Structure of the Allosteric Regulatory Enzyme of Purine Biosynthesis

Abstract

Multi-wavelength anomalous diffraction (MAD) has been used to determine the structure of the regulatory enzyme of de novo synthesis of purine nucleotides, glutamine 5-phosphoribosyl-1-pyrophosphate (PRPP) amidotransferase, from Bacillus subtilis. This allosteric enzyme, a 200-kilodalton tetramer, is subject to end product regulation by purine nucleotides. The metalloenzyme from B. subtilis is a paradigm for the higher eukaryotic enzymes, which have been refractory to isolation in stable form. The two folding domains of the polypeptide are correlated with functional domains for glutamine binding and for transfer of ammonia to the substrate PRPP. Eight molecules of the feedback inhibitor adenosine monophosphate (AMP) are bound to the tetrameric enzyme in two types of binding sites: the PRPP catalytic site of each subunit and an unusual regulatory site that is immediately adjacent to each active site but is between subunits. An oxygen-sensitive [4Fe-4S] cluster in each subunit is proposed to regulate protein turnover in vivo and is distant from the catalytic site. Oxygen sensitivity of the cluster is diminished by AMP, which blocks a channel through the protein to the cluster. The structure is representative of both glutamine amidotransferases and phosphoribosyltransferases.

The quorum-quenching N -acyl homoserine lactone acylase PvdQ is an Ntn-hydrolase with an unusual substrate-binding pocket

“…While their catalytic mechanism and the amino acid residues surrounding the N-terminal nucleophile are strictly conserved, their substrate specificity has been shown to be quite broad, ranging from small hydrophilic or hydrophobic compounds to large proteins. Consequently, Ntn-hydrolases are utilized in a wide variety of cellular processes from protein degradation (26) to nucleotide biosynthesis (34). The discovery of PvdQ introduces yet another important role for Ntn-hydrolases, the hydrolysis of long acyl amides involved in quorum sensing.…”

Crystal structure of a bifunctional aldolase–dehydrogenase: Sequestering a reactive and volatile intermediate

“…Channeling processes are particularly advantageous over the free diffusion of reaction products through the bulk solvent because they can protect chemically labile intermediates from breakdown, prevent loss of nonpolar intermediates by diffusion across cell membranes, or protect the cell from toxic intermediates. Crystallographic studies on a number of different enzyme systems involved in substrate channeling (3)(4)(5)(6)(7)(8)(9)(10)(11) have revealed important structural factors that mediate intersubunit or interdomain communication and facilitate the efficient transfer of intermediates between distant active sites.…”

Biochemical and structural characterization ofKlebsiella pneumoniaeoxamate amidohydrolase in the uric acid degradation pathway

“…GGT is a relatively recent addition to the Ntn-hydrolase superfamily (Suzuki & Kumagai, 2002). Many members of the Ntn-hydrolase superfamily have been structurally characterized, including B. subtilis glutamine 5-phosphoribosyl-1-pyrophosphate amidotransferase (Smith et al, 1994), E. coli penicillin G acylases and the E. coli, H. pylori and B. subtilis GGTs (Boanca et al, 2007;Okada et al, 2006;Wada et al, 2010). BLAST results suggested that HpxW is a member of the GGT family, which has modest sequence identity among its members ($30%; Suzuki et al, 1989).…”