Molecular determinants of substrate specificity in plant 5'-methylthioadenosine nucleosidases

5′-Methylthioadenosine (MTA)/S-adenosylhomocysteine (SAH) nucleosidase (MTAN) is essential for cellular metabolism and development in many bacterial species. While the enzyme is found in plants, plant MTANs appear to select for MTA preferentially, with little or no affinity for SAH. To understand what determines substrate specificity in this enzyme, MTAN homologues from Arabidopsis thaliana (AtMTAN1 and AtMTAN2, which are referred to as AtMTN1 and AtMTN2 in the plant literature) have been characterized kinetically. While both homologues hydrolyze MTA with comparable kinetic parameters, only AtMTAN2 shows activity towards SAH. AtMTAN2 also has higher catalytic activity towards other substrate analogues with longer 5′-substituents. The structures of apo AtMTAN1 and its complexes with the substrate- and transition-state-analogues, 5′-methylthiotubercidin and formycin A, respectively, have been determined at 2.0-1.8 Å resolution. A homology model of AtMTAN2 was generated using the AtMTAN1 structures. Comparison of the AtMTAN1 and AtMTAN2 structures reveals that only three residues in the active site differ between the two enzymes. Our analysis suggests that two of these residues, Leu181/Met168 and Phe148/Leu135 in AtMTAN1/AtMTAN2, likely account for the divergence in specificity of the enzymes. Comparison of the AtMTAN1 and available Escherichia coli MTAN (EcMTAN) structures suggests that a combination of differences in the 5′-alkylthio binding region and reduced conformational flexibility in the AtMTAN1 active site likely contribute to its reduced efficiency in binding substrate analogues with longer 5′-substituents. In addition, in contrast to EcMTAN, the active site of AtMTAN1 remains solvated in its ligand-bound forms. As the apparent pK_a of an amino acid depends on its local environment, the putative catalytic acid Asp225 in AtMTAN1 may not be protonated at physiological pH and this suggests the transition state of AtMTAN1, like human MTA phosphorylase and Streptococcus pneumoniae MTAN, may be different from that found in EcMTAN.     

Crystallographic structure of phosphofructokinase-2 from Escherichia coli in complex with two ATP molecules. Implications for substrate inhibition

Phosphofructokinase-1 and -2 (Pfk-1 and Pfk-2, respectively) from Escherichia coli belong to different homologous superfamilies. However, in spite of the lack of a common ancestor, they share the ability to catalyze the same reaction and are inhibited by the substrate MgATP. Pfk-2, an ATP-dependent 6-phosphofructokinase member of the ribokinase-like superfamily, is a homodimer of 66 kDa subunits whose oligomerization state is necessary for catalysis and stability. The presence of MgATP favors the tetrameric form of the enzyme. In this work, we describe the structure of Pfk-2 in its inhibited tetrameric form, with each subunit bound to two ATP molecules and two Mg ions. The present structure indicates that substrate inhibition occurs due to the sequential binding of two MgATP molecules per subunit, the first at the usual site occupied by the nucleotide in homologous enzymes and the second at the allosteric site, making a number of direct and Mg-mediated interactions with the first. Two configurations are observed for the second MgATP, one of which involves interactions with Tyr23 from the adjacent subunit in the dimer and the other making an unusual non-Watson-Crick base pairing with the adenine in the substrate ATP. The oligomeric state observed in the crystal is tetrameric, and some of the structural elements involved in the binding of the substrate and allosteric ATPs are also participating in the dimer-dimer interface. This structure also provides the grounds to compare analogous features of the nonhomologous phosphofructokinases from E. coli.     

Structure of N-acetyl-beta-D-glucosaminidase (GcnA) from the endocarditis pathogen Streptococcus gordonii and its complex with the mechanism-based inhibitor NAG-thiazoline

The crystal structure of GcnA, an N-acetyl-β-d-glucosaminidase from Streptococcus gordonii, was solved by multiple wavelength anomalous dispersion phasing using crystals of selenomethionine-substituted protein. GcnA is a homodimer with subunits each comprised of three domains. The structure of the C-terminal α-helical domain has not been observed previously and forms a large dimerisation interface. The fold of the N-terminal domain is observed in all structurally related glycosidases although its function is unknown. The central domain has a canonical (β/α)₈ TIM-barrel fold which harbours the active site. The primary sequence and structure of this central domain identifies the enzyme as a family 20 glycosidase. Key residues implicated in catalysis have different conformations in two different crystal forms, which probably represent active and inactive conformations of the enzyme. The catalytic mechanism for this class of glycoside hydrolase, where the substrate rather than the enzyme provides the cleavage-inducing nucleophile, has been confirmed by the structure of GcnA complexed with a putative reaction intermediate analogue, N-acetyl-β-d-glucosamine-thiazoline. The catalytic mechanism is discussed in light of these and other family 20 structures.     

