Substrate Recognition by the Hetero-Octameric ATP Phosphoribosyltransferase from Lactococcus lactis

Abstract

Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms, and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisG S catalytic subunits related to periplasmic binding proteins, and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs, and among phosphoribosyltransferases in general, we determined the steady state kinetics for the heterooctameric form, and characterized the active site by mutagenesis. The K m PRPP (18.4 ± 3.5 μM) and k cat (2.7 ± 0.3 sec −1 ) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the K m for ATP (2.7 ± 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be non-competitive (K i = 81.1 μM) and competitive (K i = 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments investigating the side chains recognizing PRPP showed that 5′ phosphate contacts (T159A and T162A) had the largest (25-and 155-fold) decreases in k cat /K m , while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety, or contacts to the 2′ OH. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters, and employ mechanistic strategies reminiscent of the broader PRT superfamily.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 † This work was supported by N.I.H. grant GM54899 (CSF) histidine biosynthesis is genetic regulation of the expression of histidine biosynthetic enzymes, by use of the classic attenuation mechanism (reviewed in (5)).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 cofac...

Independent catalysis of the short form HisG from Lactococcus lactis

“…Previous work suggested that LlaHisG S is inactive without LlaHisZ [3]. By performing in vitro enzyme kinetics assays with a LlaHisG S concentration greater than previously reported [15], we detected phosphoribosyltransferase activity of LlaHisG S in vitro. ATP-PRT activity was also observed in vivo, as an E. coli hisG knockout strain transformed with a plasmid encoding LlaHisG S was able to proliferate in the absence of an exogenous histidine source.…”

“…histidine [12,13,15]. Despite the structural similarity between a single chain of HisG S and HisG L , the short form and long form ATP-PRTs differ in their quaternary structure.…”

Phosphoribosyl Diphosphate (PRPP): Biosynthesis, Enzymology, Utilization, and Metabolic Significance

“…PRPP-binding loops have been identified in ATP phosphoribosyltransferases of L. lactis (148-Gly-Leu-Ala-Asp-Ala-Ile-Val-Asp-IleVal-Glu-Thr-Gly-Asn-Thr-162) (281) and of the hyperthermophilic bacterium T. maritima (140-Gly-Leu-Ser-Asp-Leu-Ile-Val-Asp-Ile-Thr-Glu-Thr-Gly-Arg-Thr-155) (282). Mutations that replaced Thr159 or Thr162 of L. lactis ATP phosphoribosyltransferase confirmed the importance of this sequence in PRPP binding (283). (Fig.…”

Biochemical Characterization of Quinolinic Acid Phosphoribosyltransferase from Mycobacterium tuberculosis H37Rv and Inhibition of Its Activity by Pyrazinamide

“…PRTases are classified into four subclasses (types I, II, III, and IV) according to their structures [15], [22]–[26]. QAPRTase is a type II PRTase that participates in the de novo pathway of the pyridine coenzyme NAD [7], [15].…”