Sculpting of the spliceosomal branch site recognition motif by a conserved pseudouridine

Pairing of a consensus sequence of the precursor (pre)-mRNA intron with a short region of the U2 small nuclear (sn)RNA during assembly of the eukaryotic spliceosome results in formation of a complementary helix of seven base pairs with a single unpaired adenosine residue. The 2' OH of this adenosine, called the branch site, brings about nucleophilic attack at the pre-mRNA 5' splice site in the first step of splicing. Another feature of this pairing is the phylogenetic conservation of a pseudouridine (psi) residue in U2 snRNA nearly opposite the branch site. We show that the presence of this psi in the pre-mRNA branch-site helix of Saccharomyces cerevisiae induces a dramatically altered architectural landscape compared with that of its unmodified counterpart. The psi-induced structure places the nucleophile in an accessible position for the first step of splicing.     

The structural basis for tRNA recognition and pseudouridine formation by pseudouridine synthase I

Pseudouridine synthases catalyze the isomerization of specific uridines to pseudouridine in a variety of RNAs, yet the basis for recognition of the RNA sites or how they catalyze this reaction is unknown. The crystal structure of pseudouridine synthase I from Escherichia coli, which, for example, modifies positions 38, 39 and/or 40 in tRNA, reveals a dimeric protein that contains two positively charged, RNA-binding clefts along the surface of the protein. Each cleft contains a highly conserved aspartic acid located at its center. The structural domains have a topological similarity to those of other RNA-binding proteins, though the mode of interaction with tRNA appears to be unique. The structure suggests that a dimeric enzyme is required for binding transfer RNA and subsequent pseudouridine formation.     

Kinetic and structural studies on roles of the serine ligand and a strictly conserved tyrosine residue in nitrile hydratase

Nitrile hydratases (NHase), which catalyze the hydration of nitriles to amides, have an unusual Fe³⁺ or Co³⁺ center with two modified Cys ligands: cysteine sulfininate (Cys-SO₂ ⁻) and either cysteine sulfenic acid or cysteine sulfenate [Cys-SO(H)]. Two catalytic mechanisms have been proposed. One is that the sulfenyl oxygen activates a water molecule, enabling nucleophilic attack on the nitrile carbon. The other is that the Ser ligand ionizes the strictly conserved Tyr, activating a water molecule. Here, we characterized mutants of Fe-type NHase from Rhodococcus erythropolis N771, replacing the Ser and Tyr residues, αS113A and βY72F. The αS113A mutation partially affected catalytic activity and did not change the pH profiles of the kinetic parameters. UV–vis absorption spectra indicated that the electronic state of the Fe center was altered by the αS113A mutation, but the changes could be prevented by a competitive inhibitor, n-butyric acid. The overall structure of the αS113A mutant was similar to that of the wild type, but significant changes were observed around the catalytic cavity. Like the UV–vis spectra, the changes were compensated by the substrate or product. The Ser ligand is important for the structure around the catalytic cavity, but is not essential for catalysis. The βY72F mutant exhibited no activity. The structure of the βY72F mutant was highly conserved but was found to be the inactivated state, with αCys114-SO(H) oxidized to Cys-SO₂ ⁻, suggesting that βTyr72 affected the electronic state of the Fe center. The catalytic mechanism is discussed on the basis of the results obtained.     

Formation of the conserved pseudouridine at position 55 in archaeal tRNA

Pseudouridine (Ψ) located at position 55 in tRNA is a nearly universally conserved RNA modification found in all three domains of life. This modification is catalyzed by TruB in bacteria and by Pus4 in eukaryotes, but so far the Ψ55 synthase has not been identified in archaea. In this work, we report the ability of two distinct pseudouridine synthases from the hyperthermophilic archaeon Pyrococcus furiosus to specifically modify U55 in tRNA in vitro. These enzymes are _pfuCbf5, a protein known to play a role in RNA-guided modification of rRNA, and _pfuPsuX, a previously uncharacterized enzyme that is not a member of the TruB/Pus4/Cbf5 family of pseudouridine synthases. _pfuPsuX is hereafter renamed _pfuPus10. Both enzymes specifically modify tRNA U55 in vitro but exhibit differences in substrate recognition. In addition, we find that in a heterologous in vivo system, _pfuPus10 efficiently complements an Escherichia coli strain deficient in the bacterial Ψ55 synthase TruB. These results indicate that it is probable that _pfuCbf5 or _pfuPus10 (or both) is responsible for the introduction of pseudouridine at U55 in tRNAs in archaea. While we cannot unequivocally assign the function from our results, both possibilities represent unexpected functions of these proteins as discussed herein.     

