Mutation of alphaPhe55 of methylamine dehydrogenase alters the reorganization energy and electronic coupling for its electron transfer reaction with amicyanin

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe55 of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis of alphaPhe55 has revealed roles for this residue in determining substrate specificity and binding monovalent cations at the active site. It is now shown that the alphaF55A mutation also increases the rate of the true electron transfer (ET) reaction from O-quinol MADH to amicyanin. The reorganization energy associated with the ET reaction is decreased from 2.3 to 1.8 eV. The electronic coupling associated with the ET reaction is decreased from 12 to 3 cm(-1). The crystal structure of alphaF55A MADH in complex with its electron acceptors, amicyanin and cytochrome c-551i, has been determined. Little difference in the overall structure is seen, relative to the native complex; however, there are significant changes in the solvent content of the active site and substrate channel. The crystal structure of alphaF55A MADH has also been determined with phenylhydrazine covalently bound to TTQ in the active site. Phenylhydrazine binding significantly perturbs the orientation of the TTQ rings relative to each other. The ET results are discussed in the context of the new and old crystal structures of the native and mutant enzymes.     

Use of indirect site-directed mutagenesis to alter the substrate specificity of methylamine dehydrogenase.

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone-dependent enzyme that catalyzes the oxidative deamination of primary amines. Native MADH exhibits a strong preference for methylamine over longer carbon chain amines. Residue alphaPhe(55) controls this substrate specificity. When alphaPhe(55) is replaced with Ala, this preference is reversed with alphaF55A MADH preferring long-chain amines with at least seven carbons (Zhu, Z., Sun, D., and Davidson, V. L. (2000) Biochemistry 39, 11184-11186). To further modulate the substrate specificity of MADH, the side-chain of alphaPhe(55) was repositioned by site-directed mutagenesis of residue betaIle(107). This residue makes close contact with alphaPhe(55) and restricts its movement. betaI107V MADH exhibits a strong preference for propylamine, and betaI107N MADH exhibits a preference for 1-aminopentane. Thus, it has been possible to create forms of MADH that exhibit a preference for amines with carbon chain lengths of one, three, five, or seven carbons. The ability to discriminate between amines of different chain length was essentially abolished by an alphaF55I mutation. Molecular modeling studies with the known crystal structure are described that provide an explanation for these results. These results provide an example of a design-based approach to protein engineering in which site-directed mutagenesis on one residue can be used to reposition another residue to specifically alter enzyme specificity.     

Probing mechanisms of catalysis and electron transfer by methylamine dehydrogenase by site-directed mutagenesis of alpha Phe55

Methylamine dehydrogenase (MADH) possesses an alpha(2)beta(2) subunit structure with each smaller beta subunit possessing a tryptophan tryptophylquinone (TTQ) prosthetic group. Phe(55) of the alpha subunit is located where the substrate channel from the enzyme surface opens into the active site. Site-directed mutagenesis studies have revealed several roles for this residue in catalysis and electron transfer (ET) by MADH. Site-directed mutagenesis of either alpha Phe(55) or beta Ile(107) (a residue in the beta subunit which interacts with alpha Phe(55)) converts MADH into enzymes with specificities for long-chain amines, amylamine or propylamine. Mutation of alpha Phe(55) also affects monovalent cation binding to the active site. alpha F55A MADH exhibits an increased K(d) for cation-dependent spectral changes and a decreased K(d) for cation-dependent stimulation of the rate of gated ET from N-quinol MADH to amicyanin. These results demonstrate that alpha Phe(55) is able to directly participate in a wide range of biochemical processes not typically observed for a phenylalanine residue.     

Molecular basis for complex formation between methylamine dehydrogenase and amicyanin revealed by inverse mutagenesis of an interprotein salt bridge

