Microbial transcription modulator NusG interacts with RNA polymerase and termination factor rho, displaying striking functional homology to eukaryotic Spt5. The protein is also a translational regulator. We have determined crystal structures of Aquifex aeolicus NusG showing a modular design: an N-terminal RNP-like domain, a C-terminal element with a KOW sequence motif and a species-specific immunoglobulin-like fold. The structures reveal bona fide nucleic acid binding sites, and nucleic acid binding activities can be detected for NusG from three organisms and for the KOW element alone. A conserved KOW domain is defined as a new class of nucleic acid binding folds. This module is a close structural homolog of tudor protein-protein interaction motifs. Putative protein binding sites for the RNP and KOW domains can be deduced, which differ from the areas implicated in nucleic acid interactions. The results strongly argue that both protein and nucleic acid contacts are important for NusG's functions and that the factor can act as an adaptor mediating indirect protein-nucleic acid associations.

The gene encoding human cystathionine ␥-lyase was cloned from total cellular Hep G2 RNA. Fusion to a T7 promoter allowed expression in Escherichia coli, representing the first mammalian cystathionine ␥-lyase overproduced in a bacterial system. About 90% of the heterologous gene product was insoluble, and renaturation experiments from purified inclusion bodies met with limited success. About 5 mg/liter culture of human cystathionine ␥-lyase could also be extracted from the soluble lysis fraction, employing a three-step native procedure. While the enzyme showed high ␥-lyase activity toward L-cystathionine (K m ‫؍‬ 0.5 mM, V max ‫؍‬ 2.5 units/ mg) with an optimum pH of 8.2, no residual cystathionine ␤-lyase behavior and only marginal reactivity toward L-cystine and L-cysteine were detected. Inhibition studies were performed with the mechanism-based inactivators propargylglycine, trifluoroalanine, and aminoethoxyvinylglycine. Propargylglycine inactivated human cystathionine ␥-lyase much more strongly than trifluoroalanine, in agreement with the enzyme's preference for C-␥-S bonds. Aminoethoxyvinylglycine showed slow and tight binding characteristics with a K i of 10.5 M, comparable with its effect on cystathionine ␤-lyase. The results have important implications for the design of specific inhibitors for transsulfuration components.Transsulfuration and reverse transsulfuration constitute part of the metabolic interconversion of the sulfur-containing amino acids cysteine and methionine (Fig. 1). The forward pathway, the transformation of cysteine into homocysteine via the intermediate L-cystathionine is catalyzed by the sequential action of the enzymes cystathionine ␤-lyase (CBL) 1 and cystathionine ␥-synthase (CGS) and has been identified in bacteria, fungi, and plants. Conversely, reverse transsulfuration, catalyzed by the enzymes cystathionine ␤-synthase and cystathionine ␥-lyase (CGL), is known only in fungi and mammals (1, 2). Actinomycetes species present a notable exception to this rule (1). The four enzymatic transsulfuration components are all pyridoxal 5Ј-phosphate (PLP)-dependent enzymes, but they pertain to different structural groups; CBL, CGS, and CGL show extensive sequence homology and are members of the PLP ␥-family (Ref. 3; Fig. 2), while cystathionine ␤-synthase is unrelated and belongs to the ␤-family.The high resolution crystal structures of Escherichia coli CBL (4) and CGS (5), together with crystallographic (6) and kinetic investigations (6 -9) on inhibitors, allowed the suggestion and evaluation of reaction mechanisms (4, 10). Both CBL and CGS are homotetramers composed of ϳ40 -45-kDa subunits and carry one PLP cofactor per monomer covalently bound via a Schiff base to an active site lysine. A similar situation has been found for CGL (1,11,12). In the present paper, we extend our structure-function analyses to human CGL (EC 4.4

