Substitution of asparagine 76 by a tyrosine residue induces domain swapping in Helicobacter pylori phosphopantetheine adenylyltransferase

Phosphopantetheine adenylyltransferase (PPAT) catalyses the penultimate step in coenzyme A biosynthesis in bacteria and is therefore a candidate target for antibacterial drug development. We randomly mutated the residues in the Helicobacter pylori PPAT sequence to identify those that govern protein folding and ligand binding, and we describe the crystal structure of one of these mutants (I4V/N76Y) that contains the mutations I4?→?V and N76?→?Y. Unlike other PPATs, which are homohexamers, I4V/N76Y is a domain-swapped homotetramer. The protomer structure of this mutant is an open conformation in which the 65 C-terminal residues are intertwined with those of a neighbouring protomer. Despite structural differences between wild-type PPAT and IV4/N76Y, they had similar ligand-binding properties. ATP binding to these two proteins was enthalpically driven, whereas that for Escherichia coli PPAT is entropically driven. The structural packing of the subunits may affect the thermal denaturation of wild-type PPAT and I4V/N76Y. Mutations in hinge regions often induce domain swapping, i.e. the spatial exchange of portions of adjacent protomers, but residues 4 and 76 of H. pylori PPAT are not located in or near to the hinge region. However, one or both of these residues is responsible for the large conformational change in the C-terminal region of each protomer. To identify the residue(s) responsible, we constructed the single-site mutant, N76Y, and found a large displacement of α-helix 4, which indicated that its flexibility allowed the domain swap to occur.     

Conserved aromatic residues of the C-terminus of human butyrylcholinesterase mediate the association of tetramers.

Human butyrylcholinesterase (BChE) in serum is composed predominantly of tetramers. The tetramerization domain of each subunit is contained within 40 C-terminal residues. To identify key residues within this domain participating in tetramer stabilization, the interaction between C-terminal 46 residue peptides was quantitated in the yeast two-hybrid system. The wild-type peptide interacted strongly with another wild-type peptide in the yeast two-hybrid system. The C571A mutant peptides interacted to a similar degree as the wild-type. However, the mutant in which seven conserved aromatic residues (Trp 543, Phe 547, Trp 550, Tyr 553, Trp 557, Phe 561, and Tyr 564) and C571 were altered to alanines showed only 12% of the interaction seen with the wild-type peptide. The seven mutations (aromatics-off) were incorporated into the complete BChE molecule, with or without the C571A mutation, and expressed in 293T and CHO-K1 cells. Expression of wild-type BChE in these cell lines yielded 10% tetramers. The aromatics-off mutant formed dimers and monomers but no tetramers. The aromatics-off/C571A mutant yielded only monomers. Addition of poly-L-proline to culture medium, or coexpression with the N-terminus of COLQ including the proline-rich attachment domain (Q(N)PRAD), increased the amount of tetrameric wild-type BChE from 10 to 70%, but had no effect on the G534stop (lacking 41 C-terminal residues) and the aromatics-off mutants. Recombinant BChE produced by coexpression with Q(N)PRAD was purified by column chromatography. The purified tetramers contained the FLAG-tagged Q(N)PRAD peptide. These observations suggest that the stabilization of BChE tetramers is mediated through the interaction of the seven conserved aromatic residues and that poly-L-proline and PRAD act through these aromatic residues to induce tetramerization.     

Analysis of a conserved hydrophobic pocket important for the thermostability of Bacillus pumilus chloramphenicol acetyltransferase (CAT-86)

