Kinetic characterization of the monofunctional glycosyltransferase from Staphylococcus aureus

The glycosyltransferase (GT) module of class A penicillin-binding proteins (PBPs) and monofunctional GTs (MGTs) belong to the GT51 family in the sequence-based classification of GTs. They both possess five conserved motifs and use lipid II precursor (undecaprenyl-pyrophosphate-N-acetylglucosaminyl-N-acetylmuramoyl- pentapeptide) to synthesize the glycan chain of the bacterial wall peptidoglycan. MGTs appear to be dispensable for growth of some bacteria in vitro. However, new evidence shows that they may be essential for the infection process and development of pathogenic bacteria in their hosts. Only a small number of class A PBPs have been characterized so far, and no kinetic data are available on MGTs. In this study, we present the principal enzymatic properties of the Staphylococcus aureus MGT. The enzyme catalyzes glycan chain polymerization with an efficiency of approximately 5,800 M(-1) s(-1) and has a pH optimum of 7.5, and its activity requires metal ions with a maximum observed in the presence of Mn2+. The properties of S. aureus MGT are distinct from those of S. aureus PBP2 and Escherichia coli MGT, but they are similar to those of E. coli PBP1b. We examined the role of the conserved Glu100 of S. aureus MGT (equivalent to the proposed catalytic Glu233 of E. coli PBP1b) by site-directed mutagenesis. The Glu100Gln mutation results in a drastic loss of GT activity. This shows that Glu100 is also critical for catalysis in S. aureus MGT and confirms that the conserved glutamate of the first motif EDXXFXX(H/N)X(G/A) is likely the key catalytic residue in the GT51 active site.     

Differential functionalities of amphiphilic peptide segments of the cell-septation penicillin-binding protein 3 of Escherichia coli

The class B M1-V577 penicillin-binding protein (PBP) 3 of Escherichia coli consists of a M1-L39 membrane anchor (bearing a cytosolic tail) that is linked via a G40-S70 intervening peptide to an R71-I236 non-catalytic module (containing the conserved motifs 1-3) itself linked via motif 4 to a D237-V577 catalytic module (containing the conserved motifs 5-7 of the penicilloyl serine transferases superfamily). It has been proposed that during cell septation the peptidoglycan crosslinking activity of the acyl transferase module of PBP3 is regulated by the associated M1-I236 polypeptide itself in interaction with other components of the divisome. The fold adopted by the R71-V577 polypeptide of PBP3 has been modelled by reference to the corresponding R76-S634 polypeptide of the class B Streptococcus pneumoniae PBP2x. Based on these data and the results of site-directed mutagenesis of motifs 1-3 and of peptide segments of high amphiphilicity (identified from hydrophobic moment plots), the M1-I236 polypeptide of PBP3 appears to be precisely designed to work in the way proposed. The membrane anchor and the G40-S70 sequence (containing the G57-Q66 peptide segment) upstream from the non-catalytic module have the information ensuring that PBP3 undergoes proper insertion within the divisome at the cell septation site. Motif 1 and the I74-L82 overlapping peptide segment, motif 2 and the H160-G172 overlapping peptide segment, and the G188-D197 motif 3 are located at or close to the intermodule junction. They contain the information ensuring that PBP3 folds correctly and the acyl transferase catalytic centre adopts the active configuration. The E206-V217 peptide segment is exposed at the surface of the non-catalytic module. It has the information ensuring that PBP3 fulfils its cell septation activity within the fully complemented divisome.     

Site-directed mutagenesis experiments on the putative deprotonation site of squalene-hopene cyclase from Alicyclobacillus acidocaldarius

To provide insight into the catalytic mechanism for the final deprotonation reaction of squalene-hopene cyclase (SHC) from Alicyclobacillus acidocaldarius, mutagenesis experiments were conducted for the following ten residues: Thr41, Glu45, Glu93, Arg127, Trp133, Gln262, Pro263, Tyr267, Phe434 and Phe437. An X-ray analysis of SHC has revealed that two types of water molecules ("front water" and "back waters") were involved around the deprotonation site. The results of these mutagenesis experiments allow us to propose the functions of these residues. The two residues of Gln262 and Pro263 probably work to keep away the isopropyl group of the hopanyl cation intermediate from the "front water molecule," that is, to place the "front water" in a favorable position, leading to the minimal production of by-products, i.e., hopanol and hop-21(22)-ene. The five residues of Thr41, Glu45, Glu93, Arg127 and Trp133, by which the hydrogen-bonded network incorporating the "back waters" is constructed, increase the polarization of the "front water" to facilitate proton elimination from the isopropyl moiety of the hopanyl cation, leading to the normal product, hop-22(29)-ene. The three aromatic residues of Tyr267, Phe434 and Phe437 are likely to play an important role in guiding squalene from the enzyme surface to the reaction cavity (substrate channeling) by the strong affinity of their aromatic residues to the squalene substrate.     

