Alteration of regiospecificity in biphenyl dioxygenase by active-site engineering

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation step during the metabolism of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in the determination of the substrate specificity of biphenyl-related compounds, including polychlorinated biphenyls (PCBs). Based on crystallographic analyses of naphthalene dioxygenase (B. Kauppi, K. Lee, E. Carredano, R. E. Parales, D. T. Gibson, H. Eklund, and S. Ramaswamy, Structure 6:571-586, 1998), we developed a three-dimensional model of KF707 BphA1 of Pseudomonas pseudoalcaligenes KF707. Based on structural information about the amino acids which coordinate the catalytic nonheme iron center, we constructed 12 site-directed BphA1 mutants with changes at positions 227, 332, 335, 376, 377, and 383 and expressed these enzymes in Escherichia coli. The Ile335Phe, Thr376Asn, and Phe377Leu Bph Dox mutants exhibited altered regiospecificities for various PCBs compared with wild-type Bph Dox. In particular, the Ile335Phe mutant acquired the ability to degrade 2,5,2',5'-tetrachlorobiphenyl by 3,4-dioxygenation and showed bifunctional 2,3-dioxygenase and 3,4-dioxygenase activities for 2,5,2'-trichlorobiphenyl and 2,5,4'-trichlorobiphenyl. Furthermore, two mutants, the Phe227Val and Phe377Ala mutants, introduced molecular oxygen at the 2,3 position, forming 3-chloro-2',3'-dihydroxy biphenyl with concomitant dechlorination.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

Emergence of multifunctional oxygenase activities by random priming recombination

Biphenyl dioxygenase (Bph Dox) is responsible for the initial dioxygenation of biphenyl. The large subunit (BphA1) of Bph Dox plays a crucial role in determination of substrate specificity of biphenyl-related compounds including polychlorinated biphenyls (PCBs). Functional evolution of Bph Dox of Pseudomonas pseudoalcaligenes KF707 was accomplished by random priming recombination of the bphA1 gene, involving two rounds of in vitro recombination and mutation followed by selection for increased activity in vivo. Evolved Bph Dox acquired novel and multifunctional degradation capabilities not only for PCBs but also for dibenzofuran, dibenzo-p-dioxin, dibenzothiophene, and fluorene, the compounds scarcely attacked by the original KF707 Bph Dox. The modes of oxygenation were angular and lateral dioxygenation for dibenzofuran and dibenzo-p-dioxin, sulfoxidation for dibenzothiophene, and mono-oxygenation for fluorene. These enzymes also exhibited enhanced degradation abilities for PCB congeners, retaining 2,3-dioxygenase activity and gaining 3,4-dioxygenase activity, depending on the chlorine substitution of PCB congeners. Further mutation analysis revealed that the amino acid at position 376 in BphA1 is significantly involved in the acquisition of multifunctional oxygenase activities and mode of oxygenation.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Functional analyses of a variety of chimeric dioxygenases constructed from two biphenyl dioxygenases that are similar structurally but different functionally.

The biphenyl dioxygenases (BP Dox) of strains Pseudomonas pseudoalcaligenes KF707 and Pseudomonas cepacia LB400 exhibit a distinct difference in substrate ranges of polychlorinated biphenyls (PCB) despite nearly identical amino acid sequences. The range of congeners oxidized by LB400 BP Dox is much wider than that oxidized by KF707 BP Dox. The PCB degradation abilities of these BP Dox were highly dependent on the recognition of the chlorinated rings and the sites of oxygen activation. The KF707 BP Dox recognized primarily the 4'-chlorinated ring (97%) of 2,5,4'-trichlorobiphenyl and introduced molecular oxygen at the 2',3' position. The LB400 BP Dox recognized primarily the 2,5-dichlorinated ring (95%) of the same compound and introduced O2 at the 3,4 position. It was confirmed that the BphA1 subunit (iron-sulfur protein of terminal dioxygenase encoded by bphA1) plays a crucial role in determining the substrate selectivity. We constructed a variety of chimeric bphA1 genes by exchanging four common restriction fragments between the KF707 bphA1 and the LB400 bphA1. Observation of Escherichia coli cells expressing various chimeric BP Dox revealed that a relatively small number of amino acids in the carboxy-terminal half (among 20 different amino acids in total) are involved in the recognition of the chlorinated ring and the sites of dioxygenation and thereby are responsible for the degradation of PCB. The site-directed mutagenesis of Thr-376 (KF707) to Asn-376 (LB400) in KF707 BP Dox resulted in the expansion of the range of biodegradable PCB congeners.     