Site-directed mutagenesis was carried out on Bacillus pumilus chloramphenicol acetyltransferase (CAT-86) to determine the effects of substitution at a conserved hydrophobic pocket identified earlier as important for thermostability. Mutations were introduced that would substitute residues at consensus positions 33, 191 and 203 in the enzyme, both individually and in combination. Two mutants, SDM1 (CAT-86 Y33F, A203V) and SDM5 (CAT-86 A203I), were more thermostable than wild-type and two mutants, SDM4 (CAT-86 I191V) and SDM7 (CAT-86 A203G), were less stable. Reconstruction of the residues of this hydrophobic pocket to that of a more thermostable CAT-R387 enzyme pocket (as a Y33F, I191V, A203V triple mutant) increased the thermostability of the enzyme above the wild-type, but its stability was less than that of SDM1 and SDM5. The K(m) values of the mutant enzymes for chloramphenicol and acetyl-CoA were essentially unaltered (in the ranges 15-30 and 26-35 microM respectively) and the specific activity of purified enzyme was in the range 270-710 units/mg protein. The possible effects of the amino acid substitutions on the CAT-86 structure were determined by homology modelling. A reduction in conformational strain and optimized hydrophobic interactions are predicted to be responsible for the increased thermostability of the SDM1 and SDM5 mutants.     

Identification of residues critical for toxicity in Clostridium perfringens phospholipase C, the key toxin in gas gangrene.

Clostridium perfringens phospholipase C (PLC), also called alpha-toxin, is the major virulence factor in the pathogenesis of gas gangrene. The toxic activities of genetically engineered alpha-toxin variants harboring single amino-acid substitutions in three loops of its C-terminal domain were studied. The substitutions were made in aspartic acid residues which bind calcium, and tyrosine residues of the putative membrane-interacting region. The variants D269N and D336N had less than 20% of the hemolytic activity and displayed a cytotoxic potency 103-fold lower than that of the wild-type toxin. The variants in which Tyr275, Tyr307, and Tyr331 were substituted by Asn, Phe, or Leu had 11-73% of the hemolytic activity and exhibited a cytotoxic potency 102- to 105-fold lower than that of the wild-type toxin. The results demonstrated that the sphingomyelinase activity and the C-terminal domain are required for myotoxicity in vivo and that the variants D269N, D336N, Y275N, Y307F, and Y331L had less than 12% of the myotoxic activity displayed by the wild-type toxin. This work therefore identifies residues critical for the toxic activities of C. perfringens PLC and provides new insights toward understanding the mechanism of action of this toxin at a molecular level.     

A monomeric human apolipoprotein E carboxyl-terminal domain

ApoE plays a critical role in lipoprotein metabolism and plasma lipid homeostasis through its high-affinity binding to the LDL-receptor family. In solution, apoE is an oligomeric protein and the C-terminal domain causes apoE's aggregation. The aggregation property presents a major difficulty for the structural determination of this protein. A high-level expression system of the apoE C-terminal domain is reported here. Using protein engineering techniques, we identified a monomeric, biologically active apoE C-terminal domain mutant. This mutant replaces five bulky hydrophobic residues in the region of residues 253-289 with either smaller hydrophobic or polar/charged residues (F257A, W264R, V269A, L279Q, and V287E). The solubility of the mutant is significantly increased ( approximately 10-fold). Cross-linking experiments indicate that this mutant is 100% monomeric even at 5 mg/mL. CD and guanidine hydrochloride denaturation results indicate that the mutant maintains an identical alpha-helical secondary structure and stability as compared with those of the wild-type protein. DMPC-binding assays demonstrate an identical vesicle clearance rate shared by both the mutant and the wild-type apoE C-terminal domain. In addition, electron microscopic results show identical recombinant HDL particles prepared with both the mutant and the wild-type proteins. These results indicate that residues F257, W264, V269, L279, and V287 are critical residues for aggregation but may not be important in maintaining the structure, stability, and lipid-binding activity of this apoE domain, suggesting that apoE may use different "epitopes" for its aggregation property, helical structure/stability, and lipid-binding activity. Finally, preliminary NMR data demonstrated that we have collected high-quality NMR spectra, allowing for an NMR structural determination of the apoE C-terminal domain.     