Structure-function studies of yeast C-4 sphingolipid long chain base hydroxylase

The roles of putative active site residues of the Saccharomyces cerevisiae sphingolipid C-4 long chain base hydroxylase (Sur2p) were investigated by site-directed mutagenesis. The replacement of any one of conserved His residues of three histidine-rich motifs with an alanine eliminated hydroxylase activity in vivo and in vitro, indicating that they are all essential elements of the active site. An additional conserved His residue (His 249) outside of the histidine-rich cluster region was also found to be crucial for activity. Additional mutants altered in residues in close proximity to the histidine-rich cluster were generated. In order to determine their roles in hydroxylase vs. desaturase activities, residues were replaced with conserved residues from the yeast Delta7-sterol-C5(6)-desaturase, Erg3p. Residues Phe 174, Asn 182, Ser 191, Leu 196, Pro 199, Asn 266, Tyr 269, Asp 271 and Gln 275 appear to be additionally important elements of the active site but their conversion into corresponding Erg3p residues did not lead to a gain in desaturase activity. It is concluded that Sur2p is a membrane-bound hydroxylase that belongs to the diiron family of eight-histidine motif enzymes.     

The catalytic, glycosyl transferase and acyl transferase modules of the cell wall peptidoglycan-polymerizing penicillin-binding protein 1b of Escherichia coli

The penicillin-binding protein (PBP) 1b of Escherichia coli catalyses the assembly of lipid-transported N-acetyl glucosaminyl-beta-1, 4-N-acetylmuramoyl-L-alanyl-gamma-D-glutamyl-(L)-meso-diaminopimelyl+ ++- (L)-D-alanyl-D-alanine disaccharide pentapeptide units into polymeric peptidoglycan. These units are phosphodiester linked, at C1 of muramic acid, to a C55 undecaprenyl carrier. PBP1b has been purified in the form of His tag (M46-N844) PBP1bgamma. This derivative provides the host cell in which it is produced with a functional wall peptidoglycan. His tag (M46-N844) PBP1bgamma possesses an amino-terminal hydrophobic segment, which serves as transmembrane spanner of the native PBP. This segment is linked, via an congruent with 100-amino-acid insert, to a D198-G435 glycosyl transferase module that possesses the five motifs characteristic of the PBPs of class A. In in vitro assays, the glycosyl transferase of the PBP catalyses the synthesis of linear glycan chains from the lipid carrier with an efficiency of congruent with 39 000 M-1 s-1. Glu-233, of motif 1, is central to the catalysed reaction. It is proposed that the Glu-233 gamma-COOH donates its proton to the oxygen atom of the scissile phosphoester bond of the lipid carrier, leading to the formation of an oxocarbonium cation, which then undergoes attack by the 4-OH group of a nucleophile N-acetylglucosamine. Asp-234 of motif 1 or Glu-290 of motif 3 could be involved in the stabilization of the oxocarbonium cation and the activation of the 4-OH group of the N-acetylglucosamine. In turn, Tyr-310 of motif 4 is an important component of the amino acid sequence-folding information. The glycosyl transferase module of PBP1b, the lysozymes and the lytic transglycosylase Slt70 have much the same catalytic machinery. They might be members of the same superfamily. The glycosyl transferase module is linked, via a short junction site, to the amino end of a Q447-N844 acyl transferase module, which possesses the catalytic centre-defining motifs of the penicilloyl serine transferases superfamily. In in vitro assays with the lipid precursor and in the presence of penicillin at concentrations sufficient to derivatize the active-site serine 510 of the acyl transferase, the rate of glycan chain synthesis is unmodified, showing that the functioning of the glycosyl transferase is acyl transferase independent. In the absence of penicillin, the products of the Ser-510-assisted double-proton shuttle are glycan strands substituted by cross-linked tetrapeptide-pentapeptide and tetrapeptide-tetrapeptide dimers and uncross-linked pentapeptide and tetrapeptide monomers. The acyl transferase of the PBP also catalyses aminolysis and hydrolysis of properly structured thiolesters, but it lacks activity on D-alanyl-D-alanine-terminated peptides. This substrate specificity suggests that carbonyl donor activity requires the attachment of the pentapeptides to the glycan chains made by the glycosyl transferase, and it implies that one and the same PBP molecule catalyses transglycosylation and peptide cross-linking in a sequential manner. Attempts to produce truncated forms of the PBP lead to the conclusion that the multimodular polypeptide chain behaves as an integrated folding entity during PBP1b biogenesis.     