The crystal structure of beta-alanine synthase from Drosophila melanogaster reveals a homooctameric helical turn-like assembly

β-Alanine synthase (βAS) is the third enzyme in the reductive pyrimidine catabolic pathway, which is responsible for the breakdown of the nucleotide bases uracil and thymine in higher organisms. It catalyzes the hydrolysis of N-carbamyl-β-alanine and N-carbamyl-β-aminoisobutyrate to the corresponding β-amino acids. βASs are grouped into two phylogenetically unrelated subfamilies, a general eukaryote one and a fungal one. To reveal the molecular architecture and understand the catalytic mechanism of the general eukaryote βAS subfamily, we determined the crystal structure of Drosophila melanogaster βAS to 2.8 Å resolution. It shows a homooctameric assembly of the enzyme in the shape of a left-handed helical turn, in which tightly packed dimeric units are related by 2-fold symmetry. Such an assembly would allow formation of higher oligomers by attachment of additional dimers on both ends. The subunit has a nitrilase-like fold and consists of a central β-sandwich with a layer of α-helices packed against both sides. However, the core fold of the nitrilase superfamily enzymes is extended in D. melanogaster βAS by addition of several secondary structure elements at the N-terminus. The active site can be accessed from the solvent by a narrow channel and contains the triad of catalytic residues (Cys, Glu, and Lys) conserved in nitrilase-like enzymes.     

Crystal structure of tRNA N2,N2-guanosine dimethyltransferase Trm1 from Pyrococcus horikoshii

Trm1 catalyzes a two-step reaction, leading to mono- and dimethylation of guanosine at position 26 in most eukaryotic and archaeal tRNAs. We report the crystal structures of Trm1 from Pyrococcus horikoshii liganded with S-adenosyl-l-methionine or S-adenosyl-l-homocysteine. The protein comprises N-terminal and C-terminal domains with class I methyltransferase and novel folds, respectively. The methyl moiety of S-adenosyl-l-methionine points toward the invariant Phe27 and Phe140 within a narrow pocket, where the target G26 might flip in. Mutagenesis of Phe27 or Phe140 to alanine abolished the enzyme activity, indicating their role in methylating G26. Structural analyses revealed that the movements of Phe140 and the loop preceding Phe27 may be involved in dissociation of the monomethylated tRNA•Trm1 complex prior to the second methylation. Moreover, the catalytic residues Asp138, Pro139, and Phe140 are in a different motif from that in DNA 6-methyladenosine methyltransferases, suggesting a different methyl transfer mechanism in the Trm1 family.     

Assessing side-chain perturbations of the protein backbone: a knowledge-based classification of residue Ramachandran space

Grouping the 20 residues is a classic strategy to discover ordered patterns and insights about the fundamental nature of proteins, their structure, and how they fold. Usually, this categorization is based on the biophysical and/or structural properties of a residue's side-chain group. We extend this approach to understand the effects of side chains on backbone conformation and to perform a knowledge-based classification of amino acids by comparing their backbone phi, psi distributions in different types of secondary structure. At this finer, more specific resolution, torsion angle data are often sparse and discontinuous (especially for nonhelical classes) even though a comprehensive set of protein structures is used. To ensure the precision of Ramachandran plot comparisons, we applied a rigorous Bayesian density estimation method that produces continuous estimates of the backbone phi, psi distributions. Based on this statistical modeling, a robust hierarchical clustering was performed using a divergence score to measure the similarity between plots. There were seven general groups based on the clusters from the complete Ramachandran data: nonpolar/beta-branched (Ile and Val), AsX (Asn and Asp), long (Met, Gln, Arg, Glu, Lys, and Leu), aromatic (Phe, Tyr, His, and Cys), small (Ala and Ser), bulky (Thr and Trp), and, lastly, the singletons of Gly and Pro. At the level of secondary structure (helix, sheet, turn, and coil), these groups remain somewhat consistent, although there are a few significant variations. Besides the expected uniqueness of the Gly and Pro distributions, the nonpolar/beta-branched and AsX clusters were very consistent across all types of secondary structure. Effectively, this consistency across the secondary structure classes implies that side-chain steric effects strongly influence a residue's backbone torsion angle conformation. These results help to explain the plasticity of amino acid substitutions on protein structure and should help in protein design and structure evaluation.     