Multiple Roles of the Extracellular Vestibule Amino Acid Residues in the Function of the Rat P2X4 Receptor

The binding of ATP to trimeric P2X receptors (P2XR) causes an enlargement of the receptor extracellular vestibule, leading to opening of the cation-selective transmembrane pore, but specific roles of vestibule amino acid residues in receptor activation have not been evaluated systematically. In this study, alanine or cysteine scanning mutagenesis of V47–V61 and F324–N338 sequences of rat P2X4R revealed that V49, Y54, Q55, F324, and G325 mutants were poorly responsive to ATP and trafficking was only affected by the V49 mutation. The Y54F and Y54W mutations, but not the Y54L mutation, rescued receptor function, suggesting that an aromatic residue is important at this position. Furthermore, the Y54A and Y54C receptor function was partially rescued by ivermectin, a positive allosteric modulator of P2X4R, suggesting a rightward shift in the potency of ATP to activate P2X4R. The Q55T, Q55N, Q55E, and Q55K mutations resulted in non-responsive receptors and only the Q55E mutant was ivermectin-sensitive. The F324L, F324Y, and F324W mutations also rescued receptor function partially or completely, ivermectin action on channel gating was preserved in all mutants, and changes in ATP responsiveness correlated with the hydrophobicity and side chain volume of the substituent. The G325P mutant had a normal response to ATP, suggesting that G325 is a flexible hinge. A topological analysis revealed that the G325 and F324 residues disrupt a β-sheet upon ATP binding. These results indicate multiple roles of the extracellular vestibule amino acid residues in the P2X4R function: the V49 residue is important for receptor trafficking to plasma membrane, the Y54 and Q55 residues play a critical role in channel gating and the F324 and G325 residues are critical for vestibule widening.     

Identification of amino acid residues responsible for taurocyamine binding in mitochondrial taurocyamine kinase from Arenicola brasiliensis

In order to investigate the residues associated with binding of the substrate taurocyamine in Arenicola mitochondrial taurocyamine kinase (TK), we performed Ala-scanning of the amino acid sequence HTKTV at positions 67-71 on the GS loop, and determined apparent K(m) and V(max) (appK(m) and appV(max), respectively) of the mutant forms for the substrates taurocyamine and glycocyamine. The appK(m) values for taurocyamine of the K69A, T70A and V71A mutants were significantly increased as compared with wild-type, suggesting that these residues are associated with taurocyamine binding. Of special interest is a property of V71A mutant: its catalytic efficiency for glycocyamine was twice that for taurocyamine, indicating that the V71A mutant acts like a glycocyamine kinase, rather than a TK. The role of the amino acid residue K95 of Arenicola MiTK was also examined. K95 was replaced with R, H, Y, I, A and E. K95R, K95H and K95I have a 3-fold higher affinity for taurocyamine, and activity was largely lost in K95E. On the other hand, the K95Y mutant showed a rather unique feature; namely, an increase in substrate concentration caused a decrease in initial velocity of the reaction (substrate inhibition). This is the first report on the key amino acid residues responsible for taurocyamine binding in mitochondrial TK.     

Catalysis and binding in L-ribulose-5-phosphate 4-epimerase: a comparison with L-fuculose-1-phosphate aldolase.

L-Ribulose-5-phosphate (L-Ru5P) 4-epimerase and L-fuculose-1-phosphate (L-Fuc1P) aldolase are evolutionarily related enzymes that display 26% sequence identity and a very high degree of structural similarity. They both employ a divalent cation in the formation and stabilization of an enolate during catalysis, and both are able to deprotonate the C-4 hydroxyl group of a phosphoketose substrate. Despite these many similarities, subtle distinctions must be present which allow the enzymes to catalyze two seemingly different reactions and to accommodate substrates differing greatly in the position of the phosphate (C-5 vs C-1). Asp76 of the epimerase corresponds to the key catalytic acid/base residue Glu73 of the aldolase. The D76N mutant of the epimerase retained considerable activity, indicating it is not a key catalytic residue in this enzyme. In addition, the D76E mutant did not show enhanced levels of background aldolase activity. Mutations of residues in the putative phosphate-binding pocket of the epimerase (N28A and K42M) showed dramatically higher values of K(M) for L-Ru5P. This indicates that both enzymes utilize the same phosphate recognition pocket, and since the phosphates are positioned at opposite ends of the respective substrates, the two enzymes must bind their substrates in a reversed or "flipped" orientation. The epimerase mutant D120N displays a 3000-fold decrease in the value of k(cat), suggesting that Asp120' provides a key catalytic acid/base residue in this enzyme. Analysis of the D120N mutant by X-ray crystallography shows that its structure is indistinguishable from that of the wild-type enzyme and that the decrease in activity was not simply due to a structural perturbation of the active site. Previous work [Lee, L. V., Poyner, R. R., Vu, M. V., and Cleland, W. W. (2000) Biochemistry 39, 4821-4830] has indicated that Tyr229' likely provides the other catalytic acid/base residue. Both of these residues are supplied by an adjacent subunit. Modeling of L-Ru5P into the active site of the epimerase structure suggests that Tyr229' is responsible for deprotonating L-Ru5P and Asp120' is responsible for deprotonating its epimer, D-Xu5P.     

