Oxidizing side of the cyanobacterial photosystem I. Evidence for interaction between the electron donor proteins and a luminal surface helix of the PsaB subunit.

Photosystem I (PSI) interacts with plastocyanin or cytochrome c6 on the luminal side. To identify sites of interaction between plastocyanin/cytochrome c6 and the PSI core, site-directed mutations were generated in the luminal J loop of the PsaB protein from Synechocystis sp. PCC 6803. The eight mutant strains differed in their photoautotrophic growth. Western blotting with subunit-specific antibodies indicated that the mutations affected the PSI level in the thylakoid membranes. PSI proteins could not be detected in the S600R/G601C/N602I, N609K/S610C/T611I, and M614I/G615C/W616A mutant membranes. The other mutant strains contained different levels of PSI proteins. Among the mutant strains that contained PSI proteins, the H595C/L596I, Q627H/L628C/I629S, and N638C/N639S mutants showed similar levels of PSI-mediated electron transfer activity when either cytochrome c6 or an artificial electron donor was used. In contrast, cytochrome c6 could not function as an electron donor to the W622C/A623R mutant, even though the PSI activity mediated by an artificial electron donor was detected in this mutant. Thus, the W622C/A623R mutation affected the interaction of the PSI complex with cytochrome c6. Biotin-maleimide modification of the mutant PSI complexes indicated that His-595, Trp-622, Leu-628, Tyr-632, and Asn-638 in wild-type PsaB may be exposed on the surface of the PSI complex. The results presented here demonstrate the role of an extramembrane loop of a PSI core protein in the interaction with soluble electron donor proteins.     

Independent, spontaneous mutants of adenovirus type 2-simian virus 40 hybrid Ad2+ND3 that grow efficiently in monkey cells possess indentical mutations in the adenovirus type 2 DNA-binding protein gene.

Four independent, spontaneous mutants of the adenovirus type 2-simian virus 40 hybrid Ad2+ND3 that allow efficient growth in monkey cells were isolated previously (C. W. Anderson, Virology 111:263-269, 1981). All four mutations have been mapped within the coding sequence for the adenovirus DNA-binding protein by marker rescue analysis. DNA sequence analysis of a region of ca. 1,000 base pairs shown by marker rescue to contain the host range mutations demonstrated that the host range mutant hr602 differs from its parent, Ad2+ND3, at only a single nucleotide. Mutant hr602 has a thymine in place of a cytosine at the first position of the 130th codon, as measured from the initiation site for the DNA-binding protein. This change results in the replacement of a histidine by a tyrosine in mutant hr602 DNA-binding protein. Each of the other three Ad2+ND3 host range mutants have exactly the same nucleotide alteration as does hr602. This same nucleotide change was recently reported for a similarly derived host range mutant of adenovirus 5.     

Investigation of the selectivity of maltoporin channels using mutant LamB proteins: mutations changing the maltodextrin binding site.

Wild-type and seven mutant maltoporins were purified and their channel-forming activities studied after reconstitution into black lipid membranes. The proteins were assayed for alterations at the maltodextrin binding site by measuring the sugar-dependent blockage of ion flux through these channels. Some substitutions (R8H, W74R) caused reduced channel affinity for all maltodextrins without changing single channel conductivities. The channel with a GlySer insertion after residue 9 was also poorly blocked by sugars but unique to this protein, the channel showed a striking, almost exponential increase of affinity with increasing maltodextrin chain length. In mutants with AspPro insertions after residues 79 and 183, there was an increase in affinity for glucose and maltose but not longer maltodextrins. The additional negative charge in the AspPro insertion mutants increased the cation selectivity of maltoporin channels, as did the decrease in positive charge resulting from the R8H substitution. A mutant with a W120C substitution also showed an increased affinity for glucose and maltose but reduced affinity for longer maltosaccharides. In contrast, a Y118F substitution resulted in an 8-fold increase in maltotriose affinity, but lesser improvements for other sugars. These results are interpreted to reflect changes in subsites contributing to an extended binding site within the channel, which in turn determines the overall sugar affinity of maltoporin.     