Sequences of tryptic peptides containing the five cysteinyl residues of ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum

Tryptic peptides which account for all five cysteinyl residues in ribulosebisphosphate carboxylase/oxygenase from Rhodospirillum rubrum have been purified and sequenced. Collectively, these peptides contain 94 of the approximately 500 amino acid residues per molecule of subunit. Due to one incomplete cleavage at a site for trypsin and two incomplete chymotryptic-like cleavages, eight major radioactive peptides (rather than five as predicted) were recovered from tryptic digests of the enzyme that had been carboxymethylated with [³H]iodoacetate. The established sequences are: GlyTyrThrAlaPheValHisCys¹Lys TyrValAspLeuAlaLeuLysGluGluAspLeuIleAla GlyGlyGluHisValLeuCys¹AlaTyr AlaGlyTyrGlyTyrValAlaThrAlaAlaHisPheAla AlaGluSerSerThrGlyThrAspValGluValCys¹ ThrThrAsxAsxPheThrArg AlaCys¹ThrProIleIleSerGlyGlyMetAsnAla LeuArg ProPheAlaGluAlaCys¹HisAlaPheTrpLeuGly GlyAsnPheIleLys In these peptides, radioactive carboxymethylcysteinyl residues are denoted with asterisks and the sites of incomplete cleavage with vertical wavy lines. None of the peptides appear homologous with either of two cysteinyl-containing, active-site peptides previously isolated from spinach ribulosebisphosphate carboxylase/oxygenase.     

Directed evolution of biphenyl dioxygenase: emergence of enhanced degradation capacity for benzene, toluene, and alkylbenzenes

Biphenyl dioxygenase (Bph Dox) catalyzes the initial oxygenation of biphenyl and related compounds. Bph Dox is a multicomponent enzyme in which a large subunit (encoded by the bphA1 gene) is significantly responsible for substrate specificity. By using the process of DNA shuffling of bphA1 of Pseudomonas pseudoalcaligenes KF707 and Burkholderia cepacia LB400, a number of evolved Bph Dox enzymes were created. Among them, an Escherichia coli clone expressing chimeric Bph Dox exhibited extremely enhanced benzene-, toluene-, and alkylbenzene-degrading abilities. In this evolved BphA1, four amino acids (H255Q, V258I, G268A, and F277Y) were changed from the KF707 enzyme to those of the LB400 enzyme. Subsequent site-directed mutagenesis allowed us to determine the amino acids responsible for the degradation of monocyclic aromatic hydrocarbons.     

Effects of orally administrated amino acids on myofibrillar proteolysis in chicks

We examined the effects of orally administrated amino acids on myfibrillar proteolysis in food-deprived chicks. Plasma N(tau)-methylhistidine concentration, as an index of myofibrillar proteolysis, was decreased by the administration of Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg but not by Asp, Val, Phe, Tyr or His to chicks. Orally administrated Cys was fatal to chicks. These results indicate that oral Glu, Gly, Ala, Leu, Ile, Ser, Thr, Met, Trp, Asn, Gln, Pro, Lys and Arg administration suppressed myofibrillar proteolysis in chicks.     

Synthesis of highly hydroxylated aromatics by evolved biphenyl dioxygenase and subsequent dihydrodiol dehydrogenase

The evolved bphA1 (2049) gene, in which nine amino acids from the Pseudomonas pseudoalcaligenes KF707 BphA1 were changed to those from the Burkholderia xenovorans LB400 BphA1 (M247I, H255Q, V258I, G268A, D303E, -313G, S324T, V325I, and T376N), was expressed in Escherichia coli along with the bphA2A3A4 and bphB genes derived from strain KF707. This recombinant E. coli cells converted biphenyl and several heterocyclic aromatic compounds into the highly hydroxylated products such as biphenyl-2,3,2′,3′-tetraol (from biphenyl), 2-(2,3-dihydroxyphenyl)benzoxazole-4,5-diol (from 2-phenylbenzoxazole), and 2-(2,5-dihydroxyphenyl)benzoxazole-4,5-diol [from 2-(2-hydroxyphenyl)benzoxazole]. The antioxidative activity of these generated compounds was markedly higher than that of the original substrate used.     

