Abstract
3-Dehydroquinate dehydratase (DHQase) catalyzes the conversion of 3-dehydroquinic acid to 3-dehydroshikimic acid of the shikimate pathway. In this study, 3180 prokaryotic genomes were examined and 459 DHQase sequences were retrieved. Based on sequence analysis and their original hosts, 38 DHQase genes were selected for chemical synthesis. The selected DHQases were translated into new DNA sequences according to the genetic codon usage bias by both Escherichia coli and Corynebacterium glutamicum. The new DNA sequences were customized for synthetic biological applications by adding Biobrick adapters at both ends and by removal of any related restriction endonuclease sites. The customized DHQase genes were successfully expressed in E. coli, and functional DHQases were obtained. Kinetic parameters of Km, kcat, and Vmax of DHQases were determined with a newly established high-throughput method for DHQase activity assay. Results showed that DHQases possessed broad strength of substrate affinities and catalytic capacities. In addition to the DHQase kinetic diversities, this study generated a DHQase library with known catalytic constants that could be applied to design artificial modules of shikimate pathway for metabolic engineering and synthetic biology.
Highlights
Shikimate pathway widely exists in microbes and plants, but not animals
Genome data-mining for DHQase genes The amino acid sequences of the type II DHQase (NP_599670) from Corynebacterium glutamicum ATCC13032 and of the type I DHQase (NP_416208) from E. coli K-12 were used as seed sequences to retrieve putative DHQase sequences from NCBI genome database with a filter condition of threshold E value ≤ 10−10
Kinetic diversity of enzymes is the results of natural revolution in life, similar to their phylogenetic diversity (Zhi et al 2014)
Summary
Shikimate pathway widely exists in microbes and plants, but not animals. This pathway is involved in the synthesis of aromatic amino acids, vitamins, as well as lignin (Herrmann and Weaver 1999; Vanholme et al 2012). Many investigations of DHQases have been focused on their structures and catalytic mechanisms (Blomberg et al 2009; Bottomley et al 1996; Devi et al 2013; Lee et al 2002; Pan et al 2012; Roszak et al 2002), or on structure-based design of inhibitors to DHQase activity (Blanco et al 2012; 2014; Dias et al 2011; Peon et al 2010). These investigations have generated increasing numbers of DHQase structures with high resolution and have significantly advanced the understanding of DHQase catalytic mechanisms
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