Crystal Structure of the Reaction Complex of 3-Deoxy-d-arabino-heptulosonate-7-phosphate Synthase from Thermotoga maritima Refines the Catalytic Mechanism and Indicates a New Mechanism of Allosteric Regulation

Abstract

3-Deoxy-d-arabino-heptulosonate-7-phosphate synthase (DAHPS) catalyzes the first reaction of the aromatic biosynthetic pathway in bacteria, fungi, and plants, the condensation of phosphoenolpyruvate (PEP) and d-erythrose-4-phosphate (E4P) with the formation of DAHP. Crystals of DAHPS from Thermotoga maritima (DAHPSTm) were grown in the presence of PEP and metal cofactor, Cd2+, and then soaked with E4P at 4 °C where the catalytic activity of the enzyme is negligible. The crystal structure of the “frozen” reaction complex was determined at 2.2Å resolution. The subunit of the DAHPSTm homotetramer consists of an N-terminal ferredoxin-like (FL) domain and a (β/α)8-barrel domain. The active site located at the C-end of the barrel contains Cd2+, PEP, and E4P, the latter bound in a non-productive conformation. The productive conformation of E4P is suggested and a catalytic mechanism of DAHPS is proposed. The active site of DAHPSTm is nearly identical to the active sites of the other two known DAHPS structures from Escherichia coli (DAHPSEc) and Saccharomyces cerevisiae (DAHPSSc). However, the secondary, tertiary, and quaternary structures of DAHPSTm are more similar to the functionally related enzyme, 3-deoxy-d-manno-octulosonate-8-phosphate synthase (KDOPS) from E.coli and Aquiflex aeolicus, than to DAHPSEc and DAHPSSc. Although DAHPSTm is feedback-regulated by tyrosine and phenylalanine, it lacks the extra barrel segments that are required for feedback inhibition in DAHPSEc and DAHPSSc. A sequence similarity search revealed that DAHPSs of phylogenetic family Iβ possess a FL domain like DAHPSTm while those of family Iα have extra barrel segments similar to those of DAHPSEc and DAHPSSc. This indicates that the mechanism of feedback regulation in DAHPSTm and other family Iβ enzymes is different from that of family Iα enzymes, most likely being mediated by the FL domain.