Methylamine dehydrogenase (MADH) and amicyanin form a physiologic complex which is required for interprotein electron transfer. The crystal structure of this protein complex is known, and the importance of certain residues on amicyanin in its interaction with MADH has been demonstrated by site-directed mutagenesis. In this study, site-directed mutagenesis of MADH, kinetic data, and thermodynamic analysis are used to probe the molecular basis for stabilization of the protein complex by an interprotein salt bridge between Arg99 of amicyanin and Asp180 of the alpha subunit of MADH. This paper reports the first site-directed mutagenesis of MADH, as well as the construction, heterologous expression, and characterization of a six-His-tagged MADH. alpha Asp180 of MADH was converted to arginine to examine the effect on complex formation with native and mutant amicyanins. This mutation had no effect on the parameters for methylamine oxidation by MADH, but significantly affected its interaction with amicyanin. Of the native and mutant proteins that were studied, their observed order of affinity for each other was as follows: native MADH and native amicyanin > native MADH and R99D amicyanin > alpha D180R MADH and native amicyanin > alpha D180R MADH and R99D amicyanin, and alpha D180R MADH and R99L amicyanin. The alpha D180R mutation also eliminated the ionic strength dependence of the reaction of MADH with amicyanin that is observed with wild-type MADH. Interestingly, the inverse mutation pair of alpha D180R MADH and R99D amicyanin did not restore the favorable salt bridge, but instead disrupted complex formation much more severely than did either individual mutation. These results are explained using molecular modeling and thermodynamic analysis of the kinetic data to correlate the energy contributions of specific stabilizing and destabilizing interactions that are present in the wild-type and mutant complexes. A model is also proposed to describe the sequence of events that leads to stable complex formation between MADH and amicyanin.     

Re-engineering monovalent cation binding sites of methylamine dehydrogenase: effects on spectral properties and gated electron transfer

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ)-dependent enzyme that catalyzes the oxidative deamination of primary amines. Monovalent cations are known to affect the spectral properties of MADH and to influence the rate of the gated electron transfer (ET) reaction from substrate-reduced MADH to amicyanin. Two putative monovalent cation binding sites in MADH have been identified by X-ray crystallography [Labesse, G., Ferrari, D., Chen, Z.-W., Rossi, G.-L., Kuusk, V., McIntire, W. S., and Mathews, F. S. (1998) J. Biol. Chem. 273, 25703-25712]. One requires cation-pi interactions involving residue alpha Phe55. An alpha F55A mutation differentially affects these two monovalent cation-dependent phenomena. The apparent K(d) associated with spectral perturbations increases 10-fold. The apparent K(d) associated with enhancement of the gated ET reaction becomes too small to measure, indicating that either it has decreased more than 1000-fold or the mutation has caused a conformational change that eliminates the requirement for the cation for the gated ET. These results show that of the two binding sites revealed in the structure, cation binding to the distal site, which is stabilized by the cation-pi interactions, is responsible for the spectral perturbations. Cation binding to the proximal site, which is stabilized by several oxygen ligands, is responsible for the enhancement of the rate of gated ET. Another site-directed mutant, alpha F55E MADH, exhibited cation binding properties that were the same as those of the native enzyme, indicating that interactions with the carboxylate of Glu can effectively replace the cation-pi interactions with Phe in stabilizing monovalent cation binding to the distal site.     

Conversion of methylamine dehydrogenase to a long-chain amine dehydrogenase by mutagenesis of a single residue

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) dependent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalytic alpha subunit line a substrate channel that leads from the protein surface to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alphaPhe55 to alanine dramatically alters the substrate preference of MADH. The K(m) for methylamine increases from 9 microM to 15 mM. The preferred substrates are now primary amines with chain lengths of at least seven carbons. The K(m) for 1, 10-diaminodecane is 11 microM, compared to 1.2 mM for wild-type MADH. Despite the large variation in K(m) values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.     

Aromatic amine dehydrogenase, a second tryptophan tryptophylquinone enzyme.