Effects of point mutations in a hinge region on the stability, folding, and enzymatic activity of Escherichia coli dihydrofolate reductase

The role of a hinge region in the folding, stability, and activity of Escherichia coli dihydrofolate reductase was investigated with three site-directed mutants at valine-88, the central residue of the hinge. The three mutants, V88A and V88I and a valine-88 deletion, were created to perturb the packing of hydrophobic residues in the interior of a loose turn formed by residues 85-91. Deleting the valine-88 residue destabilized the protein by 2.93 +/- 0.6 kcal/mol as determined by equilibrium unfolding transitions in urea monitored by circular dichroism at 20 degrees C. Substitution of alanine for valine-88 stabilized the protein by -0.20 +/- 0.02 kcal/mol, and the isoleucine substitution was mildly destabilizing by 1.73 +/- 0.2 kcal/mol. Although there was no clear correlation between side-chain volume and stability, these results suggest that side-chain interactions in the interior of the turn influence the folding and stability of dihydrofolate reductase. The specific activity of the valine deletion mutant was approximately twice that of the wild-type protein while the specific activities of the V88A and V88I proteins were only slightly greater than the wild type. The full time courses of the reactions catalyzed by the mutants were almost identical with that for the wild type, indicating no major changes in the kinetic mechanism. Additionally, the rate constants associated with interconversion between various forms of the apoenzyme were identical for the mutant and wild-type enzymes. The rate constants for refolding transitions were examined by dilution of urea-inactivated protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutations in two distinct regions of acetolactate synthase regulatory subunit from Streptomyces cinnamonensis result in the lack of sensitivity to end-product inhibition

Acetolactate synthase small subunit encoding ilvN genes from the parental Streptomyces cinnamonensis strain and mutants resistant either to valine analogues or to 2-ketobutyrate were cloned and sequenced. The wild-type IlvN from S. cinnamonensis is composed of 175 amino acid residues and shows a high degree of similarity with the small subunits of other valine-sensitive bacterial acetolactate synthases. Changes in the sequence of ilvN conferring the insensitivity to valine in mutant strains were found in two distinct regions. Certain point mutations were located in the conserved domain near the N terminus, while others resulting in the same phenotype shortened the protein at V(104) or V(107). To confirm whether the described mutations were responsible for the changed biochemical properties of the native enzyme, the wild-type large subunit and the wild-type and mutant forms of the small one were expressed separately in E. coli and combined in vitro to reconstitute the active enzyme.     

