Browsing by Author "Liang, Jing"

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    3,4-Dihydroxyphenylacetaldehyde synthase and cuticle formation in insects
    Liao, Chenghong; Liang, Jing; Han, Qian; Li, Jianyong (2018-06-02)
    Cuticle is the most important structure that protects mosquitoes and other insect species from adverse environmental conditions and infections of microorganism. The physiology and biochemistry of insect cuticle formation have been studied for many years and our understanding of cuticle formation and hardening has increased considerably. This is especially true for flexible cuticle. The recent discovery of a novel enzyme that catalyzes the production of 3,4-dihydroxyphenylacetaldehyde (DOPAL) in insects provides intriguing insights concerning the flexible cuticle formation in insects. For convenience, the enzyme that catalyzes the production DOPAL from L-dopa is named DOPAL synthase. In this mini-review, we summarize the biochemical pathways of cuticle formation and hardening in general and discuss DOPAL synthase-mediated protein crosslinking in insect flexible cuticle in particular.
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    Biochemical Studies of Aromatic Amino Acid Decarboxylases and Acetaldehyde Synthases
    Liang, Jing (Virginia Tech, 2018-07-09)
    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.
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    Current Advances on Structure-Function Relationships of Pyridoxal 5'-Phosphate-Dependent Enzymes
    Liang, Jing; Han, Qian; Tan, Yang; Ding, Haizhen; Li, Jianyong (Frontiers, 2019-03-05)
    Pyridoxal 5'-phosphate (PLP) functions as a coenzyme in many enzymatic processes, including decarboxylation, deamination, transamination, racemization, and others. Enzymes, requiring PLP, are commonly termed PLP-dependent enzymes, and they are widely involved in crucial cellular metabolic pathways in most of (if not all) living organisms. The chemical mechanisms for PLP-mediated reactions have been well elaborated and accepted with an emphasis on the pure chemical steps, but how the chemical steps are processed by enzymes, especially by functions of active site residues, are not fully elucidated. Furthermore, the specific mechanism of an enzyme in relation to the one for a similar class of enzymes seems scarcely described or discussed. This discussion aims to link the specific mechanism described for the individual enzyme to the same types of enzymes from different species with aminotransferases, decarboxylases, racemase, aldolase, cystathionine beta-synthase, aromatic phenylacetaldehyde synthase, et al. as models. The structural factors that contribute to the reaction mechanisms, particularly active site residues critical for dictating the reaction specificity, are summarized in this review.
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    Identification of aaNAT5b as a spermine N-acetyltransferase in the mosquito, Aedes aegypti
    Guan, Huai; Wang, Maoying; Liao, Chenghong; Liang, Jing; Mehere, Prajwalini; Tian, Meiling; Liu, Hairong; Robinson, Howard; Li, Jianyong; Han, Qian (PLOS, 2018-03-19)
    Mosquitoes transmit a number of diseases in animals and humans, including Dengue, Chikungunya and Zika viruses that affect millions of people each year. Controlling the disease-transmitting mosquitoes has proven to be a successful strategy to reduce the viruses transmission. Polyamines are required for the life cycle of the RNA viruses, Chikungunya virus and Zika virus, and a depletion of spermidine and spermine in the host via induction of spermine N-acetyltransferase restricts their replication. Spermine N-acetyltransferase is a key catabolic enzyme in the polyamine pathway, however there is no information of the enzyme identification in any insects. Aliphatic polyamines play a fundamental role in tissue growth and development in organisms. They are acetylated by spermidine/spermine N-1-acetyl-transferase (SAT). In this study we provided a molecular and biochemical identification of SAT from Aedes aegypti mosquitoes. Screening of purified recombinant proteins against polyamines established that aaNAT5b, named previously based on sequence similarity with identified aaNAT1 in insects, is active to spermine and spermidine. A crystal structure was determined and used in molecular docking in this study. Key residues were identified to be involved in spermine binding using molecular docking and simulation. In addition, SAT transcript was down regulated by blood feeding using a real time PCR test. Based on its substrate profile and transcriptional levels after blood feeding, together with previous reports for polyamines required in arboviruses replication, SAT might be potentially used as a target to control arboviruses with human interference.