Browsing by Author "Han, Qian"
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- 3,4-Dihydroxyphenylacetaldehyde synthase and cuticle formation in insectsLiao, 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.
- AvrRxo1 Is a Bifunctional Type III Secreted Effector and Toxin-Antitoxin System Component with Homologs in Diverse Environmental ContextsTriplett, Lindsay R.; Shidore, Teja; Long, John J.; Miao, Jiamin; Wu, Shuchi; Han, Qian; Zhou, Changhe; Ishihara, Hiromichi; Li, Jianyong; Zhao, Bingyu Y.; Leach, Jan E. (PLOS, 2016-07-08)Toxin-antitoxin (TA) systems are ubiquitous bacterial systems that may function in genome maintenance and metabolic stress management, but are also thought to play a role in virulence by helping pathogens survive stress. We previously demonstrated that the Xanthomonas oryzae pv. oryzicola protein AvrRxo1 is a type III-secreted virulence factor that has structural similarities to the zeta family of TA toxins, and is toxic to plants and bacteria in the absence of its predicted chaperone Arc1. In this work, we confirm that AvrRxo1 and its binding partner Arc1 function as a TA system when expressed in Escherichia coli. Sequences of avrRxo1 homologs were culled from published and newly generated phytopathogen genomes, revealing that avrRxo1:arc1 modules are rare or frequently inactivated in some species and highly conserved in others. Cloning and functional analysis of avrRxo1 from Acidovorax avenae, A. citrulli, Burkholderia andropogonis, Xanthomonas translucens, and Xanthomonas euvesicatoria showed that some AvrRxo1 homologs share the bacteriostatic and Rxo1-mediated cell death triggering activities of AvrRxo1 from X. oryzae. Additional distant putative homologs of avrRxo1 and arc1 were identified in genomic or metagenomic sequence of environmental bacteria with no known pathogenic role. One of these distant homologs was cloned from the filamentous soil bacterium Cystobacter fuscus. avrRxo1 from C. fuscus caused watersoaking and triggered Rxo1-dependent cell collapse in Nicotiana benthamiana, but no growth suppression in E. coli was observed. This work confirms that a type III effector can function as a TA system toxin, and illustrates the potential of microbiome data to reveal new environmental origins or reservoirs of pathogen virulence factors.
- Congenital infection of mice with toxoplasma gondii induces minimal change in behavior and no change in neurotransmitter concentrationsGoodwin, David G.; Hrubec, Terry C.; Klein, Bradley G.; Strobl, Jeannine S.; Werre, Stephen R.; Han, Qian; Zajac, Anne M.; Lindsay, David S. (American Society of Parasitology, 2012-08-01)We examined the effect of maternal Toxoplasma gondii infection on behavior and the neurotransmitter concentrations of congenitally infected CD-I mice at 4 and 8 wk of age when latent tissue cysts would be present in their brains. Because of sex-associated behavioral changes that develop during aging, infected female mice were compared with control females and infected male mice were compared with control males. Only the short memory behavior (distance between goal box and first hole investigated) of male mice congenitally infected with T. gondii was significantly different (P < 0.05) from that of uninfected control males at both 4 and 8 wk by using the Barnes maze test. The other parameters examined in the latter test, i.e., functional observational battery tests, virtual cliff, visual placement, and activity tests, were not significantly different (P > 0.05) at 4 and 8 wk. Concentrations of neurotransmitters and their metabolites (dopamine; 3,4-dihydroxyphenylacetic acid; homovanillic acid; norepinephrine; epinephrine; 3-methoxy-4-hydroxyphenylglycol; serotonin; and 5-hydroxyindoleacetic acid) in the frontal cortex and striatum were not different (P > 0.05) between infected and control mice at 8 wk of age. The exact mechanism for the observed effect on short-term memory in male mice is not known, and further investigation may help elucidate the molecular mechanisms associated with the proposed link between behavioral changes and T gondil infection in animals. We were not able, however, to confirm the widely held belief that changes in neurotransmitters result from chronic T. gondii infection of the brain.
- Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine DecarboxylaseHan, Qian; Ding, Haizhen; Robinson, Howard; Christensen, Bruce M.; Li, Jianyong (PLOS, 2010-01-21)Background 3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses. Principal Findings In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine. Conclusions The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.
- Current Advances on Structure-Function Relationships of Pyridoxal 5'-Phosphate-Dependent EnzymesLiang, 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.
- Editorial: Aromatic Amino Acid MetabolismHan, Qian; Phillips, Robert S.; Li, Jianyong (Frontiers, 2019-04-10)
- From L-Dopa to Dihydroxyphenylacetaldehyde: A Toxic Biochemical Pathway Plays a Vital Physiological Function in InsectsVavricka, Christopher J.; Han, Qian; Huang, Yongping; Erickson, Sara M.; Harich, Kim; Christensen, Bruce M.; Li, Jianyong (PLOS, 2011-01-24)One protein in Aedes aegypti, classified into the aromatic amino acid decarboxylase (AAAD) family based on extremely high sequence homology (∼70%) with dopa decarboxylase (Ddc), was biochemically investigated. Our data revealed that this predicted AAAD protein use L-dopa as a substrate, as does Ddc, but it catalyzes the production of 3,4-dihydroxylphenylacetaldehyde (DHPAA) directly from L-dopa and apparently has nothing to do with the production of any aromatic amine. The protein is therefore named DHPAA synthase. This subsequently led to the identification of the same enzyme in Drosophila melanogaster, Anopheles gambiae and Culex quinquefasciatus by an initial prediction of putative DHPAA synthase based on sequence homology and subsequent verification of DHPAA synthase identity through protein expression and activity assays. DHPAA is highly toxic because its aldehyde group readily reacts with the primary amino groups of proteins, leading to protein crosslinking and inactivation. It has previously been demonstrated by several research groups that Drosophila DHPAA synthase was expressed in tissues that produce cuticle materials and apparent defects in regions of colorless, flexible cuticular structures have been observed in its gene mutants. The presence of free amino groups in proteins, the high reactivity of DHPAA with the free amino groups, and the genetically ascertained function of the Drosophila DHPAA synthase in the formation of colorless, flexible cuticle, when taken together, suggest that mosquito and Drosophila DHPAA synthases are involved in the formation of flexible cuticle through their reactive DHPAA-mediated protein crosslinking reactions. Our data illustrate how a seemingly highly toxic pathway can serve for an important physiological function in insects.
