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The eukaryotic translation initiation factor 4A (eIF4A) is a member of the DEA(D͞H)-box RNA helicase family, a diverse group of proteins that couples an ATPase activity to RNA binding and unwinding. Previous work has provided the structure of the amino-terminal, ATP-binding domain of eIF4A. Extending those results, we have solved the structure of the carboxyl-terminal domain of eIF4A with data to 1.75 Å resolution; it has a parallel ␣-␤ topology that superimposes, with minor variations, on the structures and conserved motifs of the equivalent domain in other, distantly related helicases. Using data to 2.8 Å resolution and molecular replacement with the refined model of the carboxyl-terminal domain, we have completed the structure of full-length eIF4A; it is a ''dumbbell'' structure consisting of two compact domains connected by an extended linker. By using the structures of other helicases as a template, compact structures can be modeled for eIF4A that suggest (i) helicase motif IV binds RNA; (ii) Arg-298, which is conserved in the DEA(D͞H)-box RNA helicase family but is absent from many other helicases, also binds RNA; and (iii) motifs V and VI ''link'' the carboxyl-terminal domain to the amino-terminal domain through interactions with ATP and the DEA(D͞H) motif, providing a mechanism for coupling ATP binding and hydrolysis with conformational changes that modulate RNA binding.

The 2.5-Å resolution crystal structure of recombinant aristolochene synthase from the blue cheese mold, Penicillium roqueforti, is the first of a fungal terpenoid cyclase. The structure of the enzyme reveals active site features that participate in the cyclization of the universal sesquiterpene cyclase substrate, farnesyl diphosphate, to form the bicyclic hydrocarbon aristolochene. Metal-triggered carbocation formation initiates the cyclization cascade, which proceeds through multiple complex intermediates to yield one exclusive structural and stereochemical isomer of aristolochene. Structural homology of this fungal cyclase with plant and bacterial terpenoid cyclases, despite minimal amino acid sequence identity, suggests divergence from a common, primordial ancestor in the evolution of terpene biosynthesis.Aristolochene synthase is a terpenoid cyclase from the blue cheese mold, Penicillium roqueforti, that catalyzes the metal-dependent cyclization of farnesyl diphosphate to form the bicyclic hydrocarbon aristolochene (Fig. 1) (1). Farnesyl diphosphate is the universal precursor of myriad cyclic sesquiterpenes, so each sesquiterpene cyclase plays a critical role in governing the structural and stereochemical outcome of its particular cyclization reaction. Accordingly, sesquiterpene cyclase reactions maximize product diversity starting from a minimal substrate pool, indeed, a single substrate, and the structural basis of this catalytic diversity comprises a growing question at the interface of chemistry and biology.Aristolochene synthase is a 38-kDa monomeric sesquiterpene cyclase that has been cloned (2) and overexpressed (3) in Escherichia coli. Numerous enzymological studies of P. roqueforti and Aspergillus terreus aristolochene synthases using stereospecifically labeled substrates (4 -6), a mechanism-based inhibitor (7), and the anomalous substrate (7R)-6,7-dihydrofarnesyldiphosphate (8) indicate a complex cyclization cascade proceeding through at least two discrete intermediates. Aristolochene formation is the first committed step in the biosynthesis of a large group of sesquiterpenoid fungal toxins, the most lethal of which is the novel bis-epoxide PR-toxin (4). Interestingly, the (ϩ)-enantiomer of aristolochene is generated by the fungi P. roqueforti and A. terreus, but the (Ϫ)-enantiomer is generated by the plants Aristolochia indica (9) and Bixa orella (10). Accordingly, each aristolochene synthase must provide a different template for binding the flexible polyisoprenoid substrate and subsequent intermediates in productive conformations leading to correct stereoisomer formation.The diastereomeric sesquiterpene epi-aristolochene (4-epieremophila-9,11-diene) has been identified in tobacco (Nicotiana tabacum) (11-13) and results from the cyclization of farnesyl diphosphate by epi-aristolochene synthase (Fig. 1) (14). This enzyme catalyzes the cyclization of farnesyl diphosphate by a mechanism similar in some respects to that of P. roqueforti aristolochene synthase despite only 16% amino acid sequence ident...