The covalent structure of pig kidney fructose 1,6-bisphosphatase: sequence of the 60-residue NH2-terminal peptide produced by digestion with subtilisin

Digestion of the native pig kidney fructose 1,6-bisphosphatase tetramer with subtilisin cleaves each of the 35,000-molecular-weight subunits to yield two major fragments: the S-subunit (M_{r ca.} 29,000), and the S-peptide (M_r 6,500). The following amino acid sequence has been determined for the S peptide: AcThrAspGlnAlaAlaPheAspThrAsnIle Val ThrLeuThrArgPheValMetGluGlnGlyArgLysAla ArgGlyThrGlyGlu MetThrGlnLeuLeuAsnSerLeuCysThrAlaValLys AlaIleSerThrAla z.sbnd;ValArgLysAlaGlyIleAlaHisLeuTyrGlyIleAla. Comparison of this sequence with that of the NH₂-terminal 60 residues of the enzyme from rabbit liver (El-Dorry et al., 1977, Arch. Biochem. Biophys.182, 763) reveals strong homology with 52 identical positions and absolute identity in sequence from residues 26 to 60.Although subtilisin cleavage of fructose 1,6-bisphosphatase results in diminished sensitivity of the enzyme to AMP inhibition, we have found no AMP inhibition-related amino acid residues in the sequenced S-peptide. The loss of AMP sensitivity that occurs upon pyridoxal-P modification of the enzyme does not result in the modification of lysyl residues in the S-peptide. Neither photoaffinity labeling of fructose 1,6-bisphosphatase with 8-azido-AMP nor modification of the cysteinyl residue proximal to the AMP allosteric site resulted in the modification of residues located in the NH₂-terminal 60-amino acid peptide.     

Steady-state kinetic characterization of evolved biphenyl dioxygenase, which acquired novel degradation ability for benzene and toluene

Biphenyl dioxygenase (Bph Dox) catalyzes initial oxygenation in the bacterial biphenyl degradation pathway. Bph Dox in Pseudomonas pseudoalcaligenes KF707 is a Rieske type three-component enzyme in which a large subunit (encoded by the bphA1 gene) plays an important role in the substrate specificity of Bph Dox. Steady-state kinetic assays using purified enzyme components demonstrated that KF707 Bph Dox had a kcat/Km of 33.1 x 10(3) (M(-1) s(-1)) for biphenyl. Evolved 1072 Bph Dox generated by the process of DNA shuffling (Suenaga, H. et al., J. Bacteriol., 184, 3682-3688 (2002)) exhibited enhanced degradation activity not only for biphenyl (kcat/Km of 62.2 x 10(3) [M(-1) s(-1)]) but also for benzene and toluene, compounds that are rarely attacked by KF707 Bph Dox. These results suggest that evolved 1072 Bph Dox acquires higher affinities and catalytic efficiencies for various substrates than the original KF707 enzyme.     

Comparison of the monomer structure of the FMN-binding protein from Desulfovibrio vulgaris obtained by NMR and molecular dynamics simulation approaches

Flavin mononucleotide (FMN)-binding proteins (FBPs) play an important role in the electron transport process in bacteria. In this study, the structures of the FBP from Desulfovibrio vulgaris (DvFBP) (Miyazaki F) were compared between those obtained experimentally by nuclear magnetic resonance (NMR) spectroscopy and those derived from molecular dynamics simulations (MDSs). A high-residue root of mean square deviation (RMSD) was observed in residues located at both sides of the wings (Gly22, Glu23, Asp24, Ala59, Arg60, Asp61, Glu62, Gly75, Arg76, Asn77, Gly78 and Pro79), while a low-residue RMSD was found in residues located in a hollow of the structure (Asn12, Glu13, Gly14, Val15, Val16, Asn30, Thr31, Trp32, Asn33, Ser34, Gly69, Ser70, Arg71 and Lys72). Inter-planar angles between the Phe7 and Iso and between the Phe7 and Trp106 residues were remarkably different between the MDS- and NMR-derived DvFBP structures. Distribution of the torsion angles around the covalent bonds in the aliphatic chain of FMN was similar in the MDS- and NMR-derived structures, except for those around the C1′–C2′ and C5′–O5′ bonds. Hydrogen bond formation between IsoO2 and the Gly49 or Gly50 peptide NH was formed in both the NMR- and MDS-derived structures. Overall, the MDS-derived structures were found to be considerably different from the NMR-derived structures, which must be considered when the photoinduced electron transfer in flavoproteins is analysed with MDS-derived structures.     