Structural studies of mutants of T4 lysozyme that alter hydrophobic stabilization

Multiple replacements at amino acid position 3 of bacteriophage T4 lysozyme have shown that the conformational stability of the protein is directly governed by the hydrophobicity of the residue substituted (Matsumura, M., Becktel, W. J., and Matthews, B. W. (1988) Nature 334, 406-410). Of the 13 mutant lysozymes made by site-directed mutagenesis, two variants, one with valine (I3V) and the other with tyrosine (I3Y), were crystallized and their structures solved. In this report we describe the crystal structures of these variants at 1.7 A resolution. While the structure of the I3V mutant is essentially the same as that of wild-type lysozyme, the I3Y mutant has substantial changes in its structure. The most significant of these are that the side chain of the tyrosine is not accommodated within the interior of the protein and the amino-terminal polypeptide (residues 1-9) moves 0.6-1.1 A relative to the wild-type structure. Using coordinates based on the wild-type and available mutant structures, solvent accessible surface area of residue 3 as well as the adjacent 9 residues in the folded form were calculated. The free energy of stabilization based on the transfer of these residues from a fully extended form to the interior to the folded protein was found to correlate well with the protein stability determined by thermodynamic analysis. The enhanced thermostability of the variant Ile-3----Leu, relative to wild-type lysozyme, can also be rationalized by surface-area calculations based on a model-built structure. Noncrystallization of most lysozyme variants at position 3 appears to be due to disruption of intermolecular contacts in the crystal. The Ile-3----Val variant is closely isomorphous with wild-type and maintains the same crystal contacts. In the Ile-3----Tyr variant, however, a new set of contacts is made in which direct protein-protein hydrogen bonds are replaced by protein-water-protein hydrogen bonds as well as a novel hydrogen bond involving the phenolic hydroxyl of the substituted tyrosine.     

Controlling the substrate selectivity of deacetoxycephalosporin/deacetylcephalosporin C synthase

Deacetoxycephalosporin/deacetylcephalosporin C synthase (DAOC/DACS) is an iron(II) and 2-oxoglutarate-dependent oxygenase involved in the biosynthesis of cephalosporin C in Cephalosporium acremonium. It catalyzes two oxidative reactions, oxidative ring-expansion of penicillin N to deacetoxycephalosporin C, and hydroxylation of the latter to give deacetylcephalosporin C. The enzyme is closely related to deacetoxycephalosporin C synthase (DAOCS) and DACS from Streptomyces clavuligerus, which selectively catalyze ring-expansion or hydroxylation reactions, respectively. In this study, structural models based on DAOCS coupled with site-directed mutagenesis were used to identify residues within DAOC/DACS that are responsible for controlling substrate and reaction selectivity. The M306I mutation abolished hydroxylation of deacetylcephalosporin C, whereas the W82A mutant reduced ring-expansion of penicillin G (an "unnatural" substrate). Truncation of the C terminus of DAOC/DACS to residue 310 (Delta310 mutant) enhanced ring-expansion of penicillin G by approximately 2-fold. A double mutant, Delta310/M306I, selectively catalyzed the ring-expansion reaction and had similar kinetic parameters to the wild-type DAOC/DACS. The Delta310/N305L/M306I triple mutant selectively catalyzed ring-expansion of penicillin G and had improved kinetic parameters (K(m) = 2.00 +/- 0.47 compared with 6.02 +/- 0.97 mm for the wild-type enzyme). This work demonstrates that a single amino acid residue side chain within the DAOC/DACS active site can control whether the enzyme catalyzes ring-expansion, hydroxylation, or both reactions. The catalytic efficiency of mutant enzymes can be improved by combining active site mutations with other modifications including C-terminal truncation and modification of Asn-305.     

Active-site residues are critical for the folding and stability of methylamine dehydrogenase.

Site-directed mutagenesis was used to alter active-site residues of methylamine dehydrogenase (MADH) from Paracoccus denitrificans. Four residues of the beta subunit of MADH which are in close proximity to the tryptophan tryptophylquinone (TTQ) prosthetic group were modified. The crystal structure of MADH reveals that each of these residues participates in hydrogen bonding interactions with other active-site residues, TTQ or water. Relatively conservative mutations which removed the potentially reactive oxygens on the side chains of Thr122, Tyr119, Asp76 and Asp32 each resulted in greatly reduced or undetectable levels of MADH production. The reduction of MADH levels was determined by assays of activity and Western blots of crude extracts with antisera specific for the MADH beta subunit. No activity or cross-reactive protein was detected in extracts of cells expressing D76N, T122A and T122C MADH mutants. Very low levels of active MADH were produced by cells expressing D32N, Y119F, Y119E and Y119K MADH mutants. The Y119F and D32N mutants were purified from cell extracts and found to be significantly less stable than wild-type MADH. Only the T122S MADH mutant was produced at near wild-type levels. Possible roles for these amino acid residues in stabilizing unusual structural features of the MADH beta subunit, protein folding and TTQ biosynthesis are discussed.     

