Developing a microbial chassis with efficient enzymes is key to the synthesis of products by metabolic engineering. The wide distribution of desired pathway enzymes across several species and categories is posing major challenges in screening and selection of the same for pathway reconstruction. One such key enzyme is isopropylmalate isomerase (IPMI) of leucine/isoleucine biosynthetic pathway. The enzymes reported earlier as citraconase and maleate hydratase in Arthrobacter sp. and Pseudomonas sp. respectively, were found to have the characteristics of IPMI. If a systematic study is undertaken to show that these orphan enzymes indeed are part of the aconitase family of enzymes, these reported ones will add to t... More
Developing a microbial chassis with efficient enzymes is key to the synthesis of products by metabolic engineering. The wide distribution of desired pathway enzymes across several species and categories is posing major challenges in screening and selection of the same for pathway reconstruction. One such key enzyme is isopropylmalate isomerase (IPMI) of leucine/isoleucine biosynthetic pathway. The enzymes reported earlier as citraconase and maleate hydratase in Arthrobacter sp. and Pseudomonas sp. respectively, were found to have the characteristics of IPMI. If a systematic study is undertaken to show that these orphan enzymes indeed are part of the aconitase family of enzymes, these reported ones will add to the repertoire of enzymes available for branch-chained amino acid pathway engineering. This work is focused on functional characterisation of the enzymes citraconase and maleate hydratase based on the properties of IPMI. The partially sequenced gene of maleate hydratase reported earlier served as a template to identify the respective genes in these organisms which is found to be that of IPMI with conserved regions in the active site. The native enzymes and the IPMI of A. globiformis and P. pseudoalcaligenes, expressed in E. coli acted upon all the substrates in the forward direction comprising of D-citramalate, citraconate & D-erythro-3-methylmalate. In the reverse direction all the enzymes converted citraconate to D-citramalate with high activity. The estimated equilibrium ratio was same for both the native enzyme and the over-expressed IPMI which is 96:1.5:2.5 for D-citramalate: citraconate: D-erythro-3-methylmalate. The iron requirement for both enzymes which is characteristic of IPMI is ascertained by chelation and reconstitution of the same. Therefore, this work elucidated the broad specificity and the reactions in equilibrium catalysed by these enzymes like that of IPMI, paving way for the integration of these two efficient candidates into aconitase family of enzymes facilitating pathway engineering.
