Molecular Evolution Of Threonine Dehydratase Bacteria

There are usually two types of threonine dehydratase (TD) in bacteria: the biosyntheticthreonine dehydratase (BTD) and the catabolic threonine dehydratase (CTD). They bothconvert L-threonine to2-ketobutyrate, while having the different structures, properties, andparticipating in different pathways. There are also two types of BTD: BTD1and BTD2. Theytake part in the same pathway, but have different structures and properties. The similarfunction among CTDs, BTD1s and BTD2s reveals there exists revolutionary relationshipamong them, while the diversity of structures, properties and pathways shows they havedifferent roles on bacteria adapting the environment. So far, numbers of work have been doneto study the structures and properties of CTDs, BTD1s and BTD2s, but their evolutionaryrelationships, pathways and promotors are still not clear. In this work, the revolutionaryrelationships, pathways and possible promotors of the three different TDs were analyzed bybioinformatics and molecular biological methods, and a possible revolutionary model for TDswas proposed. Furthermore, the ACT-like subdomains of BTDs were directly removed toresist the feedback inhibition of isoleucine. Our research values in both theoretical andpractical applications. The main achievements are listed below:(1) Sequential features of CTDs from328species and BTDs from546species werecompared, and structural features of StCTD (2GN2) and EcBTD2(1TDJ) were aligned. Theresults revealed both sequential and structural features of CTDs and BTDs were accordant,which implied that though CTDs and BTDs participated in different pathways, they were notthe analogous proteins, but the homologous proteins from the same ancestor.(2)82strains from Proteobacteria, Firmicutes and Actinobacteria were selected and thephylogenetic tree of TDs protein sequences was constructed. The tree revealed CTDs andBTD2s were both distributed within only one clusters respectively, while BTD1s spreaded intwo clusters (BTD1-A and BTD1-B), and the cluster of BTD1-A was very close to that ofBTD2. The results indicated genes coding for BTD1s duplicated and BTD1-As were muchcloser to BTD2s than BTD1-Bs.(3) Phylogenetic tree by the16s rDNAsequences of the82genomes was constructed, andthe distributions of CTDs, BTD1s and BTD2s were analyzed. CTDs widely spreaded amongthese three phyla, and BTD1s also widely spreaded among the three phyla exceptγ-proteobacteria. Only8genomes had both BTD1-A and BTD1-B, while others onlycontained one of them. Besides, BTD2s only existed in Proteobacteria. These results showedCTDs might appear earliest, and the deletion event should happen after the duplication ofBTD1coding genes, and BTD2s were generated latest within Proteobacteria.(4)3CTDs,2BTD1-As,2BTD1-Bs and2BTD2s were overexpressed within Escherichiacoli, and9TD expression strains were constructed to study the promoters of TD evolution.9TDs from E. coli were purified and their activities and stabilities under different pHs andtemperatures were compared. BTD1s were more stable than CTDs and BTD2s, and theactivities of BTD2s were highest. These results declared that ACT-like subdomains influencedthe stabilities and flexibilities of TDs, which possibly promoted the evolution of TDs. (5) ACT-like subdomain of BTD1from Corynebacterium glutamicum and BTD2from E.coli were removed, and2recombinant expression strains were constructed. The2mutantenzymes were purified from E. coli, and the activities and feedback resistant abilities of themwere assayed. The activities of mutant enzymes were decreased, while they were no longerinhibited by isoleucine, which demonstrated that the deletion of ACT-like subdomain could letBTD1s free from the inhibition of isoleucine.