Substrate specificity of Escherichia coli thymidine phosphorylase

Substrate specificity of Escherichia coli thymidine phosphorylase to thymidine derivatives modified at 5' -, 3' -, and 2' ,3' - positions of the sugar moiety was studied. Equilibrium and kinetic constants (K(m), K(I), k(cat)) of the phosphorolysis reaction have been determined for 20 thymidine analogs. The results are compared with X-ray and molecular dynamics data. The most important hydrogen bonds in the enzyme-substrate complex are revealed.     

Mapping of the dimer interface of the Escherichia coli mannitol permease by cysteine cross-linking

A cysteine cross-linking approach was used to identify residues at the dimer interface of the Escherichia coli mannitol permease. This transport protein comprises two cytoplasmic domains and one membrane-embedded C domain per monomer, of which the latter provides the dimer contacts. A series of single-cysteine His-tagged C domains present in the native membrane were subjected to Cu(II)-(1,10-phenanthroline)(3)-catalyzed disulfide formation or cysteine cross-linking with dimaleimides of different length. The engineered cysteines were at the borders of the predicted membrane-spanning alpha-helices. Two residues were found to be located in close proximity of each other and capable of forming a disulfide, while four other locations formed cross-links with the longer dimaleimides. Solubilization of the membranes did only influence the cross-linking behavior at one position (Cys(73)). Mannitol binding only effected the cross-linking of a cysteine at the border of the third transmembrane helix (Cys(134)), indicating that substrate binding does not lead to large rearrangements in the helix packing or to dissociation of the dimer. Upon mannitol binding, the Cys(134) becomes more exposed but the residue is no longer capable of forming a stable disulfide in the dimeric IIC domain. In combination with the recently obtained projection structure of the IIC domain in two-dimensional crystals, a first proposal is made for alpha-helix packing in the mannitol permease.     

Substrate specificity of CTP synthetase from Escherichia coli

The stoichiometry of the enzymatic reaction catalyzed by CTP synthetase from Escherichia coli was analyzed by high-performance liquid chromatography. The results revealed that for every mole of UTP transformed to CTP, one mole of ATP was converted to ADP. The substrate specificity of CTP synthetase from E. coli was investigated by means of UTP analogs. Chemical modification of UTP involved either the uracil, ribose or 5'-triphosphate part. None of the UTP analogs studied proved to be a substrate. The capacity of the UTP analogs to inhibit CTP synthetase was investigated. From the UTP derivatives employed only 2-thiouridine 5'-triphosphate was found to inhibit the enzyme competitively with reasonable affinity: Ki/Km(UTP) = 1. This study indicated that the three main structural elements of the UTP molecule: uracil, ribose and 5'-triphosphate moiety, contribute to substrate specificity. The behaviour of a limited number of CTP analogs as product-like inhibitors supported this view.     

Membrane disposition of the Escherichia coli mannitol permease: identification of membrane-bound and cytoplasmic domains

Two proteolytic fragments of the Escherichia coli mannitol permease (EIImtl) have been identified on autoradiograms of sodium dodecyl sulfate-polyacrylamide gels and mapped with respect to the membrane. EIImtl was selectively radiolabeled with either [35S]methionine or a mixture of 14C-labeled amino acids in E. coli minicells harboring a plasmid containing the mannitol operon. The intact permease (Mr 65,000) in everted vesicles derived from labeled minicells was cleaved by mild trypsinolysis into two smaller fragments (Mr 34,000 and 29,000). The 34,000-dalton fragment remained in the membrane and was insensitive to further proteolysis by trypsin. This fragment was identified as the N-terminal half of the protein by comparing the amount of the original [35S]methionine label that it retained with the known differential distribution of methionine in the two halves of EIImtl. The 29,000-dalton fragment, which was released into the soluble fraction and was sensitive to further trypsinolysis, therefore corresponds to the C-terminal half of the mannitol permease. Both fragments were shown to be antigenically related to EIImtl by immunoblotting with anti-EIImtl antibody. The 34,000-dalton fragment was further shown to form an oligomer under conditions which allow the intact enzyme to dimerize, suggesting that this domain plays an important role in EIImtl subunit interactions. These results support a model in which EIImtl consists of two domains of approximately equal size: a membrane-bound, N-terminal domain with a tendency to self-associate, and a cytoplasmic C-terminal domain.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substrate specificity of the Escherichia coli outer membrane protease OmpT

OmpT is a surface protease of gram-negative bacteria that has been shown to cleave antimicrobial peptides, activate human plasminogen, and degrade some recombinant heterologous proteins. We have analyzed the substrate specificity of OmpT by two complementary substrate filamentous phage display methods: (i) in situ cleavage of phage that display protease-susceptible peptides by Escherichia coli expressing OmpT and (ii) in vitro cleavage of phage-displayed peptides using purified enzyme. Consistent with previous reports, OmpT was found to exhibit a virtual requirement for Arg in the P1 position and a slightly less stringent preference for this residue in the P1' position (P1 and P1' are the residues immediately prior to and following the scissile bond). Lys, Gly, and Val were also found in the P1' position. The most common residues in the P2' position were Val or Ala, and the P3 and P4 positions exhibited a preference for Trp or Arg. Synthetic peptides based upon sequences selected by bacteriophage display were cleaved very efficiently, with kcat/Km values up to 7.3 x 10(6) M(-1) s(-1). In contrast, a peptide corresponding to the cleavage site of human plasminogen was hydrolyzed with a kcat/Km almost 10(6)-fold lower. Overall, the results presented in this work indicate that in addition to the P1 and P1' positions, additional amino acids within a six-residue window (between P4 and P2') contribute to the binding of substrate polypeptides to the OmpT binding site.     