Molecular basis of calmodulin binding to cardiac muscle Ca(2+) release channel (ryanodine receptor)

Calmodulin (CaM) is a ubiquitous Ca2+-binding protein that regulates the ryanodine receptors (RyRs) by direct binding. CaM inhibits the skeletal muscle ryanodine receptor (RyR1) and cardiac muscle receptor (RyR2) at >1 microm Ca2+ but activates RyR1 and inhibits RyR2 at <1 microm Ca2+. Here we tested whether CaM regulates RyR2 by binding to a highly conserved site identified previously in RyR1. Deletion of RyR2 amino acid residues 3583-3603 resulted in background [35S]CaM binding levels. In single channel measurements, deletion of the putative CaM binding site eliminated CaM inhibition of RyR2 at Ca2+ concentrations below and above 1 microm. Five RyR2 single or double mutants in the CaM binding region (W3587A, L3591D, F3603A, W3587A/L3591D, L3591D/F3603A) eliminated or greatly reduced [35S]CaM binding and inhibition of single channel activities by CaM depending on the Ca2+ concentration. An RyR2 mutant, which assessed the effects of 4 amino acid residues that differ between RyR1 and RyR2 in or flanking the CaM binding domain, bound [35S]CaM and was inhibited by CaM, essentially identical to wild type (WT)-RyR2. Three RyR1 mutants (W3620A, L3624D, F3636A) showed responses to CaM that differed from corresponding mutations in RyR2. The results indicate that CaM regulates RyR1 and RyR2 by binding to a single, highly conserved CaM binding site and that other RyR type-specific sites are likely responsible for the differential functional regulation of RyR1 and RyR2 by CaM.     

Two nuclear export signals of Cdc6 are differentially associated with CDK-mediated phosphorylation residues for cytoplasmic translocation

Cdc6 is cleaved at residues 442 and 290 by caspase-3 during apoptosis producing p49-tCdc6 and p32-tCdc6, respectively. While p32-tCdc6 is unable to translocate into the cytoplasm, p49-tCdc6 retains cytoplasmic translocation activity, but it has a lower efficiency than wild-type Cdc6. We hypothesized that a novel nuclear export signal (NES) sequence exists between amino acids 290 and 442. Cdc6 contains a novel NES in the region of amino acids 300–315 (NES2) that shares sequence similarity with NES1 at residues 462–476. In mutant versions of Cdc6, we replaced leucine with alanine in NES1 and NES2 and co-expressed the mutant constructs with cyclin A. We observed that the cytoplasmic translocation of these mutants was reduced in comparison to wild-type Cdc6. Moreover, the cytoplasmic translocation of a mutant in which all four leucine residues were mutated to alanine was significantly inhibited in comparison to the translocation of wild-type Cdc6. The Crm1 binding activities of Cdc6 NES mutants were consistent with the efficiency of its cytoplasmic translocation. Further studies have revealed that L468 and L470 of NES1 are required for cytoplasmic translocation of Cdc6 phosphorylated at S74, while L311 and L313 of NES2 accelerate the cytoplasmic translocation of Cdc6 phosphorylated at S54. These results suggest that the two NESs of Cdc6 work cooperatively and distinctly for the cytoplasmic translocation of Cdc6 phosphorylated at S74 and S54 by cyclin A/Cdk2.     

