.date.available | 2020-01-01T07:00:47Z | en |
| dc.date.issued | 2018-07-09 | en |
| dc.description.abstract | Pyridoxal 5'-phosphate (PLP)-dependent enzymes widely exist in most living organisms from bacteria to human. Among different types of PLP-dependent enzymes, aromatic amino acid decarboxylases play critical physiological roles because many aromatic amines are essential neurotransmitters. This dissertation concerns the biochemical characterization of several PLP-dependent decarboxylases and aims to understand the structure-function relationships, especially critical residues involved in their catalysis. We first present an overview of the current opinions and recent advances in structure-function relationships of several PLP-dependent enzymes with the first reaction step at substrate Cα position, including decarboxylase and acetaldehyde synthase. L-3, 4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC) is a model enzyme we use as a reference because the structures and functions of DDC are relatively well established. We previously identified two annotated DDC-like proteins from Drosophila indeed catalyzing a decarboxylation-oxidative deamination reaction of L-dopa to form 3,4-dihydroxyphenylacetaldehyde (DHPA), CO2, NH3, and H2O2 and we named these proteins as DHPA synthases due to the physiological importance of DHPA for cuticle protein crosslinking. Our results provide an efficient way to identify more DHPA synthase enzymes from DDC based on sequence identity and the signature residues we identified (Asn192 in DHPA synthase versus His192 in DDC), and we also propose a reasonable explanation of the mechanism. The results that H2O2 produced by the reaction can be reused in the reaction as an oxidizing agent suggest a way to avoid the oxidative stress of H2O2. We then compared tyrosine decarboxylase (TyDC) with DDC. As the enzyme catalyzing the first step of insect neurotransmitter tyramine/octopamine synthesis, the biochemical characteristics of insect TyDC have not been thoroughly elucidated yet because of the expression difficulty. We expressed one insect TyDC and analyzed its biochemical properties. Our enzyme analyses reveal that insect TyDC prefers tyrosine as a substrate, but it also displays some activity to L-dopa. Spectral analysis also shows that the absorbance spectra of insect TyDC have major differences as compared to those of DDC. Site-directed mutagenesis indicates that the interactions between residue Asn304 with PLP is primarily responsible for its spectra differences of TyDC as compared to those of DDC and also is involved in higher substrate affinity to L-tyrosine. Another active site residue (Ser353) has the main effect on substrate selectivity. Our results show the biochemical properties of TyDC for the first time and also provide some insights into the mechanism of its substrate selectivity. | en |
| dc.description.abstractgeneral | Pyridoxal 5’-phosphate (PLP) is a coenzyme for many enzymatic reactions. Among PLP-dependent enzymes, aromatic amino acid decarboxylase plays critical physiological roles, because many aromatic amines produced by decarboxylation of aromatic amino acids are essential neurotransmitters. L-3, 4-dihydroxyphenylalanine (L-dopa) decarboxylase (DDC) is one well-established decarboxylase, and we use DDC as a reference enzyme for comparative study with other decarboxylases. We analyzed the catalytic characteristics of several PLPdependent decarboxylases, with a focus on the active site residues important for the catalysis and substrate specificity. We previously identified that two annotated Drosophila L-3, 4- dihydroxyphenylalanine (L-dopa) decarboxylase (DDC) indeed catalyzed a decarboxylation-oxidative deamination reaction of L-dopa to form 3,4-dihydroxyphenylacetaldehyde (DHPA), CO₂, NH₃, and H₂O₂ and we named these proteins as DHPA synthases due to the physiological importance of DHPA for cuticle protein crosslinking. We also functionally expressed one insect tyrosine decarboxylase (TyDC) and analyzed its biochemical properties. Our results indicate that Asn192 in DHPA synthase versus His192 in DDC are important for DHPA synthase activity and DDC activity respectively, and the roles of these two residues in both enzymes were also proposed. In addition, DHPA synthase can reuse H₂O₂ as the oxidizing agent, and the oxidative stress of accumulated H₂O₂ may be avoided in this way. Our biochemical analyses of TyDC reveal its substrate specificity and spectra characteristics. Site-directed mutagenesis suggests the interactions between Asn304 with PLP is primarily responsible for TyDC spectra differences compared with those of DDC, and Asn304 is also involved in higher substrate affinity of TyDC to L-tyrosine. Another active site residue (Ser353) has the main effect on the substrate selectivity. Our results show the biochemical properties of insect TyDC for the first time, including the substrate specificity, spectra characteristics, and suggest some key residues involved in the substrate specificity and spectra of insect TyDC. | en |
| dc.description.degree | PHD | en |
| dc.format.medium | ETD | en |
| dc.identifier.other | vt_gsexam:15205 | en |
| dc.identifier.uri | http://hdl.handle.net/10919/96242 | en |
| dc.publisher | Virginia Tech | en |
| dc.rights | In Copyright | en |
| dc.rights.uri | http://rightsstatements.org/vocab/InC/1.0/ | en |
| dc.subject | Chemistry | en |
| dc.subject | Biochemistry | en |
| dc.title | Biochemical Studies of Aromatic Amino Acid Decarboxylases and Acetaldehyde Synthases | en |
| dc.type | Dissertation | en |
| thesis.degree.discipline | Biochemistry | en |
| thesis.degree.grantor | Virginia Polytechnic Institute and State University | en |
| thesis.degree.level | doctoral | en |
| thesis.degree.name | PHD | en |