Aromatic amine dehydrogenase (AADH) catalyzes the oxidative deamination of aromatic amines including tyramine and dopamine. AADH is structurally similar to methylamine dehydrogenase (MADH) and possesses the same tryptophan tryptophylquinone (TTQ) prosthetic group. AADH exhibits an alpha 2 beta 2 structure with subunit molecular weights of 39,000 and 18,000 and with a quinone covalently attached to each beta subunit. Neither subunit cross-reacted immunologically with antibodies to the corresponding subunits of MADH, and the N-terminal amino acid sequence of the beta subunit of AADH exhibited no homology with the highly conserved beta subunits of MADH. The absorption spectra for the oxidized, semiquinone, and reduced forms of AADH have been characterized, and extinction coefficients for the absorption maxima of each redox form have been determined. These spectra are very similar to those for MADH, indicating the likelihood of a TTQ cofactor. This was verified by the near identity of the vibrational frequencies and intensities in the resonance Raman spectra for the oxidized forms of AADH and MADH. A stable semiquinone of AADH could be observed during a reductive titration with dithionite, whereas titration with tyramine proceeded directly from the oxidized to the reduced form. AADH was very stable against denaturation by heat and exposure to guanidine. The individual subunits could be separated by gel filtration after incubation in guanidine hydrochloride, and partial reconstitution of activity was observed on recombination of the subunits. Steady-state kinetic analysis of AADH yielded a Vmax of 17 mumol/min/mg and a Km for tyramine of 5.4 microM. Substrate inhibition by tyramine was observed. AADH was irreversibly inhibited by hydrazine, phenylhydrazine, hydroxylamine, semicarbazide, and aminoguanidine. Isonicotinic acid hydrazide (isoniazid) and isonicotinic acid 2-isopropyl hydrazide (iproniazid) were reversible noncompetitive inhibitors of AADH and exhibited K(i) values of 8 and 186 microM, respectively. The similarities and differences between AADH and other amine oxidizing enzymes are also discussed.     

Active site hydrophobicity is critical to the bioluminescence activity of Vibrio harveyi luciferase

Vibrio harveyi luciferase is an alphabeta heterodimer containing a single active site, proposed earlier to be at a cleft in the alpha subunit. In this work, six conserved phenylalanine residues at this proposed active site were subjected to site-directed mutations to investigate their possible functional roles and to delineate the makeup of luciferase active site. After initial screening of Phe --> Ala mutants, alphaF46, alphaF49, alphaF114, and alphaF117 were chosen for additional mutations to Asp, Ser, and Tyr. Comparisons of the general kinetic properties of wild-type and mutated luciferases indicated that the hydrophobic nature of alphaF46, alphaF49, alphaF114, and alphaF117 was important to luciferase V(max) and V(max)/K(m), which were reduced by 3-5 orders of magnitude for the Phe --> Asp mutants. Both alphaF46 and alphaF117 also appeared to be involved in the binding of reduced flavin substrate. Additional studies on the stability and yield of the 4a-hydroperoxyflavin intermediate II and measurements of decanal substrate oxidation by alphaF46D, alphaF49D, alphaF114D, and alphaF117D revealed that their marked reductions in the overall quantum yield (phi( degrees )) were a consequence of diminished yields of luciferase intermediates and, with the exception of alphaF114D, emission quantum yield of the excited emitter due to the replacement of the hydrophobic Phe by the anionic Asp. The locations of these four critical Phe residues in relation to other essential and/or hydrophobic residues are depicted in a refined map of the active site. Functional implications of these residues are discussed.     

Mechanistic contributions of residues in the M1 transmembrane domain of the nicotinic receptor to channel gating

The nicotinic receptor (AChR) is a pentamer of homologous subunits with an alpha(2)betaepsilondelta composition in adult muscle. Each subunit contains four transmembrane domains (M1-M4). Position 15' of the M1 domain is phenylalanine in alpha subunits while it is isoleucine in non-alpha subunits. Given this peculiar conservation pattern, we studied its contribution to muscle AChR activation by combining mutagenesis with single-channel kinetic analysis. AChRs containing the mutant alpha subunit (alphaF15'I) as well as those containing the reverse mutations in the non-alpha subunits (betaI15'F, deltaI15'F, and epsilonI15'F) show prolonged lifetimes of the diliganded open channel resulting from a slower closing rate with respect to wild-type AChRs. The kinetic changes are not equivalent among subunits, the beta subunit, being the one that produces the most significant stabilization of the open state. Kinetic analysis of betaI15'F of AChR channels activated by the low-efficacious agonist choline revealed a 10-fold decrease in the closing rate, a 2.5-fold increase in the opening rate, a 28-fold increase in the gating equilibrium constant in the diliganded receptor, and a significant increase opening in the absence of agonist. Mutations at betaI15' showed that the structural bases of its contribution to gating is complex. Rate-equilibrium linear free-energy relationships suggest an approximately 70% closed-state-like environment for the beta15' position at the transition state of gating. The overall results identify position 15' as a subunit-selective determinant of channel gating and add new experimental evidence that gives support to the involvement of the M1 domain in the operation of the channel gating apparatus.     