Phlorizin recognition in a C-terminal fragment of SGLT1 studied by tryptophan scanning and affinity labeling

SGLT1 as a sodium/glucose cotransporter is strongly inhibited by phlorizin, a phloretin 2'-glucoside that has strong interactions with the C-terminal loop 13. We have examined phlorizin recognition by the protein by site-directed single Trp scanning mutagenesis experiments. Six mutants (Q581W, E591W, R601W, D611W, E621W, and L630W) of truncated loop 13 (amino acids 564-638) were expressed in Escherichia coli and purified to homogeneity. Changes in Trp quenching and positions of the emission maxima were determined after addition of phlorizin. D611W displayed the largest quenching of 80%, followed by R601W (67%). It also exhibited the maximum red shift in Trp fluorescence ( approximately 14 nm), indicating an exposure of this region to a more hydrophilic environment. Titration experiments performed for each mutant showed a similar affinity for all mutants, except for D611W, which exhibited a significantly lower affinity (Kd approximately 54 microm). Also the maximum change in the collisional quenching constant by acrylamide was noted for D611W (KSV = 11 m-1 in the absence of phlorizin and 55 m-1 in its presence). Similar results were obtained with phloretin. CD measurements and computer modeling revealed that D611W is positioned in a random coil situated between two alpha-helical segments. By combining gel electrophoresis, enzymatic fragmentation, and matrix-assisted laser desorption ionization mass spectrometry, we also analyzed truncated loop 13 photolabeled with 3-azidophlorizin. The attachment site of the ortho-position of aromatic ring B of phlorizin was localized to Arg-602. Taken together, these data indicate that phlorizin binding elicits changes in conformation leading to a less ordered state of loop 13. Modeling suggests an interaction of the 4- and 6-OH groups of aromatic ring A of phlorizin with the region between amino acids 606 and 611 and an interaction of ring B at or around amino acid 602. Phloretin seems to interact with the same region of the protein.     

D-Glucose-recognition and phlorizin-binding sites in human sodium/D-glucose cotransporter 1 (hSGLT1): a tryptophan scanning study

In order to gain a better understanding of the structure-function relation in hSGLT1, single Trp residues were introduced into a functional hSGLT1 mutant devoid of Trps at positions that previously had been postulated to be involved in sugar recognition/translocation and/or phlorizin binding. The mutant proteins were expressed in Pichia pastoris, purified, and reconstituted into liposomes. In transport experiments the putative sugar binding site mutants W457hSGLT1 and W460hSGLT1 showed a drastic decrease in affinity toward alpha-methyl-d-glucopyranoside with Km values of 13.3 and 5.26 mM compared to 0.4 mM of the Trp-less hSGLT1. In addition, a strong decrease in the inhibitory effect of phlorizin was observed. In Trp fluorescence studies the position of the emission maxima of the mutants, their sensitivity to N-bromosuccinimide oxidation, and their interaction with water soluble quenchers demonstrate that Trp457 and Trp460 are in contact with the hydrophilic extravesicular environment. In both mutants Trp fluorescence was quenched significantly, but differently, by various glucose analogues. They also show significant protection by d-glucose and phlorizin against acrylamide, KI, or TCE quenching. W602hSGLT1 and W609hSGLT1, the putative aglucone binding site mutants, exhibit normal sugar and phlorizin affinity, and show fluorescence properties which indicate that these residues are located in a very hydrophilic environment. Phlorizin and phloretin, but not d-glucose, protect both mutants against collisional quenchers. Depth-calculations using the parallax method suggest a location of Trp457 and Trp460 at an average distance of 10.8 A and 7.4 A from the center of the bilayer, while Trp602 and Trp609 are located outside the membrane. These results suggest that in the native carrier residues Gln at position 457 and Thr at position 460 reside in a hydrophilic access pathway extending 5-7 A into the membrane to which sugars as well as the sugar moiety of inhibitory glucosides bind. Residues Phe602 and Phe609 contribute by their hydrophobic aromatic residues toward binding of the aglucone part of phlorizin. Thereby in the phlorizin-carrier complex a close vicinity between these two subdomains of the transporter is established creating a phlorizin binding pocket with the previously estimated dimensions of 10 x 17 x 7 A.     