Detailed characterization of an anti-factor IX monoclonal antibody that neutralizes the prolonged ox brain prothrombin time of hemophilia B(M) by synthetic peptides

In a previous study, we prepared a monoclonal antibody (MoAb) to coagulation factor IX (FIX), designated 65-10, which interfered with the activation of FIX by the activated factor XI/Ca(2+) and neutralized the prolonged ox brain prothrombin time of hemophilia B(M) [11,12]. The location of the epitope on the FIX for 65-10 MoAb is (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) [21]. In this paper, we studied in more detail an epitope on FIX using the systematic substitution of different amino acids at each residue of the epitope peptides and the influence of the epitope peptide on the prolonged ox brain prothrombin time of the hemophilia B(M) plasma of 65-10 MoAb. In the replacement set of amino acids, peptides showing low or no reactivity to 65-10 were (175)Phe --> Asp, Glu, Gly, Lys, Arg, Thr, Val, (176)Asn --> Asp, Glu, Phe, Ile, Lys, Leu, Pro, Val, Tyr, (177)Asp --> Cys, Glu, Phe, Ile, Lys, Leu, Met, Pro, Gln, Arg, Ser, Thr, Val, Trp, Tyr, and (178) Phe --> Pro. These results imply that a hydrophobic molecule of (175) Phe, a hydrophilic molecule of (176)Asn, and a negative charge molecule of (177)Asp were important to the epitope. The 65-10 MoAb antibody neutralized the prolonged ox brain prothrombin time of hemophilia B(M) Nagoya 2 ((180)Arg -->Trp) and Kashihara ((181)Val --> Phe) as well as B(M) Kiryu ((313)Val --> Asp) and Niigata ((390)Ala --> Val). This reaction was inhibited by preincubation with a (168) Ile-Thr-Gln-Ser-Thr-Gln-Ser-Phe-Asn-Asp-Phe-Thr-Arg-Val-Val(182) peptide conjugated with bovine serum albumin (BSA). 65-10 MoAb that has been useful in detailing epitopes will be useful for qualitative analysis of hemophilia B(M).     

Amino acid sequence of seminalplasmin, an antimicrobial protein from bull semen

Analytical ultracentrifugation of highly purified seminalplasmin revealed a molecular mass of 6300. Amino acid analysis of the protein preparation indicated the absence of sulfur-containing amino acids cysteine and methionine. The amino acid sequence of seminalplasmin was determined by manual Edman degradation of peptides obtained by proteolytic enzymes trypsin, chymotrypsin and thermolysin: NH₂-Ser Asp Glu Lys Ala Ser Pro Asp Lys His His Arg Phe Ser Leu Ser Arg Tyr Ala Lys Leu Ala Asn Arg Leu Ser Lys Trp Ile Gly Asn Arg Gly Asn Arg Leu Ala Asn Pro Lys Leu Leu Glu Thr Phe Lys Ser Val-COOH. The number of amino acids according to the sequence were 48, the molecular mass 6385. As predicted from the sequence, seminalplasmin very likely contains two α-helical domains in which residues 8-17 and 40-48 are involved. No evidence for the existence of β-sheet structures was obtained. Treatment of seminalplasmin with the above proteases as well as with amino peptidase M and carboxypeptidase Y completely eliminated biological activity.     

Subcellular distribution of aminoacyl-transfer RNA synthetases in Chinese hamster ovary cell culture

Chinese hamster ovary cells grown in cell culture were broken and fractionated by differential centrifugation. Four principal fractions: nuclear and membrane, microsomal, postribosomal, and supernatant were obtained. The distribution of aminoacyl-tRNA synthetases in these four fractions was determined for all twenty amino acids.It was shown that there is a differential distribution of synthetases. Activities specific for eight amino acids: Ala, Ser, Gly, Cys, His, Arg, Thr and Pro were found mainly in the supernatant fraction. Activities specific for eleven amino acids: Asp, Asn, Glu, Gln, Ile, Leu, Lys, Met, Phe, Tyr and Val were found mainly in the postribosomal fraction. Four activities were found at significant levels in the microsomal fraction: Asp, Phe, Lys and Pro. The nuclear and membrane fraction contained activity for Lys, His, Asp and Thr.Changes in aminoacyl-tRNA synthetase activities in various fractions from preparations made by breaking cells with a membrane-dissociating detergent showed that some of the aminoacyl-tRNA synthetase activities may be membrane bound.     