- Identification of aaNAT5b as a spermine N-acetyltransferase in the mosquito, Aedes aegyptiGuan, 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.
- Select Small Core Structure Carbamates Exhibit High Contact Toxicity to "Carbamate-Resistant" Strain Malaria Mosquitoes, Anopheles gambiae (Akron)Wong, Dawn M.; Li, Jianyong; Chen, Qiao-Hong; Han, Qian; Mutunga, James M.; Wysinski, Ania; Anderson, Troy D.; Ding, Haizhen; Carpenetti, Tiffany L.; Verma, Astha; Islam, Rafique; Paulson, Sally L.; Lam, Polo Chun Hung; Totrov, Maxim M.; Bloomquist, Jeffrey R.; Carlier, Paul R. (PLOS, 2012-10-01)Acetylcholinesterase (AChE) is a proven target for control of the malaria mosquito (Anopheles gambiae). Unfortunately, a single amino acid mutation (G119S) in An. gambiae AChE-1 (AgAChE) confers resistance to the AChE inhibitors currently approved by the World Health Organization for indoor residual spraying. In this report, we describe several carbamate inhibitors that potently inhibit G119S AgAChE and that are contact-toxic to carbamate-resistant An. gambiae. PCR-RFLP analysis was used to confirm that carbamate-susceptible G3 and carbamate-resistant Akron strains of An. gambiae carry wild-type (WT) and G119S AChE, respectively. G119S AgAChE was expressed and purified for the first time, and was shown to have only 3% of the turnover number (kcat) of the WT enzyme. Twelve carbamates were then assayed for inhibition of these enzymes. High resistance ratios (>2,500-fold) were observed for carbamates bearing a benzene ring core, consistent with the carbamate-resistant phenotype of the G119S enzyme. Interestingly, resistance ratios for two oxime methylcarbamates, and for five pyrazol-4-yl methylcarbamates were found to be much lower (4- to 65-fold). The toxicities of these carbamates to live G3 and Akron strain An. gambiae were determined. As expected from the enzyme resistance ratios, carbamates bearing a benzene ring core showed low toxicity to Akron strain An. gambiae (LC50>5,000 μg/mL). However, one oxime methylcarbamate (aldicarb) and five pyrazol-4-yl methylcarbamates (4a–e) showed good to excellent toxicity to the Akron strain (LC50 = 32–650 μg/mL). These results suggest that appropriately functionalized “small-core” carbamates could function as a resistance-breaking anticholinesterase insecticides against the malaria mosquito.
- Thermal stability, pH dependence and inhibition of four murine kynurenine aminotransferasesHan, Qian; Cai, Tao; Tagle, Danilo A.; Li, Jianyong (2010-05-19)Background Kynurenine aminotransferase (KAT) catalyzes the transamination of kynunrenine to kynurenic acid (KYNA). KYNA is a neuroactive compound and functions as an antagonist of alpha7-nicotinic acetylcholine receptors and is the only known endogenous antagonist of N-methyl-D-aspartate receptors. Four KAT enzymes, KAT I/glutamine transaminase K/cysteine conjugate beta-lyase 1, KAT II/aminoadipate aminotransferase, KAT III/cysteine conjugate beta-lyase 2, and KAT IV/glutamic-oxaloacetic transaminase 2/mitochondrial aspartate aminotransferase, have been reported in mammalian brains. Because of the substrate overlap of the four KAT enzymes, it is difficult to assay the specific activity of each KAT in animal brains. Results This study concerns the functional expression and comparative characterization of KAT I, II, III, and IV from mice. At the applied test conditions, equimolar tryptophan with kynurenine significantly inhibited only mouse KAT I and IV, equimolar methionine inhibited only mouse KAT III and equimolar aspartate inhibited only mouse KAT IV. The activity of mouse KAT II was not significantly inhibited by any proteinogenic amino acids at equimolar concentrations. pH optima, temperature preferences of four KATs were also tested in this study. Midpoint temperatures of the protein melting, half life values at 65°C, and pKa values of mouse KAT I, II, III, and IV were 69.8, 65.9, 64.8 and 66.5°C; 69.7, 27.4, 3.9 and 6.5 min; pH 7.6, 5.7, 8.7 and 6.9, respectively. Conclusion The characteristics reported here could be used to develop specific assay methods for each of the four murine KATs. These specific assays could be used to identify which KAT is affected in mouse models for research and to develop small molecule drugs for prevention and treatment of KAT-involved human diseases.