Roles of Met-34, Cys-64, and Arg-75 in the assembly of human connexin 26. Implication for key amino acid residues for channel formation and function

Connexins form a family of membrane proteins that assemble into communication channels and directly connect the cytoplasms of adjoining cells. Malfunctioning of connexin channels often cause disease, such as the mutations M34T and R75W in human connexin 26, which are associated with hereditary deafness. Another residue known to be essential for normal channel activity in the connexin is Cys-64. To obtain structural and functional insights of connexin 26, we studied the roles of these three residues by expressing mutant connexins in insect Sf9 and HeLa cells. The M34T and M34A mutants both formed gap junction plaques, but dye transfer assays showed that the M34A mutant had a significantly reduced permeability, suggesting that for proper channel function a side chain of adequate size is required at this position. We propose that Met-34 is located in the innermost helix of the channel, where it ensures a fully open channel structure via interactions with other transmembrane helices. Gap junction channels formed by the R75W and R75D mutants dissociated upon solubilization in dodecyl maltoside, whereas the R75A mutant remained hexameric. All gap junctions formed by Arg-75 mutants also showed only negligible activity in dye transfer experiments. These results suggest that residue Arg-75 plays a role in subunit interactions needed to retain a functional and stable connexin hexamer. The C64S mutant was suggested to be defective in oligomerization and/or protein folding even in the presence of wild-type connexin.     

Sugar transport through maltoporin of Escherichia coli: role of the greasy slide

The lining of the maltodextrin-specific maltoporin (LamB) channel exhibits a string of aromatic residues, the greasy slide, part of which has been shown previously by crystallography to be involved in substrate binding. To probe the functional role of the greasy slide, alanine scanning mutagenesis has been performed on the six greasy slide residues and Y118 at the channel constriction. The mutants were characterized by an in vivo uptake assay and sugar-induced-current-noise analysis. Crystallographic analysis of the W74A mutant showed no perturbation of the structure. All mutants showed considerably decreased maltose uptake rates in vivo (<10% of the wild-type value), indicating the functional importance of the investigated residues. Substitutions at the channel center revealed appreciably increased (up to 100-fold) in vitro half-saturation concentrations for maltotriose and maltohexaose binding to the channel. Sugar association rates, however, were significantly affected also by the mutations at either end of the slide (W74A, W358A, and F227A), an effect which became most apparent upon nonsymmetrical sugar addition. The kinetic data are discussed on the basis of an asymmetric one-site two-barrier model, which suggests that, at low substrate concentrations, as are found under physiological conditions, only the heights of the extracellular and periplasmic barriers, which are reduced by the presence of the greasy slide, determine the efficiency of this facilitated diffusion channel.     

Tryptophan scanning of D1S6 and D4S6 C-termini in voltage-gated sodium channels

Recent reports suggest that four S6 C-termini may jointly close the voltage-gated cation channel at the cytoplasmic side, probably as an inverted teepee structure. In this study we substituted individually a total of 18 residues at D1S6 and D4S6 C-terminal ends of the rNav1.4 Na(+) channel alpha-subunit with tryptophan (W) and examined their corresponding gating properties when expressed in Hek293t cells along with beta1 subunit. Several W-mutants displayed significant changes in activation, fast inactivation, and/or slow inactivation gating. In particular, five S6 W-mutants showed incomplete fast inactivation with noninactivating maintained currents present. Cysteine (C) substitutions of these five residues resulted in two mutants with slightly more maintained currents. Multiple substitutions at these five positions yielded two mutants (L437C/A438W, L435W/L437C/A438W) that exhibited phenotypes with minimal fast inactivation. Unexpectedly, such inactivation-deficient mutants expressed Na(+) currents as well as did the wild-type. Furthermore, all mutants with impaired fast inactivation exhibited an enhanced slow inactivation phenotype. Implications of these results will be discussed in terms of indirect allosteric modulations via amino acid substitutions and/or a direct involvement of S6 C-termini in Na(+) channel gating.     