Mutational analysis of the ras converting enzyme reveals a requirement for glutamate and histidine residues

The Ras converting enzyme (RCE) promotes a proteolytic activity that is required for the maturation of Ras, the yeast a-factor mating pheromone, and certain other proteins whose precursors bear a C-terminal CAAX tetrapeptide motif. Despite the physiological importance of RCE, the enzymatic mechanism of this protease remains undefined. In this study, we have evaluated the substrate specificity of RCE orthologs from yeast (Rce1p), worm, plant, and human and have determined the importance of conserved residues toward enzymatic activity. Our findings indicate that RCE orthologs have conserved substrate specificity, cleaving CVIA, CTLM, and certain other CAAX motifs, but not the CASQ motif, when these motifs are placed in the context of the yeast a-factor precursor. Our mutational studies of residues conserved between the orthologs indicate that an alanine substitution at His194 completely inactivates yeast Rce1p enzymatic activity, whereas a substitution at Glu156 or His248 results in marginal activity. We have also determined that residues Glu157, Tyr160, Phe190, and Asn252 impact the substrate selectivity of Rce1p. Computational methods predict that residues influencing Rce1p function are all near or within hydrophobic segments. Combined, our data indicate that yeast Rce1p function requires residues that are invariably conserved among an extended family of prokaryotic and eukaryotic enzymes and that these residues are likely to lie within or immediately adjacent to the transmembrane segments of this membrane-localized enzyme.     

Topology of acyltransferase motifs and substrate specificity and accessibility in 1-acyl-sn-glycero-3-phosphate acyltransferase 1

1-acyl-sn-glycero-3-phosphate (AGP) acyltransferases (AGPAT) are involved in de novo biosynthesis of glycerolipids, such as phospholipids and triacylglycerol. Alignment of amino acid sequences from AGPAT, sn-glycerol-3-phosphate acyltransferase, and dihydroxyacetonephosphate acyltransferase reveals four regions with strong homology (acyltransferase motifs I-IV). The invariant amino acids within these regions may be part of a catalytically important site in this group of acyl-CoA acyltransferases. However, in human AGPAT1 a transmembrane domain is predicted to separate motif I on the cytosolic side from motifs II-III on the lumenal side, with motif IV near surface of the membrane. The topology of motifs I and III was confirmed by experiments with recombinant AGPAT1 containing potential glycosylation site near the motifs. This topology conflicts with the expectation that catalytically important sites are near one another, raising questions of whether the acyltransferase motifs really are important for AGPAT catalysis, and how substrates access motifs II-III on the lumenal side of the endoplasmic reticulum membrane. Using human AGPAT1 as a model, we have examined the catalytic roles of highly conserved residues in the four acyltransferase motifs by site-directed mutagenesis. Modifications of the sidechain structures of His104, Asp109, Phe146, Arg149, Glu178, Gly179, Thr180, Arg181 and Ile208 all affected AGPAT1 activity, indicating that the acyltransferase motifs indeed are important for AGPAT catalysis. In addition, we examined substrate accessibility to the catalytic domain of human AGPAT1 using a competition assay. Lysophosphatidic acid (LPA) with fatty acid chains shorter than 10 carbons did not access the catalytic domain, suggesting that LPA hydrophobicity is important. In contrast, short chain acyl-CoAs did access the catalytic domain but did not serve as the second substrate. These results suggest that motifs II and III are involved in LPA binding and motifs I and IV are involved in acyl-CoA binding.     

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.     