Stabilization of a sodium channel state with high affinity for saxitoxin by intramolecular cross-linking. Evidence for allosteric effects of saxitoxin binding

Incubation of purified rat brain sodium channels at 37 degrees C or at high ionic strength causes a concomitant loss of saxitoxin-binding activity and dissociation of beta 1 subunits. Reaction with hydrophilic carbodiimides produced a resistance against the loss of saxitoxin binding and caused covalent cross-linking of alpha, beta 1, and beta 2 subunits. In the presence of saxitoxin, this cross-linking reaction led to formation of a state with increased affinity for saxitoxin. However, analysis of the concentration dependence of covalent cross-linking and its inhibition by hydrophilic nucleophiles showed that the stabilization of the saxitoxin-binding activity was due to the formation of a small number of isopeptide bonds in the alpha subunit rather than to cross-linking of alpha and beta 1 subunits. In the presence of amine nucleophiles, carbodiimides caused loss of saxitoxin binding, which was prevented in the presence of the toxin. Nucleophiles yielding positively charged amide products were more effective than those forming uncharged or negatively charged products. Under conditions where saxitoxin protected the binding activity of the sodium channel from inactivation, the overall availability of carboxyl groups for reaction was increased, providing evidence for a toxin-induced conformational change on binding. These results are considered in terms of an allosteric model of saxitoxin binding, in which the functional form of the sodium channel having high affinity for saxitoxin can be stabilized against inactivation by noncovalent interactions with beta 1 subunits, binding of saxitoxin and tetrodotoxin, or intramolecular cross-linking of amino acid residues within the alpha subunit.     

Independent analysis of bait region cleavage dependent and thiolester bond cleavage dependent conformational changes by cross-linking of alpha 2-macroglobulin with cis-dichlorodiammineplatinum(II) and dithiobis(succinimidyl propionate)

Treatment of the human plasma proteinase inhibitor alpha 2-macroglobulin (alpha 2M) with proteinase results in conformational changes in the inhibitor and subsequent activation and cleavage of the internal thiolester bonds of alpha 2M. Previous studies from this laboratory have shown that cross-linking the alpha 2M subunits with cis-dichlorodiammineplatinum(II) (cis-DDP) prevents the proteinase-induced conformational changes which lead to the activation and cleavage of the internal thiolester bonds of alpha 2M. In addition, cis-DDP treatment prevents the proteinase- or CH3NH2-induced conformational changes in alpha 2M which lead to a "slow" to "fast" change in nondenaturing polyacrylamide gel electrophoresis. In this paper, we demonstrate that treatment of alpha 2M with dithiobis(succinimidyl propionate) (DSP) also results in cross-linking of the subunits of alpha 2M with concomitant loss of proteinase inhibitory activity. Although proteinase is not inhibited by DSP-treated alpha 2M, bait region specific proteolysis of the alpha 2M subunits still occurs. Unlike cis-DDP-treated alpha 2M, however, incubation of DSP-treated alpha 2M with proteinase does not prevent the bait region cleavage dependent conformational changes which lead to activation and cleavage of the internal thiolester bonds in alpha 2M. On the other hand, cross-linking of alpha 2M with DSP does prevent the conformational changes which trigger receptor recognition site exposure following cleavage of the alpha 2M thiolester bonds by CH3NH2. These conformational changes, however, occur following incubation of the CH3NH2-treated protein with proteinase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Crystal structure and interactions of the PAS repeat region of the Drosophila clock protein PERIOD

PERIOD proteins are central components of the Drosophila and mammalian circadian clock. Their function is controlled by daily changes in synthesis, cellular localization, phosphorylation, degradation, as well as specific interactions with other clock components. Here we present the crystal structure of a Drosophila PERIOD (dPER) fragment comprising two tandemly organized PAS (PER-ARNT-SIM) domains (PAS-A and PAS-B) and two additional C-terminal alpha helices (alphaE and alphaF). Our analysis reveals a noncrystallographic dPER dimer mediated by intermolecular interactions of PAS-A with PAS-B and helix alphaF. We show that alphaF is essential for dPER homodimerization and that the PAS-A-alphaF interaction plays a crucial role in dPER clock function, as it is affected by the 29 hr long-period perL mutation.     