The crystal structure of JNK2 reveals conformational flexibility in the MAP kinase insert and indicates its involvement in the regulation of catalytic activity

c-Jun N-terminal kinase (JNK) 2 is a member of the mitogen-activated protein (MAP) kinase group of signaling proteins. MAP kinases share a common sequence insertion called “MAP kinase insert”, which, for ERK2, has been shown to interact with regulatory proteins and, for p38α, has been proposed to be involved in the regulation of catalytic activity. We have determined the crystal structure of human JNK2 complexed with an indazole inhibitor by applying a high-throughput protein engineering and surface-site mutagenesis approach. A novel conformation of the activation loop is observed, which is not compatible with its phosphorylation by upstream kinases. This activation inhibitory conformation of JNK2 is stabilized by the MAP kinase insert that interacts with the activation loop in an induced-fit manner. We therefore suggest that the MAP kinase insert of JNK2 plays a role in the regulation of JNK2 activation, possibly by interacting with intracellular binding partners.     

Molecular docking for substrate identification: the short-chain dehydrogenases/reductases

Protein ligand docking has recently been investigated as a tool for protein function identification, with some success in identifying both known and unknown substrates of proteins. However, identifying a protein's substrate when cross-docking a large number of enzymes and their cognate ligands remains a challenge. To explore a more limited yet practically important and timely problem in more detail, we have used docking for identifying the substrates of a single protein family with remarkable substrate diversity, the short-chain dehydrogenases/reductases.We examine different protocols for identifying candidate substrates for 27 short-chain dehydrogenase/reductase proteins of known catalytic function. We present the results of docking > 900 metabolites from the human metabolome to each of these proteins together with their known cognate substrates and products, and we investigate the ability of docking to (a) reproduce a viable binding mode for the substrate and (b) to rank the substrate highly amongst the dataset of other metabolites. In addition, we examine whether our docking results provide information about the nature of the substrate, based on the best-scoring metabolites in the dataset. We compare two different docking methods and two alternative scoring functions for one of the docking methods, and we attempt to rationalise both successes and failures.Finally, we introduce a new protocol, whereby we dock only a set of representative structures (medoids) to each of the proteins, in the hope of characterising each binding site in terms of its ligand preferences, with a reduced computational cost. We compare the results from this protocol with our original docking experiments, and we find that although the rank of the representatives correlates well with the mean rank of the clusters to which they belong, a simple structure-based clustering is too naïve for the purpose of substrate identification. Many clusters comprise ligands with widely varying affinities for the same protein; hence important candidates can be missed if a single representative is used.     

The crystal structure of enamidase: a bifunctional enzyme of the nicotinate catabolism

The hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to 2-formylglutarate is a central step in the catabolism of nicotinate in several Clostridia and Proteobacteria. This reaction is catalyzed by the novel enzyme enamidase, a new member of the amidohydrolase superfamily as indicated by its unique reaction, sequence relationship, and the stoichiometric binding of iron and zinc. A hallmark of enamidase is its capability to catalyze a two-step reaction: the initial decyclization of 1,4,5,6-tetrahydro-6-oxonicotinate leading to 2-(enamine)glutarate followed by an additional hydrolysis step yielding (S)-2-formylglutarate. Here, we present the crystal structure of enamidase from Eubacterium barkeri at 1.9 Å resolution, providing a structural basis for catalysis and suggesting a mechanism for its exceptional activity and enantioselectivity. The enzyme forms a 222-symmetric tetramer built up by a dimer of dimers. Each enamidase monomer consists of a composite β-sandwich domain and an (α/β)₈-TIM-barrel domain harboring the active site. With its catalytic binuclear metal center comprising both zinc and iron ions, enamidase represents a special case of subtype II amidohydrolases.     

<Emphasis Type="Italic">Helitron</Emphasis> mediated amplification of cytochrome P450 monooxygenase gene in maize

The mass movement of gene sequences by Helitrons has significantly contributed to the lack of gene collinearity reported between different maize inbred lines. However, Helitron captured-genes reported to date represent truncated versions of their progenitor genes. In this report, we provide evidence that maize CYP72A27-Zm gene represents a cytochrome P450 monooxygenase (P450) gene recently captured by a Helitron and transposed into an Opie-2 retroposon. The four exons of the CYP72A27 gene contained within the element contain a putative open reading frame (ORF) for 428 amino acid residues. We provide evidence that Helitron captured CYP72A27-Zm is transcribed. To identify the progenitor gene and the evolutionary time of capture, we searched the plant genome database and discovered other closely related CYP72A27-Zm genes in maize and grasses. Our analysis indicates that CYP72A27-Zm represents an almost complete copy of maize CYP72A26-Zm gene captured by a Helitron about 3.1 million years ago (mya). The Helitron-captured gene then duplicated twice, approximately 1.5-1.6 mya giving rise to CYP72A36-Zm and CYP72A37-Zm. These data provide evidence that Helitrons can capture and mobilize intact genes that are transcribed and potentially encode biologically relevant proteins.