The crystal structure of cystathionine gamma-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 A. The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the Cys-Met-metabolism PLP-dependent family like cystathionine beta-lyase (CBL). A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity between the different family members. CGLs from yeast or human are virtually identical at their active sites to cystathionine gamma-synthase (CGS) from E. coli. Both CGLs and bacterial CGSs exhibit gamma-synthase and gamma-lyase activities depending on their position in the metabolic pathway and the available substrates. This group of enzymes has a glutamate (E333 in yeast CGL) which binds to the distal group of cystathionine (CTT) or the amino group of cysteine. Plant CGSs use homoserine phosphate instead of O-succinyl-homoserine as one substrate. This is reflected by a partially different active site structure in plant CGSs. In CGL and CBL the pseudosymmetric substrate must dock at the active site in different orientations, with S in gamma-position (CBL) or in delta-position (CGL). The conserved glutamate steers the substrate as seen in other CGLs. In CBLs this position is occupied by either tyrosine or hydrophobic residues directing binding of CTT such that S is in the in gamma-position. In methionine gamma-lyase a hydrophic patch operates as recognition site for the methyl group of the methionine substrate.

Variations in the inner ribosomal landscape determining the topology of nascent protein transport have been studied by three-dimensional cryo-electron microscopy of erythromycin-resistant Escherichia coli 70S ribosomes. Significant differences in the mouth of the 50S subunit tunnel system visualized in the present study support a simple steric-hindrance explanation for the action of the drug. Examination of ribosomes in different functional states suggests that opening and closing of the main tunnel are dynamic features of the large subunit, possibly accompanied by changes in the L7/L12 stalk region. The existence and dynamic behavior of side tunnels suggest that ribosomal proteins L4 and L22 might be involved in the regulation of a multiple exit system facilitating cotranslational processing (or folding or directing) of nascent proteins.

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

The crystal structure of Thermotoga maritima NusA, a transcription factor involved in pausing, termination, and antitermination processes, reveals a four-domain, rod-shaped molecule. An N-terminal alpha/beta portion, a five-stranded beta-barrel (S1 domain), and two K-homology (KH) modules create a continuous spine of positive electrostatic potential, suitable for nonspecific mRNA attraction. Homology models suggest how, in addition, specific mRNA regulatory sequences can be recognized by the S1 and KH motifs. An arrangement of multiple S1 and KH domains mediated by highly conserved residues is seen, creating an extended RNA binding surface, a paradigm for other proteins with similar domain arrays. Structural and mutational analyses indicate that the motifs cooperate, modulating strength and specificity of RNA binding.

Ribosomal protein L4 resides near the peptidyl transferase center of the bacterial ribosome and may, together with rRNA and proteins L2 and L3, actively participate in the catalysis of peptide bond formation. Escherichia coli L4 is also an autogenous feedback regulator of transcription and translation of the 11 gene S10 operon. The crystal structure of L4 from Thermotoga maritima at 1.7 A resolution shows the protein with an alternating alpha/beta fold and a large disordered loop region. Two separate binding sites for RNA are discernible. The N-terminal site, responsible for binding to rRNA, consists of the disordered loop with flanking alpha-helices. The C-terminal site, a prime candidate for the interaction with the leader sequence of the S10 mRNA, involves two non-consecutive alpha-helices. The structure also suggests a C-terminal protein-binding interface, through which L4 could be interacting with protein components of the transcriptional and/or translational machineries.