Correlation between the OmpG Secondary Structure and Its pH-Dependent Alterations Monitored by FTIR

The channel activity of the outer-membrane protein G (OmpG) from Escherichia coli is pH-dependent. To investigate the role of the histidine pair His231/His261 in triggering channel opening and closing, we mutated both histidines to alanines and cysteines. Fourier transform infrared spectra revealed that the OmpG mutants stay—independent of pH—in an open conformation. Temperature ramp experiments indicate that the mutants are as stable as the open state of wild-type OmpG. The X-ray structure of the alanine-substituted OmpG mutant obtained at pH 6.5 confirms the constitutively open conformation. Compared to previous structures of the wild-type protein in the open and closed conformation, the mutant structure shows a difference in the extracellular loop L6 connecting β-strands S12 and S13. A deletion of amino acids 220-228, which are thought to block the channel at low pH in wild-type OmpG, indicates conformational changes, which might be triggered by His231/His261.     

Probing the proton channel and the retinal binding site of Natronobacterium pharaonis sensory rhodopsin II

The sensory rhodopsin II from Natronobacterium pharaonis (NpSRII) was mutated to try to create functional properties characteristic of bacteriorhodopsin (BR), the proton pump from Halobacterium salinarum. Key residues from the cytoplasmic and extracellular proton transfer channel of BR as well as from the retinal binding site were chosen. The single site mutants L40T, F86D, P183E, and T204A did not display altered function as determined by the kinetics of their photocycles. However, the photocycle of each of the subsequent multisite mutations L40T/F86D, L40T/F86D/P183E, and L40T/F86D/P183E/T204A was quite different from that of the wild-type protein. The reprotonation of the Schiff base could be accelerated approximately 300- to 400-fold, to approximately two to three times faster than the corresponding reaction in BR. The greatest effect is observed for the quadruple mutant in which Thr-204 is replaced by Ala. This result indicates that mutations affecting conformational changes of the protein might be of decisive importance for the creation of BR-like functional properties.     

In vivo assay for low-activity mutant forms of Escherichia coli ribonucleotide reductase

Ribonucleotide reductase (RNR) catalyzes the essential production of deoxyribonucleotides in all living cells. In this study we have established a sensitive in vivo assay to study the activity of RNR in aerobic Escherichia coli cells. The method is based on the complementation of a chromosomally encoded nonfunctional RNR with plasmid-encoded RNR. This assay can be used to determine in vivo activity of RNR mutants with activities beyond the detection limits of traditional in vitro assays. E. coli RNR is composed of two homodimeric proteins, R1 and R2. The R2 protein contains a stable tyrosyl radical essential for the catalysis that takes place at the R1 active site. The three-dimensional structures of both proteins, phylogenetic studies, and site-directed mutagenesis experiments show that the radical is transferred from the R2 protein to the active site in the R1 protein via a radical transfer pathway composed of at least nine conserved amino acid residues. Using the new assay we determined the in vivo activity of mutants affecting the radical transfer pathway in RNR and identified some residual radical transfer activity in two mutant R2 constructs (D237N and W48Y) that had previously been classified as negative for enzyme activity. In addition, we show that the R2 mutant Y356W is completely inactive, in sharp contrast to what has previously been observed for the corresponding mutation in the mouse R2 enzyme.     

Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis

Xylanases produce xylooligosaccharides from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as the major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on the sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (k_cat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures functional diversity in a limited number of protein variants.     