Targeting the role of a key conserved motif for substrate selection and catalysis by 3-deoxy-D-manno-octulosonate 8-phosphate synthase

3-Deoxy-D-manno-octulosonate 8-phosphate synthase (KDO8PS) catalyzes the reaction between three-carbon phosphoenolpyruvate (PEP) and five-carbon d-arabinose 5-phosphate (A5P), generating KDO8P, a key intermediate in the biosynthetic pathway to 3-deoxy-D-manno-octulosonate, a component of the lipopolysaccharide of the Gram-negative bacterial cell wall. Both metal-dependent and metal-independent forms of KDO8PS have been characterized. KDO8PS is evolutionarily and mechanistically related to the first enzyme of the shikimate pathway, the obligately divalent metal ion-dependent 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS) that couples PEP and four-carbon D-erythrose 4-phosphate (E4P) to give DAH7P. In KDO8PS, an absolutely conserved KANRS motif forms part of the A5P binding site, whereas in DAH7PS, an absolutely conserved KPR(S/T) motif accommodates E4P. Here, we have characterized four mutants of this motif (AANRS, KAARS, KARS, and KPRS) in metal-dependent KDO8PS from Acidithiobacillus ferrooxidans and metal-independent KDO8PS from Neisseria meningitidis to test the roles of the universal Lys and the Ala-Asn portion of the KANRS motif. The X-ray structures, determined for the N. meningitidis KDO8PS mutants, indicated no gross structural penalty resulting from mutation, but the subtle changes observed in the active sites of these mutant proteins correlated with their altered catalytic function. (1) The AANRS mutations destroyed catalytic activity. (2) The KAARS mutations lowered substrate selectivity, as well as activity. (3) Replacing KANRS with KARS or KPRS destroyed KDO8PS activity but did not produce a functional DAH7PS. Thus, Lys is critical to catalysis, and other changes are necessary to switch substrate specificity for both the metal-independent and metal-dependent forms of these enzymes.     

The roles of the essential Asp-48 and highly conserved His-43 elucidated by the pH dependence of the pseudouridine synthase TruB

All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.     

Involvement of conserved aspartate and glutamate residues in the catalysis and substrate binding of maize starch synthase

Chemical modification of maize starch synthase IIb-2 (SSIIb-2) using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC), which modifies acidic amino acid residues, resulted in a time- and concentration-dependent inactivation of SSIIb-2. ADPGlc was found to completely protect SSIIb-2 from inactivation by EDAC. These results suggest that glutamate or aspartate is important for SS activity. On the basis of the sequence identity of SS, conserved acidic amino acids were mutagenized to identify the specific amino acid residues important for SS activity. Three amino acids (D21, D139, and E391) were found to be important for SS activity. D21N showed 4% of the wild-type enzyme activity and a 10-fold decrease in the affinity for ADPGlc, while the conservative change from D21 to E resulted in a decrease in V(max) and no change in affinity for ADPGlc, suggesting that the negative charge is important for ADPGlc binding. When sites D139 and E391 were changed to their respective amide form, no SS activity was detected. With the conservative change, D139E showed a decrease in V(max) and no changes in apparent K(m) for substrates. E391D showed a 9-fold increase in K(m) for ADPGlc, a 12-fold increase in apparent K(m) for glycogen, and a 4-fold increase in apparent K(m) for amylopectin. The circular dichroism analysis indicates that these kinetic changes may not be due to a major conformation change in the protein. These results provide the first evidence that the conserved aspartate and glutamate residues could be involved in the catalysis or substrate binding of SS.     