Molecular modelling and site-directed mutagenesis of human GALR1 galanin receptor defines determinants of receptor subtype specificity

Human galanin is a 30 amino acid neuropeptide that elicits a range of biological activities by interaction with G protein-coupled receptors. We have generated a model of the human GALR1 galanin receptor subtype (hGALR1) based on the alpha carbon maps of frog rhodopsin and investigated the significance of potential contact residues suggested by the model using site-directed mutagenesis. Mutation of Phe186 within the second extracellular loop to Ala resulted in a 6-fold decrease in affinity for galanin, representing a change in free energy consistent with hydrophobic interaction. Our model suggests interaction between Phe186 of hGALR1 and Ala7 or Leu11 of galanin. Receptor subtype specificity was investigated by replacement of residues in hGALR1 with the corresponding residues in hGALR2 and use of the hGALR2-specific ligands hGalanin(2-30) and [D-Trp2]hGalanin(1-30). The His267Ile mutant receptor exhibited a pharmacological profile corresponding to that of hGALR1, suggesting that His267 is not involved in a receptor-ligand interaction. The mutation Phe115Ala resulted in a decreased binding affinity for hGalanin and for hGALR2-specific analogues, indicating Phe115 to be of structural importance to the ligand binding pocket of hGALR1 but not involved in direct ligand interaction. Analysis of Glu271Trp suggested that Glu271 of hGALR1 interacts with the N-terminus of galanin and that the Trp residue in the corresponding position in hGALR2 is involved in receptor subtype specificity of binding. Our model supports previous reports of Phe282 of hGALR1 interacting with Trp2 of galanin and His264 of hGALR1 interacting with Tyr9 of galanin.     

Glutamates 99 and 107 in transmembrane helix III of subunit I of cytochrome bd are critical for binding of the heme b595-d binuclear center and enzyme activity

Cytochrome bd is a quinol oxidase of Escherichia coli under microaerophilic growth conditions. Coupling of the release of protons to the periplasm by quinol oxidation to the uptake of protons from the cytoplasm for dioxygen reduction generates a proton motive force. On the basis of sequence analysis, glutamates 99 and 107 conserved in transmembrane helix III of subunit I have been proposed to convey protons from the cytoplasm to heme d at the periplasmic side. To probe a putative proton channel present in subunit I of E. coli cytochrome bd, we substituted a total of 10 hydrophilic residues and two glycines conserved in helices I and III-V and examined effects of amino acid substitutions on the oxidase activity and bound hemes. We found that Ala or Leu mutants of Arg9 and Thr15 in helix I, Gly93 and Gly100 in helix III, and Ser190 and Thr194 in helix V exhibited the wild-type phenotypes, while Ala and Gln mutants of His126 in helix IV retained all hemes but partially lost the activity. In contrast, substitutions of Thr26 in helix I, Glu99 and Glu107 in helix III, Ser140 in helix IV, and Thr187 in helix V resulted in the concomitant loss of bound heme b558 (T187L) or b595-d (T26L, E99L/A/D, E107L/A/D, and S140A) and the activity. Glu99 and Glu107 mutants except E107L completely lost the heme b595-d center, as reported for heme b595 ligand (His19) mutants. On the basis of this study and previous studies, we propose arrangement of transmembrane helices in subunit I, which may explain possible roles of conserved hydrophilic residues within the membrane.     

Key role of conserved histidines in recombinant mouse beta-carotene 15,15'-monooxygenase-1 activity

Alignment of sequences of vertebrate beta-carotene 15,15'-monooxygenase-1 (BCMO1) and related oxygenases revealed four perfectly conserved histidines and five acidic residues (His172, His237, His308, His514, Asp52, Glu140, Glu314, Glu405, and Glu457 in mouse BCMO1). Because BCMO1 activity is iron-dependent, we propose that these residues participate in iron coordination and therefore are essential for catalytic activity. To test this hypothesis, we produced mutant forms of mouse BCMO1 by replacing the conserved histidines and acidic residues as well as four histidines and one glutamate non-conserved in the overall family with alanines by site-directed mutagenesis. Our in vitro and in vivo data showed that mutation of any of the four conserved histidines and Glu405 caused total loss of activity. However, mutations of non-conserved histidines or any of the other conserved acidic residues produced impaired although enzymatically active proteins, with a decrease in activity mostly due to changes in V(max). The iron bound to protein was determined by inductively coupled plasma atomic emission spectrometry. Bound iron was much lower in preparations of inactive mutants than in the wild-type protein. Therefore, the conserved histidines and Glu405 are absolutely required for the catalytic mechanism of BCMO1. Because the mutant proteins are impaired in iron binding, these residues are concluded to coordinate iron required for catalytic activity. These data are discussed in the context of the predicted structure for the related eubacterial apocarotenal oxygenase.     