Effects of mutagenesis of W343 in human annexin A6 isoform 1 on its interaction with GTP: nucleotide-induced oligomer formation and ion channel activity

Accumulated experimental evidence suggests that annexin A6 (AnxA6) is involved in ion transport in various tissues. Such a biological function is related either to the modulation of ion transport systems by AnxA6 or to the ion channel activity of the protein. While AnxA6 channel activity at low pH seems to be associated with a large conformational transition in the protein, the mechanism of GTP-induced ion channel formation remains obscure. This activity is not accompanied by changes in protein structure. The existence of a domain binding the phosphate groups of GTP in AnxA6 [Bandorowicz-Pikula, J., Kirilenko, A., van Deursen, R., Golczak, M., Kuhnel, M., Lancelin, J. M., Pikula, S., and Buchet, R. (2003) Biochemistry 42, 9137-9146] may provide some clues about the molecular mechanisms of GTP-induced ion channel formation. In addition, we observed that one of the AnxA6 tryptophan residues, W192 or W343, may be involved in GTP binding. Therefore, we created several site-directed mutants of AnxA6 in which selected amino acid residues within a consensus sequence of a putative nucleotide-binding domain of AnxA6 were replaced with other amino acid residues without affecting the overall structure of protein as examined by circular dichroism and infrared spectroscopies. Their properties were analyzed and compared to those of the native protein. In contrast to mutant W192S and wild-type annexin, mutant W343S neither bound GTP nor exhibited GTP-induced ion channel activity. In addition, we detected the likely formation of AnxA6 trimers in the presence of GTP. The ability of mutant W343S to form trimers was significantly impaired. Our findings suggest that W343 participates in the formation of AnxA6 trimers. We hypothesize that such trimers could lead to a functional unit of the GTP-induced ion channels formed by the annexin molecules.     

Anchoring effects in a wide binding pocket: the molecular basis of regioselectivity in engineered cytochrome P450 monooxygenase from B. megaterium

The molecular basis of regioselectivity of cytochrome P450 monooxygenases from Bacillus megaterium (CYP102A1) with its flexible and widely opened active site is still not well understood. In the present work (-)-alpha-pinene bound complexes with two triple mutants were modeled to elucidate the contribution of the three major factors that mediate selectivity: active site shape, protein flexibility, and chemical reactivity of the substrate. For the triple mutant A74G F87V L188Q (GVQ), one stable, productive conformation of the substrate (conformation I) was identified by multiple molecular dynamics simulations. The model predicts pinene epoxide as a major product (42% pinene oxide, 23% verbenol) which is in agreement with the experimental product profile (70% pinene oxide, 20% verbenol). In contrast, for the triple mutant A74G F87G L188Q (GGQ) two stable productive substrate conformations were identified (conformations IIa and IIb), and verbenol was predicted as major product (81% verbenol, 16% myrtenol), which is in agreement with experimental results (77% verbenol, 10% myrtenol). The effect of chemical reactivity of the substrate was demonstrated by comparison of (-)-alpha-pinene to its regioisomer (-)-beta-pinene, where the product profile is shifted from 68% pinocarveol and 32% myrtanal in mutant GVQ, to 40% pinocarveol and 60% myrtanal in mutant GGQ. Our results strongly suggest a major role of residue 87 in anchoring (-)-alpha-pinene during substrate binding which provides a simple and elegant rationalization of the dynamic structure of this enzyme-substrate complex.     

副粘病毒融合蛋白活性位点中亮氨酸基因突变分析

为了确定副粘病毒融合蛋白（Ｆ）分子上活性位点中亮氨酸在Ｆ的细胞融合作用中的作用,弄清Ｆ融合细胞的分子机理,采用基因定点突变法创造一个酶切位点,用酶切反应初步筛选突变株,然后用ＤＮＡ序列分析进一步确定,并在真核细胞内进行表达,Ｇｉｅｍｓａ染色和指示基因法检测细胞融合功能,荧光强度分析（ＦＡＣＳ）检测表达效率。结果表明,ｈＰＩＶ３等４６０位亮氨酸（Ｌ）和第４７４位异亮氨酸（Ｉ）分别突变成丙氨酸（Ａ）（     

Chemical reduction of the diferric/radical center in protein R2 from mouse ribonucleotide reductase is independent of the proposed radical transfer pathway

