The striking identification of an apparent proteasome core in Mycobacteria and allied actinomycetes suggested that additional elements of this otherwise strictly eukaryotic system for regulated protein degradation might be conserved. The genes encoding this prokaryotic proteasome are clustered in an operon with a short open reading frame that encodes a small protein of 64 amino acids resembling ubiquitin with a carboxylterminal di-glycine-glutamine motif (herein called Pup for prokaryotic ubiquitin-like protein). Expression of a polyhistidinetagged Pup followed by pulldown revealed that a broad spectrum of proteins were post-translationally modified by Pup. Two-dimensional gel electrophoresis allowed us to conclusively identify two targets of this modification as myoinositol-1-phosphate synthase and superoxide dismutase. Deletion of the penultimate di-glycine motif or the terminal glutamine completely abrogated modification of cellular proteins with Pup. Further mass spectral analysis demonstrated that Pup was attached to a lysine residue on its target protein via the carboxylterminal glutamine with deamidation of this residue. Finally, we showed that cell lysates of wild type (but not a proteasome mutant) efficiently degraded Pup-modified proteins. These data therefore establish that, despite differences in both sequence and target linkage, Pup plays an analogous role to ubiquitin in targeting proteins to the proteasome for degradation.

The substrates for Bacillus subtilis PLP synthase (YaaD and YaaE) are identified, and the first reconstitution of PLP biosynthesis using this pathway is described. Three partial reactions catalyzed by YaaD are also identified.

Summary Ubiquitin (Ub) provides the recognition and specificity required to deliver proteins to the eukaryotic proteasome for destruction. Prokaryotic ubiquitin-like protein (Pup) is functionally analogous to Ub in Mycobacterium tuberculosis (Mtb) as it dooms proteins to the Mtb proteasome. Studies suggest that Pup and Ub do not share similar mechanisms of activation and conjugation to target proteins. Dop (deamidase of Pup; Mtb Rv2112c/MT2172) deamidates the carboxyl-terminal glutamine of Pup to glutamate, preparing it for ligation to target proteins by proteasome accessory factor A (PafA). While studies have shed light on the conjugation of Pup to proteins, it was not known if Pup could be removed from substrates in a manner analogous to the deconjugation of Ub from eukaryotic proteins. Here, we show that Mycobacteria have a “depupylase” activity provided by Dop. The discovery of a depupylase strengthens the parallels between the Pup and Ub tagging systems of prokaryotes and eukaryotes, respectively.

Pyridoxal 5'-phosphate (PLP) is the biologically active form of vitamin B 6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino-sugars. The PLP synthase holoenzyme consists of two subunits: YaaD catalyzes the condensation of ribulose 5-phosphate, glyceraldehyde-3-phosphate and ammonia and YaaE catalyzes the production of ammonia from glutamine. Here we describe the structure of the PLP synthase complex (YaaD-YaaE) from Thermotoga maritima at 2.9 Å resolution. This complex consists of a core of 12 YaaD monomers with 12 noninteracting YaaE monomers attached to the core. Compared to the previously published structure of PdxS (a YaaD ortholog in Geobacillus stearothermophilus), the N-terminus (1-18), which includes helix α0, the β2-α2 loop(46-56), which includes new helix α2a, and the C-terminus (270-280) of YaaD, are ordered in the complex but disordered in PdxS. A ribulose 5-phosphate is bound to YaaD via an imine with Lys82. Previous studies have demonstrated a similar imine at Lys149 and not at Lys81 (equivalent to Lys150 and 82 in T. maritima) for the Bacillus subtilis enzyme suggesting the possibility that two separate sites on YaaD are involved in PLP formation. A phosphate from the crystallization solution is found bound to YaaD and also serves as a marker for a possible second active site. An ammonia channel that connects the active site of YaaE with the ribulose 5-phosphate binding site was identified. This channel is similar to one found in imidazole glycerol phosphate synthase; however, when the β-barrels of the two complexes are superimposed, the glutaminase domains are rotated by about 180°w ith respect to each other.Pyridoxal 5'-phosphate (4, PLP) is the biologically active form of vitamin B 6 and is an important cofactor for several of the enzymes involved in the metabolism of amine-containing natural products such as amino acids and amino-sugars (1-4). There are two distinct PLP biosynthetic pathways that have not yet been found to coexist in the same organism (5). The Escherichia coli pathway has been extensively studied (6)(7)(8)(9)(10)(11)(12). In this pathway, PdxJ catalyzes the formation of pyridoxine 5'-phosphate (3, PNP) from 3-phosphohydroxy-1-aminoacetone 2 and 1-deoxy-D-xyulose 5-phosphate 1. This is then oxidized to PLP 4 by PdxH (Scheme 1).In the alternative pathway, PLP is formed from ribose 5-phosphate (5, R5P), glyceraldehyde 3-phosphate (6, G3P) and ammonia formed by the hydrolysis of glutamine (13-18) (Scheme 1 METHODS AND MATERIALS Molecular CloningStandard methods were used for DNA restriction endonuclease digestion, ligation and transformation of DNA. Genomic DNA and plasmid DNA were purified with a Wizard Plus SV genomic DNA kit and a DNA Miniprep kit (Promega), respectively. DNA fragments were separated by agarose gel electrophoresis, excised and purified with the QiaExII (Qiagen). E. coli strain DH5 α was used as a recipient for transformation during plasmid construction ...