Structural and functional roles of deamidation and/or truncation of N- or C-termini in human alpha A-crystallin

The purpose of the study was to compare the effects of deamidation alone, truncation alone, or both truncation and deamidation on structural and functional properties of human lens alphaA-crystallin. Specifically, the study investigated whether deamidation of one or two sites in alphaA-crystallin (i.e., alphaA-N101D, alphaA-N123D, alphaA-N101/123D) and/or truncation of the N-terminal domain (residues 1-63) or C-terminal extension (residues 140-173) affected the structural and functional properties relative to wild-type (WT) alphaA. Human WT-alphaA and human deamidated alphaA (alphaA-N101D, alphaA-N123D, alphaA-N101/123D) were used as templates to generate the following eight N-terminal domain (residues 1-63) deleted or C-terminal extension (residues 140-173) deleted alphaA mutants and deamidated plus N-terminal domain or C-terminal extension deleted mutants: (i) alphaA-NT (NT, N-terminal domain deleted), (ii) alphaA-N101D-NT, (iii) alphaA-N123D-NT, (iv) alphaA-N101/123D-NT, (v) alphaA-CT (CT, C-terminal extension deleted), (vi) alphaA-N101D-CT, (vii) alphaA-N123D-CT, and (viii) alphaA-N101/123D-CT. All of the proteins were purified and their structural and functional (chaperone activity) properties determined. The desired deletions in the alphaA-crystallin mutants were confirmed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. Relative to WT-alphaA homomers, the mutant proteins exhibited major structural and functional changes. The maximum decrease in chaperone activity in homomers occurred on deamidation of N123 residue, but it was substantially restored after N- or C-terminal truncations in this mutant protein. Far-UV circular dichroism (CD) spectral analyses generally showed an increase in the beta-contents in alphaA mutants with deletions of N-terminal domain or C-terminal extension and also with deamidation plus above N- or C-terminal deletions. Intrinsic tryptophan (Trp) and total fluorescence spectral studies suggested altered microenvironments in the alphaA mutant proteins. Similarly, the ANS (8-anilino-1-naphthalenesulfate) binding showed generally increased fluorescence with blue shift on deletion of the N-terminal domain in the deamidated mutant proteins, but opposite effects were observed on deletion of the C-terminal extension. Molecular mass, polydispersity of homomers, and the rate of subunit exchange with WT-alphaB-crystallin increased on deletion of the C-terminal extension in the deamidated alphaA mutants, but on N-terminal domain deletion these values showed variable results based on the deamidation site. In summary, the data suggested that the deamidation alone showed greater effect on chaperone activity than the deletion of N-terminal domain or C-terminal extension of alphaA-crystallin. The N123 residue of alphaA-crystallin plays a crucial role in maintaining its chaperone function. However, both the N-terminal domain and C-terminal extension are also important for the chaperone activity of alphaA-crystallin because the activity was partially or fully recovered following either deletion in the alphaA-N123D mutant. The results of subunit exchange rates among alphaA mutants and WT-alphaB suggested that such exchange is an important determinant in maintenance of chaperone activity following deamidation and/or deletion of the N-terminal domain or C-terminal extension in alphaA-crystallin.     