The rates of reduction of the diferric/radical center in mouse ribonucleotide reductase protein R2 were studied by light absorption and EPR in the native protein and in three point mutants of conserved residues involved in the proposed radical transfer pathway (D266A, W103Y) or in the unstructured C terminal domain (Y370W). The pseudo-first order rate constants for chemical reduction of the tyrosyl radical and diferric center by hydroxyurea, sodium dithionite or the dihydro form of flavin adenine dinucleotide, were comparable with or higher (particularly D266A, by dithionite) than in native R2. Molecular modeling of the D266A mutant showed that the iron/radical site should be more accessible for external reductants in the mutant than in native R2. The results indicate that no specific pathway is required for the reduction. The dihydro form of flavin adenine dinucleotide was found to be a very efficient reductant in the studied proteins compared to dithionite alone. The EPR spectra of the mixed-valent Fe(II)Fe(III) sites formed by chemical reduction in the D266A and W103Y mutants were clearly different from the spectrum observed in the native protein, indicating that the structure of the diferric site was affected by the mutations, as also suggested by the modeling study. No difference was observed between the mixed-valent EPR spectra generated by chemical reduction in Y370W mutant and native mouse R2 protein.     

Molecular Dynamics Simulations Indicate that F87W,T185F-Cytochrome P450cam May Reductively Dehalogenate 1,1,1-Trichloroethane

Abstract

Cytochrome P450cam is capable of reductively dehalogenating several chlorinated alkanes at low, but measurable, rates. In previous investigations of structure-function relationships in this enzyme using molecular dynamics simulations, we noticed that 1,1,1-trichloroethane (TCA) exhibits a very high degree of mobility in the active site due to its smaller molecular volume relative to the native substrate, camphor(1,2). Several amino acid sidechains lining the active site also exhibit significant dynamic fluctuations, possibly as a result of poor steric complementarity to TCA. Guided by these results, we modeled double (F87W, T185F) and triple (F87W, T185F, V295I) mutants of P450cam, which provide additional bulk in the active site and increase the frequency of heme-substrate collision. Molecular dynamics simulations (300 ps on each protein) indicate that these mutants do not significantly perturb the three-dimensional fold of the enzyme, or local structure in the region of the active site. Both mutants bind the substrate more stably near the heme than the wild-type. Interestingly, however, the bulkier triple mutant seems to actually inhibit heme-substrate interactions relative to the double mutant. Over the final 200 ps of simulation, TCA is within 1 Å of nonbonded contact with the heme 25% more often in the double mutant versus the wild-type. The triple mutant, on the other hand, binds TCA within 1 Å of the heme only 15% as often as the wild-type. These results indicate that the double mutant may reductively dehalogenate TCA, a property not observed for the native protein. Implications for other experimentally measurable parameters are discussed.     

Structural insight into the altered substrate specificity of human cytochrome P450 2A6 mutants

Human P450 2A6 displays a small active site that is well adapted for the oxidation of small planar substrates. Mutagenesis of CYP2A6 resulted in an increased catalytic efficiency for indole biotransformation to pigments and conferred a capacity to oxidize substituted indoles (Wu, Z.-L., Podust, L.M., Guengerich, F.P. J. Biol. Chem. 49 (2005) 41090-41100.). Here, we describe the structural basis that underlies the altered metabolic profile of three mutant enzymes, P450 2A6 N297Q, L240C/N297Q and N297Q/I300V. The Asn297 substitution abolishes a potential hydrogen bonding interaction with substrates in the active site, and replaces a structural water molecule between the helix B'-C region and helix I while maintaining structural hydrogen bonding interactions. The structures of the P450 2A6 N297Q/L240C and N297Q/I300V mutants provide clues as to how the protein can adapt to fit the larger substituted indoles in the active site, and enable a comparison with other P450 family 2 enzymes for which the residue at the equivalent position was seen to function in isozyme specificity, structural integrity and protein flexibility.