Role of a conserved arginine in the mechanism of acetohydroxyacid synthase: catalysis of condensation with a specific ketoacid substrate

The thiamin diphosphate (ThDP)-dependent bio-synthetic enzyme acetohydroxyacid synthase (AHAS) catalyzes decarboxylation of pyruvate and specific condensation of the resulting ThDP-bound two-carbon intermediate, hydroxyethyl-ThDP anion/enamine (HEThDP(-)), with a second ketoacid, to form acetolactate or acetohydroxybutyrate. Whereas the mechanism of formation of HEThDP(-) from pyruvate is well understood, the role of the enzyme in control of the carboligation reaction of HEThDP(-) is not. Recent crystal structures of yeast AHAS from Duggleby's laboratory suggested that an arginine residue might interact with the second ketoacid substrate. Mutagenesis of this completely conserved residue in Escherichia coli AHAS isozyme II (Arg(276)) confirms that it is required for rapid and specific reaction of the second ketoacid. In the mutant proteins, the normally rapid second phase of the reaction becomes rate-determining. A competing alternative nonnatural but stereospecific reaction of bound HEThDP(-) with benzaldehyde to form phenylacetylcarbinol (Engel, S., Vyazmensky, M., Geresh, S., Barak, Z., and Chipman, D. M. (2003) Biotechnol. Bioeng. 84, 833-840) provides a new tool for studying the fate of HEThDP(-) in AHAS, since the formation of the new product has a very different dependence on active site modifications than does acetohydroxyacid acid formation. The effects of mutagenesis of four different residues in the site on the rates and specificities of the normal and unnatural reactions support a critical role for Arg(276) in the stabilization of the transition states for ligation of the incoming second ketoacid with HEThDP(-) and/or for the breaking of the product-ThDP bond. This information makes it possible to engineer the active site so that it efficiently and preferentially catalyzes a new reaction.     

Cocrystal structure of a tRNA Psi55 pseudouridine synthase: nucleotide flipping by an RNA-modifying enzyme.

Pseudouridine (Psi) synthases catalyze the isomerization of specific uridines in cellular RNAs to pseudouridines and may function as RNA chaperones. TruB is responsible for the Psi residue present in the T loops of virtually all tRNAs. The close homolog Cbf5/dyskerin is the catalytic subunit of box H/ACA snoRNPs. These carry out the pseudouridylation of eukaryotic rRNA and snRNAs. The 1.85 A resolution structure of TruB bound to RNA reveals that this enzyme recognizes the preformed three-dimensional structure of the T loop, primarily through shape complementarity. It accesses its substrate uridyl residue by flipping out the nucleotide and disrupts the tertiary structure of tRNA. Structural comparisons with TruB demonstrate that all Psi synthases are descended from a common molecular ancestor.     

Dissecting substrate recognition by thrombin using the inactive mutant S195A

The catalytically inactive mutant S195A was used to study the interaction of thrombin with substrates under equilibrium conditions. By monitoring changes in intrinsic fluorescence, we measured dissociation constants for a variety of synthetic substrates, PAR peptides and the inhibitor PPACK. The S195A mutant retains the Na⁺-binding properties of the wild type, and substrate binding to the mutant is enhanced by the presence of Na⁺. Temperature dependence studies allowed calculation of the thermodynamic parameters of substrate binding at the active site and showed a negligible ΔC_p. Titration of synthetic substrates carrying substitutions at the P1–P3 positions revealed energetics consistent with the specificity hierarchy identified in hydrolysis by the wild type. Titration with PAR peptides, which interact with both the active site and exosite I of thrombin, also showed consistency with the results obtained with the wild type at steady state. These findings demonstrate that inactive mutants of enzymes make it possible to dissect the equilibrium components linked to substrate binding and complement information on the kinetic properties of the wild type.     

Crystal structure of the highly divergent pseudouridine synthase TruD reveals a circular permutation of a conserved fold

The pseudouridine (Psi) synthases Pus7p and TruD define a family of RNA-modifying enzymes with no sequence similarity to previously characterized Psi synthases. The 2.2 A resolution structure of Escherichia coli TruD reveals a U-shaped molecule with a catalytic domain that superimposes closely on that of other Psi synthases. A domain that appears to be unique to TruD/Pus7p family enzymes hinges over the catalytic domain, possibly serving to clasp the substrate RNAs. The active site comprises residues that are conserved in other Psi synthases, although at least one comes from a structurally distinct part of the protein. Remarkably, the connectivity of the structural elements of the TruD catalytic domain is a circular permutation of that of its paralogs. Because the sequence of the permuted segment, a beta-strand that bisects the catalytic domain, is conserved among orthologs from bacteria, archaea and eukarya, the permutation likely happened early in evolution.     