Human carbonic anhydrase I: Effect of specific site mutations on its function

Carbonic anhydrase I (CAI) is one out of ten CA isoenzymes that have been identified in humans. X-ray crystallographic and inhibitor complex studies of human carbonic anhydrase I (HCAI) and related studies in other CA isoenzymes identified several residues, in particular Thr199, GlulO6, Tyr7, Glull7, His l07, with likely involvement in the catalytic activity of HCAI. To further study the role of these residues, we undertook, site-directed mutagenesis of HCAI. Using a polymerase chain reaction based strategy and altered oligonucleotide primers, we modified a cloned wild type hCAI gene so as to produce mutant genes encoding proteins with single amino acid substitutions. Thrl99Val, Thrl99Cys, Thr199Ser, GlulO6Ile, Glul06Gln, Tyr7Trp, Glu.117Gln, and His 107Val mutations were thus generated and the activity of each measured by ester hydrolysis. Overproduction of the Glu117Gln and HisI07Val mutant proteins inEscherichia coli resulted in a large proportion of the enzyme forming aggregates probably due to folding defect. The mutations Thr199Val, GlulO6Ile and GlulO6Gln gave soluble protein with drastically reduced enzyme activity, while the Tyr7Trp mutation had only marginal effect on the activity, thus s.uggesting important roles for Thr199 and Glu lO6 but not for Tyr7 in the catalytic function of HCAI.     

Identification of domain-domain docking sites within Clostridium symbiosum pyruvate phosphate dikinase by amino acid replacement

Potential domain-domain docking residues, identified from the x-ray structure of the Clostridium symbiosum apoPPDK, were replaced by site-directed mutagenesis. The steady-state and transient kinetic properties of the mutant enzymes were determined as a way of evaluating docking efficiency. PPDK mutants, in which one of two stringently conserved docking residues located on the N-terminal domain (Arg(219) and Glu(271)) was substituted, displayed largely unimpeded catalysis of the phosphoenolpyruvate partial reaction at the C-terminal domain, but significantly impaired catalysis (>10(4)) of the ATP pyrophosphorylation of His(455) at the N-terminal domain. In contrast, alanine mutants of two potential docking residues located on the N-terminal domain (Ser(262) and Lys(149)), which are not conserved among the PPDKs, exhibited essentially normal catalytic turnover. Arg(219) and Glu(271) were thus proposed to play an important role in guiding the central domain and, hence, the catalytic His(455) into position for catalysis. Substitution of central domain residues Glu(434)/Glu(437) and Thr(453), the respective docking partners of Arg(219) and Glu(271), resulted in mutants impaired in catalysis at the ATP active site. The x-ray crystal structure of the apo-T453A PPDK mutant was determined to test for possible misalignment of residues at the N-terminal domain-central domain interface that might result from loss of the Thr(453)-Glu(271) binding interaction. With the exception of the mutation site, the structure of T453A PPDK was found to be identical to that of the wild-type enzyme. It is hypothesized that the two Glu(271) interfacial binding sites that remain in the T453A PPDK mutant, Thr(453) backbone NH and Met(452) backbone NH, are sufficient to stabilize the native conformation as observed in the crystalline state but may be less effective in populating the reactive conformation in solution.     

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.     

Site-directed mutagenesis of active site residues of phosphite dehydrogenase

Phosphite dehydrogenase (PTDH) catalyzes the unusual oxidation of phosphite to phosphate with the concomitant reduction of NAD(+) to NADH. PTDH shares significant amino acid sequence similarity with D-hydroxy acid dehydrogenases (DHs), including strongly conserved catalytic residues His292, Glu266, and Arg237. Site-directed mutagenesis studies corroborate the essential role of His292 as all mutants of this residue were completely inactive. Histidine-selective inactivation studies with diethyl pyrocarbonate provide further evidence regarding the importance of His292. This residue is most likely the active site base that deprotonates the water nucleophile. Kinetic analysis of mutants in which Arg237 was changed to Leu, Lys, His, and Gln revealed that Arg237 is involved in substrate binding. These results agree with the typical role of this residue in D-hydroxy acid DHs. However, Glu266 does not play the typical role of increasing the pK(a) of His292 to enhance substrate binding and catalysis as the Glu266Gln mutant displayed an increased k(cat) and unchanged pH-rate profile compared to those of wild-type PTDH. The role of Glu266 is likely the positioning of His292 and Arg237 with which it forms hydrogen bonds in a homology model. Homology modeling suggests that Lys76 may also be involved in substrate binding, and this postulate is supported by mutagenesis studies. All mutants of Lys76 display reduced activity with large effects on the K(m) for phosphite, and Lys76Cys could be chemically rescued by alkylation with 2-bromoethylamine. Whereas a positively charged residue is absolutely essential for activity at the position of Arg237, Lys76 mutants that lacked a positively charged side chain still had activity, indicating that it is less important for binding and catalysis. These results highlight the versatility of nature's catalytic scaffolds, as a common framework with modest changes allows PTDH to catalyze its unusual nucleophilic displacement reaction and d-hydroxy acid DHs to oxidize alcohols to ketones.     