Crystal structure and biophysical characterisation of Helicobacter pylori phosphopantetheine adenylyltransferase

Helicobacter pylori is a bacterium that causes chronic active gastritis and peptic ulcers. Drugs targeting H. pylori phosphopantetheine adenylyltransferase (HpPPAT), which is involved in CoA biosynthesis, may be useful. Herein, we report the expression in Escherichia coli and purification of recombinant HpPPAT and describe a crystal structure for an HpPPAT/CoA complex. As is the case for E. coli PPAT (EcPPAT), HpPPAT is hexameric in solution and as a crystal. Each protomer has a well-packed dinucleotide-binding fold in which CoA binds. Structural characterisation demonstrated that CoA derived from the E. coli expression system bound tightly to HpPPAT, presumably to initiate feedback inhibition. However, the interactions between the active-site residues of HpPPAT and CoA are not identical to those of other PPATs. Finally, CoA binding affects HpPPAT thermal denaturation.     

Identification of unique amino acids that modulate CYP4A7 activity

A multifamily sequence alignment of the rabbit CYP4A members with the known structure of CYP102 indicates amino acid differences falling within the so-called substrate recognition site(s) (SRS). Chimeric proteins constructed between CYP4A4 and CYP4A7 indicate that laurate activity is affected by the residues within SRS1 and prostaglandin activity is influenced by SRS2-3. Site-directed mutant proteins of CYP4A7 found laurate and arachidonate activity markedly diminished in the R90W mutant (SRS1) and somewhat decreased in W93S. While PGE(1) activity was only slightly increased, the mutant proteins H206Y and S255F (SRS2-3), on the other hand, exhibited remarkable increases in laurate and arachidonate metabolism (3-fold) above wild-type substrate metabolism. Mutant proteins H206Y, S255F, and H206Y/S255F but not R90W/W93S, wild-type CYP4A4, or CYP4A7 metabolized arachidonic acid in the absence of cytochrome b(5). Stopped-flow kinetic experiments were performed in a CO-saturated environment performed to estimate interaction rates of the monooxygenase reaction components. The mutant protein H206Y, which exhibits 3-fold higher than wild-type substrate activity, interacts with CPR at a rate at least 10 times faster than that of wild-type CYP4A7. These experimental results provide insight regarding the residues responsible for modulation of substrate specificity, affinity, and kinetics, as well as possible localization within the enzyme structure based on comparisons with homologous, known cytochrome P450 structures.     

Asymmetric Dimeric Structure of Ferredoxin-NAD(P) Oxidoreductase from the Green Sulfur Bacterium Chlorobaculum tepidum: Implications for Binding Ferredoxin and NADP

Ferredoxin-NAD(P)⁺ oxidoreductase (FNR) catalyzes the reduction of NAD(P)⁺ to NAD(P)H with the reduced ferredoxin (Fd) during the final step of the photosynthetic electron transport chain. FNR from the green sulfur bacterium Chlorobaculum tepidum is functionally analogous to plant-type FNR but shares a structural homology to NADPH-dependent thioredoxin reductase (TrxR). Here, we report the crystal structure of C. tepidum FNR to 2.4 Å resolution, which reveals a unique structure-function relationship. C. tepidum FNR consists of two functional domains for binding FAD and NAD(P)H that form a homodimer in which the domains are arranged asymmetrically. One NAD(P)H domain is present as the open form, the other with the equivalent NAD(P)H domain as the relatively closed form. We used site-directed mutagenesis on the hinge region connecting the two domains in order to investigate the importance of the flexible hinge. The asymmetry of the NAD(P)H domain and the comparison with TrxR suggested that the hinge motion might be involved in pyridine nucleotide binding and binding of Fd. Surprisingly, the crystal structure revealed an additional C-terminal sub-domain that tethers one protomer and interacts with the other protomer by π-π stacking of Phe337 and the isoalloxazine ring of FAD. The position of this stacking Phe337 is almost identical with both of the conserved C-terminal Tyr residues of plant-type FNR and the active site dithiol of TrxR, implying a unique structural basis for enzymatic reaction of C. tepidum FNR.     