The role of tyrosine 343 in substrate binding and catalysis by human sulfite oxidase

In the crystal structure of chicken sulfite oxidase, the residue Tyr(322) (Tyr(343) in human sulfite oxidase) was found to directly interact with a bound sulfate molecule and was proposed to have an important role in mediating the substrate specificity and catalytic activity of this molybdoprotein. In order to understand the role of this residue in the catalytic mechanism of sulfite oxidase, steady-state and stopped-flow analyses were performed on wild-type and Y343F human sulfite oxidase over the pH range 6-10. In steady-state assays of Y343F sulfite oxidase using cytochrome c as the electron acceptor, k(cat) was somewhat impaired ( approximately 34% wild-type activity at pH 8.5), whereas the K(m)(sulfite) showed a 5-fold increase over wild type. In rapid kinetic assays of the reductive half-reaction of wild-type human sulfite oxidase, k(red)(heme) changed very little over the entire pH range, with a significant increase in K(d)(sulfite) at high pH. The k(red)(heme) of the Y343F variant was significantly impaired across the entire pH range, and unlike the wild-type protein, both k(red)(heme) and K(d)(sulfite) were dependent on pH, with a significant increase in both kinetic parameters at high pH. Additionally, reduction of the molybdenum center by sulfite was directly measured for the first time in rapid reaction assays using sulfite oxidase lacking the N-terminal heme-containing domain. Reduction of the molybdenum center was quite fast (k(red)(Mo) = 972 s(-1) at pH 8.65 for wild-type protein), indicating that this is not the rate-limiting step in the catalytic cycle. Reduction of the molybdenum center of the Y343F variant by sulfite was more significantly impaired at high pH than at low pH. These results demonstrate that the Tyr(343) residue is important for both substrate binding and oxidation of sulfite by sulfite oxidase.     

Cloning and characterization of a mammalian pseudouridine synthase

This report describes the cloning and characterization of a pseudouridine (psi) synthase from mouse that we have named mouse pseudouridine synthase 1 (mpus1p). The cDNA is approximately 1.5 kb and when used as a probe on a Northern blot of mouse RNA from tissues and cultured cells, several bands were detected. The open reading frame is 393 amino acids and has 35% identity over its length with yeast psi synthase 1 (pus1p). The recombinant protein was expressed in Escherichia coli and the purified protein converted specific uridines to psi in a number of tRNA substrates. The positions modified in stoichiometric amounts in vitro were 27/28 in the anticodon stem and also positions 34 and 36 in the anticodon of an intron containing tRNA. A human cDNA was also cloned and the smaller open reading frame (348 amino acids) was 92% identical over its length with mpus1p but is shorter by 45 amino acids at the amino terminus. The expressed recombinant human protein has no activity on any of the tRNA substrates, most probably the result of the truncated open reading frame.     

Multiple roles of a conserved GAF domain tyrosine residue in cyanobacterial and plant phytochromes

The phytochrome family of red/far-red photoreceptors has been optimized to support photochemical isomerization of a bound bilin chromophore, a process that triggers a conformational change and modulates biochemical output from the surrounding protein scaffold. Recent studies have established that the efficiency of this photochemical process is profoundly altered by mutation of a conserved tyrosine residue (Tyr176) within the bilin-binding GAF domain of the cyanobacterial phytochrome Cph1 [Fischer, A. J., and Lagarias, J. C. (2004) Harnessing phytochrome's glowing potential, Proc. Natl. Acad. Sci. U.S.A. 101, 17334-17339]. Here, we show that the equivalent mutation in plant phytochromes behaves similarly, indicating that the function of this tyrosine in the primary photochemical mechanism is conserved. Saturation mutagenesis of Tyr176 in Cph1 establishes that no other residue can support comparably efficient photoisomerization. The spectroscopic consequences of Tyr176 mutations also reveal that Tyr176 regulates the conversion of the porphyrin-like conformation of the bilin precursor to a more extended conformation. The porphyrin-binding ability of the Tyr176Arg mutant protein indicates that Tyr176 also regulates the ligand-binding specificity of apophytochrome. On the basis of the hydrogen-bonding ability of Tyr176 substitutions that support the nonphotochemical C15-Z,syn to C15-Z,anti interconversion, we propose that Tyr176 orients the carboxyl side chain of a conserved acidic residue to stabilize protonation of the bilin chromophore. A homology model of the GAF domain of Cph1 predicts a C5-Z,syn, C10-Z,syn, C15-Z,anti configuration for the chromophore and implicates Glu189 as the proposed acidic residue stabilizing the extended conformation, an interpretation consistent with site-directed mutagenesis of this conserved acidic residue.     