Heterologous Expression of Correctly Assembled Methylamine Dehydrogenase in Rhodobacter sphaeroides

The biosynthesis of methylamine dehydrogenase (MADH) from Paracoccus denitrificans requires four genes in addition to those that encode the two structural protein subunits, mauB and mauA. The accessory gene products appear to be required for proper export of the protein to the periplasm, synthesis of the tryptophan tryptophylquinone (TTQ) prosthetic group, and formation of several structural disulfide bonds. To accomplish the heterologous expression of correctly assembled MADH, eight genes from the methylamine utilization gene cluster of P. denitrificans, mauFBEDACJG, were placed under the regulatory control of the coxII promoter of Rhodobacter sphaeroides and introduced into R. sphaeroides by using a broad-host-range vector. The heterologous expression of MADH was constitutive with respect to carbon source, whereas the native mau promoter allows induction only when cells are grown in the presence of methylamine as a sole carbon source and is repressed by other carbon sources. The recombinant MADH was localized exclusively in the periplasm, and its physical, spectroscopic, kinetic and redox properties were indistinguishable from those of the enzyme isolated from P. denitrificans. These results indicate that mauM and mauN are not required for MADH or TTQ biosynthesis and that mauFBEDACJG are sufficient for TTQ biosynthesis, since R. sphaeroides cannot synthesize TTQ. A similar construct introduced into Escherichia coli did not produce detectable MADH activity or accumulation of the mauB and mauA gene products but did lead to synthesizes of amicyanin, the mauC gene product. This finding suggests that active recombinant MADH is not expressed in E. coli because one of the accessory gene products is not functionally expressed. This study illustrates the potential utility of R. sphaeroides and the coxII promoter for heterologous expression of complex enzymes such as MADH which cannot be expressed in E. coli. These results also provide the foundation for future studies on the molecular mechanisms of MADH and TTQ biosynthesis, as well as a system for performing site-directed mutagenesis of the MADH gene and other mau genes.     

Introduction of reactive cysteine residues in the epsilon subunit of Escherichia coli F1 ATPase, modification of these sites with tetrafluorophenyl azide-maleimides, and examination of changes in the binding of the epsilon subunit when different nucleotides are in catalytic sites.

Cysteine residues have been exchanged for serine residues at positions 10 and 108 in the epsilon subunit of the Escherichia coli F1 ATPase by site-directed mutagenesis to create two mutants, epsilon-S10C and epsilon-S108C. These two mutants and wild-type enzyme were reacted with [14C]N-ethylmaleimide (NEM) to examine the solvent accessibility of Cys residues and with novel photoactivated cross-linkers, tetrafluorophenyl azide-maleimides (TFPAM's), to examine near-neighbor relationships of subunits. In native wild-type F1 ATPase, NEM reacted with alpha subunits at a maximal level of 1 mol/mol of enzyme (1 mol/3 alpha subunits) and with the delta subunit at 1 mol/mol of enzyme; other subunits were not labeled by the reagent. In the mutants epsilon-S10C and epsilon-S108C, Cys10 and Cys108, respectively, were also labeled by NEM, indicating that these are surface residues. Reaction of wild-type enzyme with TFPAM's gave cross-linking of the delta subunit to both alpha and beta subunits. Reaction of the mutants with TFPAM's also cross-linked delta to alpha and beta and in addition formed covalent links between Cys10 of the epsilon subunit and the gamma subunit and between Cys108 of the epsilon subunit and the alpha subunit. The yield of cross-linking between sites on epsilon and other subunits depended on the nucleotide conditions used; this was not the case for delta-alpha or delta-beta cross-linked products. In the presence of ATP+EDTA the yield of cross-linking between epsilon-Cys10 and gamma was high (close to 50%) while the yield of epsilon-Cys108 and alpha was low (around 10%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Probing the agonist domain of the nicotinic acetylcholine receptor by cysteine scanning mutagenesis reveals residues in proximity to the alpha-bungarotoxin binding site