DEx Members of the DExD / H helicase family participate in many cellular processes involving rearrangement of RNA-RNA and RNA-protein interactions (1, 2). DEx D / H proteins contain a conserved catalytic core consisting of two RecA-like domains that bind ATP and single-stranded RNA (3). Similar to other RecA-like ATPases (4), the cycle of ATP binding, hydrolysis, and release is coupled to a conformational change in the core. In DEx D / H proteins, this results in translocation of the protein along single-stranded RNA and subsequent duplex destabilization and/or ribonucleoprotein disruption (5, 6). Outside of the catalytic core, DEx D / H proteins often contain ancillary N-and C-terminal extensions that correlate with their specific cellular functions. In many cases, helicases are part of multiprotein complexes, and the putative role of the ancillary domains is to recruit the helicase to the complex by proteinprotein interactions (7). Alternatively, the ancillary domains can directly bind to a specific RNA substrate, delivering the helicase to its site of action.Bacillus subtilis DEx D / H protein YxiN and Escherichia coli DbpA define a group of bacterial orthologs with a conserved C-terminal domain of ϳ80 amino acids (Fig. 1A). Both YxiN and DbpA specifically recognize the A-site region of 23 S rRNA, including hairpin 92, suggesting that the RNA specificity is conferred by the C-terminal domain (8 -11). In a domain swap experiment, the C-terminal domain of YxiN was appended to the catalytic core of SrmB, a non-sequence-specific RNA helicase from E. coli (12). The resulting chimera possessed the RNA specificity of YxiN but had catalytic properties similar to those of the parental helicase, SrmB. These results indicate that the C-terminal domain of YxiN is capable of imparting specificity in the context of another DEx D / H box protein, suggesting that the sequence-specific RNA binding and helicase activities are separable and that this group of DEx D / H proteins may be functionally modular. To prove this hypothesis, the helicase and C-terminal domains of YxiN were prepared here as separate polypeptides. This approach permits the intrinsic activities of the catalytic core and the C-terminal domain to be assayed independently and compared with the full-length protein. EXPERIMENTAL PROCEDURES Plasmids for Expression of Recombinant YxiN Fragments-Codingsequences for YxiN protein fragments were PCR-amplified from the native B. subtilis sequence in an existing plasmid (11) and cloned by restriction/ligation into the pTWIN1 vector of an intein-based expression system (New England Biolabs) using the unique NdeI and SpeI sites of the vector. Primers were designed such that the subcloned fragments deleted the first of the tandem inteins of the parent vector and placed the C-terminal residue of each encoded YxiN protein fragment flush with the N-terminal residue of the second intein. In this manner, no extraneous residues remained at the C terminus of the target after selfcleavage of the target-intein fusion. The plasmids ...

Crystal structures of two homologous peptidases from cyanobacteria Anabaena variabilis and Nostoc punctiforme at 1.05 Å and 1.60 Å resolution represent the first structures of a large class of cell-wall, cysteine peptidases that contain an N-terminal bacterial SH3-like domain (SH3b) and a C-terminal NlpC/P60 cysteine peptidase domain. The NlpC/P60 domain is a primitive, papain-like peptidase in the CA clan of cysteine peptidases with a Cys126/His176/His188 catalytic triad and a conserved catalytic core. We deduced from structure and sequence analysis, and then experimentally, that that these two proteins act as γ-D-glutamyl-L-diamino acid endopeptidases (EC 3.4.22.-). The active site is located near the interface between the SH3b and NlpC/P60 domains, where the SH3b domain may help define substrate specificity, instead of functioning as a targeting domain, so that only muropeptides with an N-terminal L-alanine can bind to the active site.