Mutation of a strictly conserved, active-site residue alters substrate specificity and cofactor biogenesis in a copper amine oxidase

The copper amine oxidases (CAOs) catalyze both the single-turnover modification of a peptidyl tyrosine to form the active-site cofactor 2,4,5-trihydroxyphenylalanine quinone (TPQ) and the oxidative deamination of primary amines using TPQ. The function of a strictly conserved tyrosine located within hydrogen-bonding distance to TPQ has been explored by employing site-directed mutagenesis on the enzyme from H. polymorpha to form the mutants Y305A, Y305C, and Y305F. Both Y305A and Y305C behave similarly with regard to aliphatic amine oxidase activity, showing 3-7-fold decreases in kinetic parameters relative to WT, while the more conservative substitution of Y305F results in a >100-fold decrease in kcat and >500-fold decrease in kcat/Km relative to WT for the reductive half-reaction. The oxidation of benzylamine by all three mutants is severely impaired, with very significant effects seen in the oxidative half-reaction. CAO activity was studied as a function of pH for WT and Y305A proteins. Profiles for WT-catalyzed methylamine oxidation and Y305A-catalyzed ethylamine oxidation are comparable, while profiles of Y305A-catalyzed methylamine oxidation suggest the pH-dependent build-up of an inhibitory intermediate, which was subsequently observed spectrophotometrically and is attributed to the product Schiff base. The relative effects of mutations at Y305 on catalytic turnover are, thus, concluded to be dependent on the nature of the amino acid which substitutes for tyrosine and the substrate used in amine oxidase assays. TPQ biogenesis experiments demonstrate a approximately 800-fold decrease in kobs for apo-Y305A compared to WT. Despite the strict conservation of Tyr305 in all CAOs, neither biogenesis nor catalytic turnover is abolished upon mutation of this residue. We propose an important, but nonessential, role for Tyr305 in the positioning of the TPQ precursor for biogenesis, and in the maintenance of the correct conformation for TPQ-derived intermediates during catalytic turnover.     

Allosteric regulation of substrate channeling and catalysis in the tryptophan synthase bienzyme complex

The tryptophan synthase α₂β₂ bi-enzyme complex catalyzes the last two steps in the synthesis of l-tryptophan (l-Trp). The α-subunit catalyzes cleavage of 3-indole-d-glycerol 3′-phosphate (IGP) to give indole and d-glyceraldehyde 3′-phosphate (G3P). Indole is then transferred (channeled) via an interconnecting 25 Å-long tunnel, from the α-subunit to the β-subunit where it reacts with l-Ser in a pyridoxal 5′-phosphate-dependent reaction to give l-Trp and a water molecule. The efficient utilization of IGP and l-Ser by tryptophan synthase to synthesize l-Trp utilizes a system of allosteric interactions that (1) function to switch the α-site on and off at different stages of the β-subunit catalytic cycle, and (2) prevent the escape of the channeled intermediate, indole, from the confines of the α- and β-catalytic sites and the interconnecting tunnel. This review discusses in detail the chemical origins of the allosteric interactions responsible both for switching the α-site on and off, and for triggering the conformational changes between open and closed states which prevent the escape of indole from the bienzyme complex.     

Histidine 282 in 5-aminolevulinate synthase affects substrate binding and catalysis

5-Aminolevulinate synthase (ALAS), the first enzyme of the heme biosynthetic pathway in mammalian cells, is a member of the alpha-oxoamine synthase family of pyridoxal 5'-phosphate (PLP)-dependent enzymes. In all structures of the enzymes of the -oxoamine synthase family, a conserved histidine hydrogen bonds with the phenolic oxygen of the PLP cofactor and may be significant for substrate binding, PLP positioning, and maintenance of the pKa of the imine nitrogen. In ALAS, replacing the equivalent histidine, H282, with alanine reduces the catalytic efficiency for glycine 450-fold and decreases the slow phase rate for glycine binding by 85%. The distribution of the absorbing 420 and 330 nm species was altered with an A420/A330 ratio increased from 0.45 to 1.05. This shift in species distribution was mirrored in the cofactor fluorescence and 300-500 nm circular dichroic spectra and likely reflects variation in the tautomer distribution of the holoenzyme. The 300-500 nm circular dichroism spectra of ALAS and H282A diverged in the presence of either glycine or aminolevulinate, indicating that the reorientation of the PLP cofactor upon external aldimine formation is impeded in H282A. Alterations were also observed in the K(Gly)d value and spectroscopic and kinetic properties, while the K(PLP)d increased 9-fold. Altogether, the results imply that H282 coordinates the movement of the pyridine ring with the reorganization of the active site hydrogen bond network and acts as a hydrogen bond donor to the phenolic oxygen to maintain the protonated Schiff base and enhance the electron sink function of the PLP cofactor.     