We have constructed a series of cysteine-substitution mutants in order to identify residues in the mouse muscle nicotinic acetylcholine receptor (AChR) that are involved in alpha-bungarotoxin (alpha-Bgtx) binding. Following transient expression in HEK 293-derived TSA-201 cells, covalent modification of the introduced cysteines with thiol-specific reagents reveals that alpha subunit residues W187, V188, F189, Y190, and P194 are solvent accessible and are in a position to contribute to the alpha-Bgtx binding site in native receptors. These results with the intact receptor are consistent with NMR studies of an alpha-Bgtx/receptor-dodecapeptide complex [Basus, V., Song., G., and Hawrot, E. (1993) Biochemistry 32, 12290-12298]. We pursued a more detailed analysis of the F189C mutant as this site varies substantially between AChRs that bind Bgtx and certain neuronal AChRs that do not. Treatment of intact cells expressing F189C with either bromoacetylcholine (BrACh) or [2-(trimethylammonium)ethyl] methane-thiosulfonate (MTSET), both methylammonium-containing thiol-modifying reagents with agonist properties, results in a marked decrease ( approximately 55-70%) in the number of alpha-Bgtx binding sites, as measured under saturating conditions. The decrease in sites appears to affect both alpha/gamma and alpha/delta sites to the same extent, as shown for alphaW187C and alphaF189C which were the two mutants examined on this issue. In contrast to the results obtained with MTSET and BrACh, modification with reagents that lack the alkylammonium entity, such as methylmethanethiosulfonate (MMTS), the negatively charged 2-sulfonatoethyl methane-thiosulfonate (MTSES), or the positively charged aminoethyl methylthiosulfonate (MTSEA), has little or no effect on the maximal binding of alpha-Bgtx to the alphaW187C, alphaV188C, or alphaF189C mutant receptors. The striking alkylammonium dependency suggests that an interaction of the tethered modifying group with the negative subsite within the agonist binding domain is primarily responsible for the observed blockade of toxin binding.     

Functional roles of conserved residues in the unstructured loop of Vibrio harveyi bacterial luciferase

Residues 257-291 of the Vibrio harveyi bacterial luciferase alpha subunit comprise a highly conserved, protease-labile, disordered loop region, most of which is unresolved in the previously determined X-ray structures of the native enzyme. This loop region has been shown to display a time- dependent proteolysis resistance upon single catalytic turnover and was postulated to undergo conformational changes during catalysis ([AbouKhair, N. K., Ziegler, M. M., and Baldwin, T. O. (1985) Biochemistry 24, 3942-3947]. To investigate the role of this region in catalysis, we have performed site-specific mutations of different conserved loop residues. In comparison with V(max) and V(max)/K(m,flavin) of the native luciferase, the bioluminescence activities of alphaG284P were decreased to 1-2% whereas those of alphaG275P and alphaF261D were reduced by 4-6 orders of magnitude. Stopped-flow results indicate that both alphaG275P and alphaF261D were able to form the 4a-hydroperoxy-FMN intermediate II but at lower yields. Both mutants also had enhanced rates for the intermediate II nonproductive dark decay and significantly compromised abilities to oxidize the decanal substrate. Additional mutations were introduced into the alphaG275 and alphaF261 positions, and the activities of the resulting mutants were characterized. Results indicate that the torsional flexibility of the alphaG275 residue and the bulky and hydrophobic nature of the alphaF261 residue were critical to the luciferase activity. Our results also support a functional role for the alpha subunit unstructured loop itself, possibly by serving as a mobile gating mechanism in shielding critical intermediates (including the excited flavin emitter) from exposure to medium.     