Mutations at the putative active cavity of styrene monooxygenase: Enhanced activity and reversed enantioselectivity

Styrene monooxygenase (SMO) catalyzes the first step of styrene degradation, and also serves as an important enzyme for the synthesis of enantiopure epoxides. To enhance its activity, molecular docking of styrene was performed based on the X-ray crystal structure of the oxygenase subunit of SMO to identify three amino acid residues (Tyr73, His76 and Ser96) being adjacent to the phenyl ring of styrene. Variants at those positions were constructed and their enzymatic activities were analyzed. Three mutants (Y73V, Y73F, and S96A) were found to exhibit higher enzymatic activities than the wild-type in the epoxidation of styrene, while retaining excellent stereoselectivity. The specific epoxidation activity of the most active mutant S96A toward styrene and trans-β-methyl styrene were 2.6 and 2.3-fold of the wild-type, respectively. In addition, the Y73V mutant showed an unexpected reversal of enantiomeric preference toward 1-phenylcyclohexene.     

Alphavirus nucleocapsid protein contains a putative coiled coil alpha-helix important for core assembly

The alphavirus nucleocapsid core is formed through the energetic contributions of multiple noncovalent interactions mediated by the capsid protein. This protein consists of a poorly conserved N-terminal region of unknown function and a C-terminal conserved autoprotease domain with a major role in virion formation. In this study, an 18-amino-acid conserved region, predicted to fold into an alpha-helix (helix I) and embedded in a low-complexity sequence enriched with basic and Pro residues, has been identified in the N-terminal region of the alphavirus capsid proteins. In Sindbis virus, helix I spans residues 38 to 55 and contains three conserved leucine residues, L38, L45, and L52, conforming to the heptad amino acid organization evident in leucine zipper proteins. Helix I consists of an N-terminally truncated heptad and two complete heptad repeats with beta-branched residues and conserved leucine residues occupying the a and d positions of the helix, respectively. Complete or partial deletion of helix I, or single-site substitutions at the conserved leucine residues (L45 and L52), caused a significant decrease in virus replication. The mutant viruses were more sensitive to elevated temperature than wild-type virus. These mutant viruses also failed to accumulate cores in the cytoplasm of infected cells, although they did not have defects in protein translation or processing. Analysis of these mutants using an in vitro assembly system indicated that the majority were defective in core particle assembly. Furthermore, mutant proteins showed a trans-dominant negative phenotype in in vitro assembly reactions involving mutant and wild-type proteins. We propose that helix I plays a central role in the assembly of nucleocapsid cores through coiled coil interactions. These interactions may stabilize subviral intermediates formed through the interactions of the C-terminal domain of the capsid protein and the genomic RNA and contribute to the stability of the virion.     

The extreme C terminus of rat liver carnitine palmitoyltransferase I is not involved in malonyl-CoA sensitivity but in initial protein folding

We previously reported that the N-terminal domain (1-147 residues) of rat liver carnitine palmitoyltransferase I (L-CPTI) was essential for import into the outer mitochondrial membrane and for maintenance of a malonyl-CoA-sensitive conformation. Malonyl-CoA binding experiments using mitochondria of Saccharomyces cerevisiae strains expressing wild-type L-CPTI or previously constructed chimeric CPTs (Cohen, I., Kohl, C., McGarry, J.D., Girard, J., and Prip-Buus, C. (1998) J. Biol. Chem. 273, 29896-29904) indicated that the N-terminal domain was unable, independently of the C-terminal domain, to bind malonyl-CoA with a high affinity, suggesting that the modulation of malonyl-CoA sensitivity occurred through N/C intramolecular interactions. To assess the role of the C terminus in malonyl-CoA sensitivity, a series of C-terminal deletion mutants was generated. The kinetic properties of Delta772-773 and Delta767-773 deletion mutants were similar to those of L-CPTI, indicating that the last two highly conserved Lys residues in all known L-CPTI species were not functionally essential. By contrast, Delta743-773 deletion mutant was totally inactive and unfolded, as shown by its sensitivity to trypsin proteolysis. Because the C terminus of the native folded L-CPTI could be cleaved by trypsin without inducing protein unfolding, we concluded that the last 31 C-terminal residues constitute a secondary structural determinant essential for the initial protein folding of L-CPTI.