Role of a strictly conserved active site tyrosine in cofactor genesis in the copper amine oxidase from Hansenula polymorpha

The copper amine oxidases catalyze the O(2)-dependent, two-electron oxidation of amines to aldehydes at an active site that contains Cu(II) and topaquinone (TPQ) cofactor. TPQ arises from the autocatalytic, post-translational oxidation of a tyrosine side chain within the same active site. The contributions of individual active site amino acids to each of these chemical processes are being delineated. Previously, using the amine oxidase from the yeast Hansenula polymorpha (HPAO), mutations of a strictly conserved and structurally pivotal active site tyrosine (Y305) were studied and their effects on the catalytic cycle demonstrated [Hevel, J. M., Mills, S. A., and Klinman, J. P. (1999) Biochemistry 38, 3683-3693]. This study examines mutations at the same position for their effects on cofactor generation. While the Y305A mutation had moderate effects on the kinetics of catalysis (2.5- and 8-fold effects on k(cat) using ethylamine and benzylamine as substrates), the same mutation slows cofactor formation by approximately 45-fold relative to that of the wild-type (WT). Additionally, the Y305A mutant forms at least two species: primarily TPQ at lower pH and a species with a blue-shifted absorbance at high pH (lambda(max) = 400 nm). The 400 nm species does not react with phenylhydrazine or ethylamine and is stable toward pH buffer exchange, long-term storage (>3 weeks), incubation at high temperatures, or incubation with reductants and colorimetric peroxide quenching reagents. A similar species accumulates appreciably even at approximately neutral pH in the Y305F mutant, despite the fact that the rate of TPQ formation is reduced only 3-fold relative to that of WT HPAO. This small impact of Y305F on the rate of biogenesis contracts with a decrease in k(cat) (using ethylamine as the substrate) of 125-fold. The opposing effects of mutations at position 305 in biogenesis versus catalysis indicate that a single residue can be recruited for different roles during these processes.     

The role of arginine residues in substrate binding and catalysis by deacetoxycephalosporin C synthase.

Deacetoxycephalosporin C synthase (DAOCS) catalyses the oxidative ring expansion of penicillin N, the committed step in the biosynthesis of cephamycin C by Streptomyces clavuligerus. Site-directed mutagenesis was used to investigate the seven Arg residues for activity (74, 75, 160, 162, 266, 306 and 307), selected on the basis of the DAOCS crystal structure. Greater than 95% of activity was lost upon mutation of Arg-160 and Arg266 to glutamine or other residues. These results are consistent with the proposed roles for these residues in binding the carboxylate linked to the nucleus of penicillin N (Arg160 and Arg162) and the carboxylate of the alpha-aminoadipoyl side-chain (Arg266). The results for mutation of Arg74 and Arg75 indicate that these residues play a less important role in catalysis/binding. Together with previous work, the mutation results for Arg306 and Arg307 indicate that modification of the C-terminus may be profitable with respect to altering the penicillin side-chain selectivity of DAOCS.     

Role of the PHTH module in protein substrate recognition by Bruton's agammaglobulinemia tyrosine kinase

Defects in Bruton's tyrosine kinase (Btk) are responsible for X chromosome-linked agammaglobulinemia in patients. Mutations in each of the structural domains of Btk have been detected in patients, yet a mechanistic explanation for most of these mutant phenotypes is lacking. To understand the possible role of the unique pleckstrin homology and Tec homology (PHTH) module of Btk, we have compared the enzymatic properties of full-length Btk and a Btk mutant lacking the PHTH module (BtkDeltaPHTH). Here we show that Btk and BtkDeltaPHTH have similar basal catalytic activity but very different abilities to recognize protein substrates. Furthermore, the catalytic domain of Btk is inactive, in contrast to the catalytic domain of the prototypical Src tyrosine kinase that retains full catalytic ability. These data suggest that the PHTH module plays an important role in protein substrate recognition, that Btk and Src likely have different interdomain organizations and regulations, and that alterations in substrate recognition might play a role in X chromosome-linked agammaglobulinemia.