Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis.

ATP phosphoribosyltransferase (ATP-PRT; E. C. 2.4.2.17) catalyzes the first and highly regulated step of histidine biosynthesis, which involves nucleophillic attack of the N1 of ATP on C1′ of 5-phosphoribosyl- 1 pyrophosphate (PRPP) to form N-1-(5′-phosphoribosyl)-ATP (PR-ATP, Figure 1A) (1). The resulting product is converted through an additional nine reactions into histidine by a pathway that is regulated both by its end product and by AMP and ADP (2). The tight regulation of this pathway (reviewed in (3)), reflects the high energetic costs associated with histidine synthesis, and the need to dramatically slow pathway flux when histidine is present in the growth media (4). Superimposed on top of metabolic control of histidine biosynthesis is genetic regulation of the expression of histidine biosynthetic enzymes, by use of the classic attenuation mechanism (reviewed in (5)). Figure 1 The ATP phosphoribosyltransferase reaction catalyzed by the hetero-octameric ATP PRT ATP-PRT is a member of the phophoribosyltransferase (PRT) superfamily of enzymes, all of whom share a common chemistry that involves transfer of the phosphoribosyl group to a nucleotide base or, in the case of glutamine phosphoribosyl pyrophosphate amidotransferase, to free ammonia generated on the enzyme (6). PRTs are found in many essential pathways, including biosynthesis and salvage of nucleotides, and the synthesis of cofactors and amino acids (7). There are at least four different structurally unrelated subfamilies of PRTs. Type I PRTs, which include the adenine, orotate (OPRT), and hypoxanthine/guanine PRTs (HGPRT), are composed of four different domains, including a core region that binds both the PRPP and the nucleotide substrate, a hood domain that recognizes the nucleotide substrate, a flexible loop that closes over PRPP, and a C-terminal arm that provides dimerization for some PRTs (8). Considerable research attention has focused on the type I PRTs, as the inherited Lesch/Nyan syndrome and orotic aciduria involve respective defects in the HPRT and orotate PRTs (7). Other type I PRTs represent targets for therapeutics directed against malaria, Chagas disease, and other pathogenic eukaryotes. Notably, type I PRTs share the PRPP binding motif, a thirteen residue signature sequence that is important for substrate recognition (9). The type II and type III enzymes are represented by quinolinate (QPRT) and anthranilate PRT, respectively, which participate in NAD+ and tryptophan biosynthesis (10, 11). ATP-PRTs represent a fourth class of PRTs, and comprise two different subfamilies with distinctly different quaternary structures. The hexameric ATP PRTs, referred to as HisGL or long form, are found in enteric bacteria and lower eukaryotes (12). Each HisGL subunit is composed of three domains (13, 14). The first two constitute a mixed α/β bilobal catalytic domain that resembles proteins of the periplasmic binding protein family (15), while the third domain provides a binding site to allow feedback inhibition by histidine (13). The two different HisGL structures available include a histidine/AMP inhibited form from Mycobacterium tuberculosis (13), and a enzyme product complex between PR-ATP and the enzyme from Escherichia coli (14). Enzymes from the second ATP-PRT family are hetero-octamers, composed of four HisGS subunits and four HisZ regulatory subunits (Figure 1B) (16, 17). The latter are structurally related to the catalytic domain of histidyl-tRNA synthetase, and provide a regulatory domain to compensate for the absence of the C-terminal domain found in HisGL. Notably, both subunits are required to form a functional complex (17). Two structures of the hetero-octameric ATP-PRT have been reported, a histidine-inhibited complex of the enzyme from Thermatoga maritima (18), and ATP-activated and PRPP-bound forms of the enzyme from Lactococcus lactis (19). The sequence conservation between HisGS and HisGL indicates a conserved active site, and suggests a similarly conserved reaction mechanism (Figure 2). Early work characterizing the steady state kinetics of the long form indicated that the reaction proceeds by an ordered Bi-Bi mechanism with ATP leading (20). Another study involving determination of kinetic isotope effects of the HisGL enzyme provided evidence that the ATP-PRT transition state has ribo-oxocarbenium character (21). In contrast, the kinetic properties of the hetero-octameric form have not yet been characterized. As part of efforts to compare the structure and function of the hexameric and hetero-octameric versions of ATP-PRT, we report here the steady state kinetics of the hetero-octameric HisG/HisZ enzyme from Lactococcus lactis, and a mutational analysis of the contacts between PRPP and the ATP-PRT based on the structure of the activated form of the complex (19). Analysis of the resulting mutant proteins highlights the importance of active site contacts with PRPP on ground state binding, as well as the interdependence of the recognition of PRPP, ATP and the inhibitor histidine. Figure 2 Multiple sequence alignment of representative HisGS and HisGL sequences, overlaid with the secondary structure assignments from L. lactis HisGS