NH2-terminal sequences of DNA-, heparin-, and gelatin-binding tryptic fragments from human plasma fibronectin

NH₂-terminal sequence analysis was performed on subregions of human plasma fibronectin including 24,000-dalton (24K) DNA-binding, 29,000-dalton (29K) gelatin-binding, and 18,000-dalton (18K) heparin-binding tryptic fragments. These fragments were obtained from fibronectin after extensive trypsin digestion followed by sequential affinity purification on gelatin-Sepharose, heparin-agarose, and DNA-cellulose columns. The gelatin-binding fragment was further purified by gel filtration on Sephadex G-100, and the DNA-binding and heparin-binding fragments were further purified by high-performance liquid chromatography. The 29K fragment had the following NH₂-terminal sequence: AlaAlaValTyrGlnProGlnProHisProGlnProPro (Pro)TyrGlyHis HisValThrAsp(His)(Thr)ValValTyrGly(Ser) ?(Ser)?-Lys. The NH₂-terminal sequence of a 50K, gelatin-binding, subtilisin fragment by L. I. Gold, A. Garcia-Pardo, B. Prangione, E. C. Franklin, and E. Pearlstein (1979, Proc. Nat. Acad. Sci. USA76, 4803–4807) is identical to positions 3–19 (with the exception of some ambiguity at position 14) of the 29K fragment. These data strongly suggest that the 29K tryptic fragment is included in the 50K subtilisin fragment, and that subtilisin cleaves fibronectin between the Ala²Val³ residues of the 29K tryptic fragment. The 18K heparin-binding fragment had the following NH₂-terminal sequence: (Glu)AlaProGlnProHisCysIleSerLysTyrIle LeuTyrTrpAspProLysAsnSerValGly?(Pro) LysGluAla?(Val)(Pro). The 29K gelatin-binding and 18K heparin-binding fragments have proline-rich NH₂-terminal sequences suggesting that they may have arisen from protease-sensitive, random coil regions of fibronectin corresponding to interdomain regions preceding macromolecular-binding domains. Both of these fragments contain the identical sequence ProGlnProHis, a sequence which may be repeated in other interdomain regions of fibronectin. The 24K DNA-binding fragment has the following NH₂-terminal sequence: SerAspThrValProSerProCysAspLeuGlnPhe ValGluValThrAspVal LysValThrIleMetTrpThrProProGluSerAla ValThrGlyTyrArgVal AspValCysProValAsnLeuProGlyGluHisGly Gln(Cys)LeuProIleSer. The sequence of positions 9–22 are homologous to positions 15–28 of the α chain of DNA-dependent RNA polymerase from Escherichia coli. The homology observed suggests that this stretch of amino acids may be a DNA-binding site.     

Evaluation of essential and variable residues of nukacin ISK-1 by NNK scanning

Nukacin ISK-1, a type-A(II) lantibiotic, comprises 27 amino acids with a distinct linear N-terminal and a globular C-terminal region. To identify the positional importance or redundancy of individual residues responsible for nukacin ISK-1 antimicrobial activity, we replaced the native codons of the parent peptide with NNK triplet oligonucleotides in order to generate a bank of nukacin ISK-1 variants. The bioactivity of each peptide variant was evaluated by colony overlay assay, and hence we identified three Lys residues (Lys1, Lys2 and Lys3) that provided electrostatic interactions with the target membrane and were significantly variable. The ring structure of nukacin ISK-1 was found to be crucially important as replacing the ring-forming residues caused a complete loss of bioactivity. In addition to the ring-forming residues, Gly5, His12, Asp13, Met16, Asn17 and Gln20 residues were found to be essential for antimicrobial activity; Val6, Ile7, Val10, Phe19, Phe21, Val22, Phe23 and Thr24 were relatively variable; and Ser4, Pro8, His15 and Ser27 were extensively variable relative to their positions. We obtained two variants, Asp13Glu and Val22Ile, which exhibited a twofold higher specific activity compared with the wild-type and are the first reported type-A(II) lantibiotic mutant peptides with increased potency.     

Effects of chronic ethanol treatment on amino acid uptake and enzyme activities in the lactating rat mammary gland

The effects of chronic ethanol consumption on mammary gland amino acid uptake at the 15th day of lactation in the rat have been studied. Ethanol treatment decreased the arterial levels of Ala, Asp, Gly, Pro, Lys and Met, and increased those of Gln and alpha-amino-butyrate. Chronic ethanol treatment produced a decrease in the arteriovenous differences of Asp, Thr, Arg, Met and Phe, and increased those of Ala, Gln, Gly, Pro and Tyr. The combination of the calculated values of relative extraction and the arteriovenous differences indicate that these alterations in amino acid uptake are related to changes in the transport process for Ala, Asp, Thr, Pro, Arg, Asn, Gly, Tyr, and Phe, and that the alterations in the arteriovenous differences of Gln, Lys and Met are due to the affected arterial levels of these amino acids. Measurements of enzymatic activities in the mammary gland show that these alterations in the amino acid transport process cannot be ascribed to changes in the gamma-glutamyl cycle.