Aspzincin, a family of metalloendopeptidases with a new zinc-binding motif. Identification of new zinc-binding sites (His(128), His(132), and Asp(164)) and three catalytically crucial residues (Glu(129), Asp(143), and Tyr(106)) of deuterolysin from Aspergillus oryzae by site-directed mutagenesis.

Deuterolysin (EC 3.4.24.39; formerly designated as neutral proteinase II) from Aspergillus oryzae, which contains 1 g atom of zinc/mol of enzyme, is a single chain of 177 amino acid residues, includes three disulfide bonds, and has a molecular mass of 19,018 Da. Active-site determination of the recombinant enzyme expressed in Escherichia coli was performed by site-directed mutagenesis. Substitutions of His(128) and His(132) with Arg, of Glu(129) with Gln or Asp, of Asp(143) with Asn or Glu, of Asp(164) with Asn, and of Tyr(106) with Phe resulted in almost complete loss of the activity of the mutant enzymes. It can be concluded that His(128), His(132), and Asp(164) provide the Zn(2+) ligands of the enzyme according to a (65)Zn binding assay. Based on site-directed mutagenesis experiments, it was demonstrated that the three essential amino acid residues Glu(129), Asp(143), and Tyr(106) are catalytically crucial residues in the enzyme. Glu(129) may be implicated in a central role in the catalytic function. We conclude that deuterolysin is a member of a family of Zn(2+) metalloendopeptidases with a new zinc-binding motif, aspzincin, defined by the "HEXXH + D" motif and an aspartic acid as the third zinc ligand.     

Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.     

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.     

浙江地区青霉素耐药肺炎链球菌PBPs基因及氨基酸序列研究

为阐明温州地区青霉素耐药肺炎链球菌（PRSP）的青霉素结合蛋白（PBPs）的基因和氨基酸序列的变异特点,对温州医学院自2000年11月～2004年1月收集的26份肺炎链球菌进行分离、鉴定及青霉素药敏实验,并对每株链球菌的PBP1A、PBP2B、PBP2X基因进行PCR扩增和直接测序,通过序列比对与生物信息学分析。结果表明,研究中的PBP1A的主要突变位点是保守基序KTG之后的4个连续氨基酸替换Thr574Ala、Ser575Thr、Gln576Gly、Phe577Tyr和保守序列STMK内的氨基酸替换Thr371Ser;PBP2B的主要突变位点是保守序列SSN之后的氨基酸替换Thr451Ala;PBP2X的主要突变位点是保守基序STMK 内的氨基酸替换Thr338Ala。以上突变类型以及菌株的青霉素耐药水平与文献报道相符。研究检测的PRSP的PBPs基因中暂未发现本地区特有的（新的）基因突变,也未检测出文献报道的某些与青霉素抗性相关的氨基酸替换。     

Contribution of active site residues to the activity and thermal stability of ribonuclease Sa

We have used site-specific mutagenesis to study the contribution of Glu 74 and the active site residues Gln 38, Glu 41, Glu 54, Arg 65, and His 85 to the catalytic activity and thermal stability of ribonuclease Sa. The activity of Gln38Ala is lowered by one order of magnitude, which confirms the involvement of this residue in substrate binding. In contrast, Glu41Lys had no effect on the ribonuclease Sa activity. This is surprising, because the hydrogen bond between the guanosine N1 atom and the side chain of Glu 41 is thought to be important for the guanine specificity in related ribonucleases. The activities of Glu54Gln and Arg65Ala are both lowered about 1000-fold, and His85Gln is totally inactive, confirming the importance of these residues to the catalytic function of ribonuclease Sa. In Glu74Lys, k(cat) is reduced sixfold despite the fact that Glu 74 is over 15 A from the active site. The pH dependence of k(cat)/K(M) is very similar for Glu74Lys and wild-type RNase Sa, suggesting that this is not due to a change in the pK values of the groups involved in catalysis. Compared to wild-type RNase Sa, the stabilities of Gln38Ala and Glu74Lys are increased, the stabilities of Glu41Lys, Glu54Gln, and Arg65Ala are decreased and the stability of His85Gln is unchanged. Thus, the active site residues in the ribonuclease Sa make different contributions to the stability.