High-Resolution Crystal Structure and Redox Properties of Chloroplastic Triosephosphate Isomerase from Chlamydomonas reinhardtii

Triosephosphate isomerase （TPI） catalyzes the interconversion of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate. Photosynthetic organisms generally contain two isoforms of TPI located in both cytoplasm and chloroplasts. While the cytoplasmic TPI is involved in the glycolysis, the chloroplastic isoform participates in the Calvin-Benson cycle, a key photosynthetic process responsible for carbon fixation. Compared with its cytoplasmic counterpart, the functional features of chloroplastic TPI have been poorly investigated and its three-dimensional structure has not been solved. Recently, several studies proposed TPI as a potential target of different redox modifications including dithiol/disulfide interchanges, glutathionylation, and nitrosylation. However, neither the effects on protein activity nor the molecular mechanisms underlying these redox modifications have been investigated. Here, we have produced recombinantly and purified TPI from the unicellular green alga Chlamydomonas reinhardtii （Cr）. The biochemical properties of the enzyme were delineated and its crystallographic structure was determined at a resolution of 1.1 A. CrTPI is a homodimer with subunits containing the typical （β/α）8-barrel fold. Although no evidence for TRX regulation was obtained, CrTPI was found to undergo glutathionylation by oxidized glutathione and trans-nitrosylation by nitrosoglutathione, confirming its sensitivity to multiple redox modifications.

Molecular epidemiology of Giardia duodenalis infection in humans in Southern Ethiopia:a triosephosphate isomerase gene-targeted analysis

Background:Giardia duodenalis is a species complex consisting of multiple genetically distinct assemblages.The species imposes a major public health crisis on developing countries.However,the molecular diversity,transmission dynamics and risk factors of the species in these countries are indeterminate.This study was conducted to determine the molecular epidemiology of G.duodenalis infection in asymptomatic individuals in Southern Ethiopia.Methods:From March to June 2014,fresh stool samples were collected from 590 randomly selected individuals.Socio-demographic data were gathered using a pre-tested structured questionnaire.The genotyping was done using triosephosphate isomerase gene-based nested polymerase chain reaction and DNA sequencing.The genetic identity and relatedness of isolates were determined using the basic local alignment search tool and phylogenetic analysis.Risk factors associated with G.duodenalis infection were analysed using binary and multinomial logistic regression models.Results:The results showed that 18.1%(92/509)of the study subjects were infected by G.duodenalis.Among the isolates,35.9%(33/92)and 21.7%(20/92)were sub-typed into assemblages A and B,respectively,whereas 42.4%(39/92)showed mixed infections of A and B.Most of the assemblage A isolates(94%,31/33)were 100%identical to sequences registered in GenBank,of which the majority belonged to sub-assemblage AII.However,the high genetic variability and frequency of double peaks made sub-genotyping of assemblage B more problematic and only 20%(4/20)of the isolates matched 100%with the sequences.The risk factors of age(P=0.032)and type of drinking water source(P=0.003)both showed a significant association with the occurrence G.duodenalis infection.Conclusions:This study established the endemicity of G.duodenalis in Southern Ethiopia.Infection with assemblage A was more frequent than with assemblage B,and the rate of infection was higher in children and in municipal/tap and open spring water consumers than the other groups.Sub-typing of assemblage B and determining the origin of double peaks were challenging.The present study confirms the need for further inclusive studies to be conducted focusing on sub-types of assemblage B and the origin of heterogeneity.

cDNA Cloning and Bioinformatic Prediction of TPI Gene from Apis mellifera

[Objective] The aim was to clone triosephosphate isomerase （TPI） gene from Apis mellifera, and predict the properties of TPI protein with bioinformatic meth- ods. [Method] The TPI gene was firstly cloned by in silico cloning based on the ex- pressed sequence tags （ESTs） from Unigene of NCBI. Some characters of the TPI protein including hydrophobicity or hydrophilicity, isoelectric point （pl） and secondary structure were analyzed and predicted by the tools of bioinformatics. [Result] The TPI gene from A. mellifera was 1 768 bp in full length and it contained a complete ORF which encoded 247 amino acids; the pl of TPI protein was 8.515; the TPI protein was a member of ~13-fold family. [Conclusion] The in silico cloning based on the expressed sequence tags is a efficient method in practice, and this study will provide more references for further study on A. mellifera at molecular level.