Threonine synthase catalyzes the final step of threonine biosynthesis, the pyridoxal 5-phosphate (PLP)-dependent conversion of O-phosphohomoserine into threonine and inorganic phosphate. Threonine is an essential nutrient for mammals, and its biosynthetic machinery is restricted to bacteria, plants, and fungi; therefore, threonine synthase represents an interesting pharmaceutical target. The crystal structure of threonine synthase from Saccharomyces cerevisiae has been solved at 2.7 Å resolution using multiwavelength anomalous diffraction. The structure reveals a monomer as active unit, which is subdivided into three distinct domains: a small N-terminal domain, a PLP-binding domain that covalently anchors the cofactor and a so-called large domain, which contains the main of the protein body. All three domains show the typical open ␣/␤ architecture. The cofactor is bound at the interface of all three domains, buried deeply within a wide canyon that penetrates the whole molecule. Based on structural alignments with related enzymes, an enzyme-substrate complex was modeled into the active site of yeast threonine synthase, which revealed essentials for substrate binding and catalysis. Furthermore, the comparison with related enzymes of the ␤-family of PLP-dependent enzymes indicated structural determinants of the oligomeric state and thus rationalized for the first time how a PLP enzyme acts in monomeric form.Threonine synthase (TS, EC 4.2.99.2) 1 is a pyridoxal 5Ј-phosphate (PLP)-dependent enzyme that catalyzes the ultimate step in threonine biosynthesis, the PLP-dependent ␤,␥-replacement reaction (Reaction 1) of O-phosphohomoserine (OPHS) yielding threonine and inorganic phosphate (1-3).Together with tryptophan synthase (TRPS), threonine deaminase (TDA), O-acetylserine sulfhydrylase (OASS), cystathione ␤-synthase (CBS), and 1-aminocyclopropane-1-carboxylate deaminase (ACCD), TS constitutes the core of the fold-type II family of PLP enzymes (4, 5) (also referred to as ␤-family (6)). Detailed amino acid sequence alignments revealed that TS can be grouped into a plant and a fungal subfamily (7). The former one (class I subfamily) comprises TS from higher plants, cyanobacteria, archaebacteria, and the eubacterial groups of Mycobacteria, Aquificaceae, and Bacillus species. The second subfamily (class II subfamily) contains the enzymes from fungi and from the eubacterial groups of Proteobacteria and corneyform bacteria. Only 5 from ϳ500 residues are invariant between both subfamilies, including the PLP-binding lysine as part of a phenylalanine-lysine-aspartate consensus sequence.During the last decades, TS was purified and characterized from several bacteria and fungi (8 -12) and from Arabidopsis thaliana (7, 13). In plants, the substrate of TS, OPHS, is the branching point for threonine and methionine biosynthesis. Flux coordination between both synthetic pathways is accomplished by allosteric activation of plant threonine synthase. The allosteric effector is S-adenosyl methionine (SAM) (14, 15), a product of methioni...

Ribosomal protein L7/L12, the only multicopy component of the ribosome, is involved in translation factor binding and in the ribosomal GTPase center. The gene for L7/L12 from Thermotoga maritima was cloned and the protein expressed at high levels in Escherichia coli. Purification of L7/L12 was achieved under non-denaturing conditions via heat treatment and two chromatographic steps. Circular dichroism melting profiles were monitored at 222 nm, showing the melting temperature of the protein at pH 7.5 around 110 degrees C, compared to approximately 60 degrees C for the highly homologous Escherichia coli protein. The unfolding was reversible and renaturation closely followed the path of the thermal melting. Dynamic light scattering, gel filtration chromatography, and crosslinking experiments suggested that under physiological buffer conditions Thermotoga maritima L7/L12 exists as a tetramer. The protein was crystallized under two conditions, yielding an orthorhombic (C222(1)) and a cubic (12(1)3) space group with an estimated two and three to four L7/L12 molecules per asymmetric unit, respectively. The crystals contained the full-length protein, and cryogenic buffers were developed which improved the mosaic spreads and the resolution limits. For the structure solution isoleucine was mutated to methionine at two separate positions, the mutant forms expressed as selenomethionine variants and crystallized.