Chemical cross-linking of bovine retinal transducin and cGMP phosphodiesterase

The bifunctional reagents para-phenyldimaleimide and maleimidobenzoyl-N-hydroxysuccinimide ester were used to chemically cross-link the subunits of the transducin and cGMP phosphodiesterase (PDE) complexes of bovine rod photoreceptor cells. The cross-linked products were identified by Western immunoblotting using antisera against purified subunits of transducin (T alpha and T beta gamma) and PDE. Oligomeric cross-linked products of transducin subunits as large as (T alpha beta gamma)3 were observed in the latent form of transducin with bound GDP. In addition to the expected T alpha beta and T beta gamma cross-linked products, a (T alpha gamma)2 structure was detected. The close proximity of T alpha and T gamma suggests that T gamma may play a role in conferring the specificity of the interaction between T alpha and rhodopsin. Most of the oligomeric cross-linked structures between T alpha and T beta gamma were diminished in the activated form of transducin, with guanosine 5'-(beta, gamma-imidotriphosphate) (Gpp(NH)p) bound. However, cross-linking between T beta and T gamma was not altered. These results suggest that transducin exists as an oligomer in solution which dissociates upon the binding of Gpp(NH)p. To identify the possible interacting domains between the T alpha, T beta, and T gamma subunits, the cross-linked products were subjected to limited tryptic proteolysis. Several cross-linked tryptic peptides of transducin subunits were found and include the cross-linked products of the N terminus 15-kDa fragment of T beta and the C terminus 5-kDa fragment of T alpha, T gamma and the 12-kDa fragment of T alpha, T gamma and the 15-kDa as well as the 23-kDa fragments of T beta, and an intra-T alpha cross-linked product of the 2- and 21-kDa fragments. These results have allowed the construction of a topographical model for the transducin subunits. The organization of the subunits of PDE (P alpha, P beta, and P gamma) was also studied. The formation of the high molecular size cross-linked products of PDE resulted in the concurrent loss of the P beta and P gamma subunits, suggesting that they are in close proximity. Finally, the interaction between transducin and PDE was examined by chemical cross-linking of transducin-Gpp(NH)p and PDE. Two additional cross-linked products of 180 and 210 kDa were obtained which could be due to the cross-linking of T alpha or T beta with P alpha beta subunits.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus denitrificans determined by molecular replacement at 2.8 A resolution.

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.     

Characterization of the beta-lactam binding site of penicillin acylase of Escherichia coli by structural and site-directed mutagenesis studies

The binding of penicillin to penicillin acylase was studied by X-ray crystallography. The structure of the enzyme-substrate complex was determined after soaking crystals of an inactive betaN241A penicillin acylase mutant with penicillin G. Binding of the substrate induces a conformational change, in which the side chains of alphaF146 and alphaR145 move away from the active site, which allows the enzyme to accommodate penicillin G. In the resulting structure, the beta-lactam binding site is formed by the side chains of alphaF146 and betaF71, which have van der Waals interactions with the thiazolidine ring of penicillin G and the side chain of alphaR145 that is connected to the carboxylate group of the ligand by means of hydrogen bonding via two water molecules. The backbone oxygen of betaQ23 forms a hydrogen bond with the carbonyl oxygen of the phenylacetic acid moiety through a bridging water molecule. Kinetic studies revealed that the site-directed mutants alphaF146Y, alphaF146A and alphaF146L all show significant changes in their interaction with the beta-lactam substrates as compared with the wild type. The alphaF146Y mutant had the same affinity for 6-aminopenicillanic acid as the wild-type enzyme, but was not able to synthesize penicillin G from phenylacetamide and 6-aminopenicillanic acid. The alphaF146L and alphaF146A enzymes had a 3-5-fold decreased affinity for 6-aminopenicillanic acid, but synthesized penicillin G more efficiently than the wild type. The combined results of the structural and kinetic studies show the importance of alphaF146 in the beta-lactam binding site and provide leads for engineering mutants with improved synthetic properties.     

Ribonucleotide reductase of Escherichia coli. Cross-linking agents as probes of quaternary and quinary structure

The quaternary structure of ribonucleotide reductase of Escherichia coli was investigated, with the use of purified B1 and B2 proteins and bifunctional cross-linking agents. The holoenzyme is known to be an alpha 2 beta 2 tetramer consisting of two dimeric proteins: B1 (alpha 2) and B2 (beta 2). The cross-linking data support a model in which both of the beta subunits interact closely with only one of the two alpha subunits. Some of the interactions involving B2 were localized to the C terminus of the protein by use of truncated B2 protein (beta',beta'), a proteolytic cleavage product of B2 in which the 30 carboxy-terminal residues are missing from each of the beta subunits. Other interactions were indicated by the ability of glutaredoxin, but not thioredoxin, to inhibit some of the cross-linking reactions. We also asked whether ribonucleotide reductase interacts closely with other proteins inside the cell, by adding cross-linkers directly to suspensions of whole bacteria. Proteins in extracts of these cross-linked bacteria were resolved electrophoretically and probed with a monoclonal antibody to the B1 protein. High-molecular-mass products were detected, supporting the utility of this method for identifying intracellular interactions among enzymes of DNA precursor biosynthesis.