Substrate specificity of Escherichia coli peptidyltransferase at the donor site

The substrate specificity of $\text{E.}\text{coli}$ peptidyltransferase at the donor site was investigated by the “50S reaction”. Seventeen N-acetylated or unacetylated aminoacyl-tRNAs and dipeptidyl-tRNAs were used as the donor substrates and puromycin as the acceptor. Results indicated that the nature of amino acid side chain of the donor tRNA has a predominant effect on the reaction rate of peptidyltransferase. Amino acids or dipeptides with high hydrophobicity were transferred faster than those with low hydrophobicity. Amino acids with alkyl side chains are better donors than those with aromatic side chains. Substrates with C-terminal proline were transferred extremely slowly which can probably be attributed to its unusual α-imino structure in addition to its low hydrophobicity.     

Substrate specificity of the pseudouridine synthase RluD in Escherichia coli

Pseudouridine synthase RluD converts uridines at positions 1911, 1915, and 1917 of 23S rRNA to pseudouridines. These nucleotides are located in the functionally important helix-loop 69 of 23S rRNA. RluD is the only pseudouridine synthase that is required for normal growth in Escherichia coli. We have analyzed substrate specificity of RluD in vivo. Mutational analyses have revealed: (a) RluD isomerizes uridine in vivo only at positions 1911, 1915, and 1917, regardless of the presence of uridine at other positions in the loop of helix 69 of 23S rRNA variants; (b) substitution of one U by C has no effect on the conversion of others (i.e. formation of pseudouridines at positions 1911, 1915, and 1917 are independent of each other); (c) A1916 is the only position in the loop of helix 69, where mutations affect the RluD specific pseudouridine formation. Pseudouridines were determined in the ribosomal particles from a ribosomal large subunit defective strain (RNA helicase DeaD(-)). An absence of pseudouridines in the assembly precursor particles suggests that RluD directed isomerization of uridines occurs as a late step during the assembly of the large ribosomal subunit.     

Substrate specificity of the Escherichia coli outer membrane protease OmpP

Escherichia coli OmpP is an F episome-encoded outer membrane protease that exhibits 71% amino acid sequence identity with OmpT. These two enzymes cleave substrate polypeptides primarily between pairs of basic amino acids. We found that, like OmpT, purified OmpP is active only in the presence of lipopolysaccharide. With optimal peptide substrates, OmpP exhibits high catalytic efficiency (k(cat)/K(m) = 3.0 x 10(6) M(-1)s(-1)). Analysis of the extended amino acid specificity of OmpP by substrate phage revealed that both Arg and Lys are strongly preferred at the P1 and P1' sites of the enzyme. In addition, Thr, Arg, or Ala is preferred at P2; Leu, Ala, or Glu is preferred at P4; and Arg is preferred at P3'. Notable differences in OmpP and OmpT specificities include the greater ability of OmpP to accept Lys at the P1 or P1', site as well as the prominence of Ser at P3 in OmpP substrates. Likewise, the OmpP P1 site could better accommodate Ser; as a result, OmpP was able to cleave a peptide substrate between Ser-Arg about 120 times more efficiently than was OmpT. Interestingly, OmpP and OmpT cleave peptides with three consecutive Arg residues at different sites, a difference in specificity that might be important in the inactivation of cationic antimicrobial peptides. Accordingly, we show that the presence of an F' episome results in increased resistance to the antimicrobial peptide protamine both in ompT mutants and in wild-type E. coli cells.     

Substrate specificity and kinetic mechanism of Escherichia coli ribulokinase.

L-ribulokinase is unusual among kinases since it phosphorylates all four 2-ketopentoses with almost the same k(cat) values. The K(m)'s differ, however, being 0.14 mM for L- and 0.39 mM for d-ribulose and 3.4 mM for l- and 16 mM for d-xylulose. In addition, L-arabitol is phosphorylated at C-5 (K(m) 4 mM) and ribitol (adonitol) is phosphorylated to D-ribitol-5-phosphate (K(m) 5.5 mM), but D-arabitol, xylitol, and aldopentoses are not substrates. The K(m)'s for MgATP depend on the substrates, being 0.02 mM with L-ribulose, 0.027 mM with D-ribulose and L-xylulose, and 0.3-0.5 mM with the other substrates. In the absence of a sugar substrate there is an ATPase with K(m) of 7 mM and k(cat) 1% of that with sugar substrates. The initial velocity pattern is intersecting, and MgAMPPNP is competitive vs MgATP and uncompetitive vs L-ribulose. L-Erythrulose is competitive vs L-ribulose and when MgATP concentration is varied induces substrate inhibition which is partial. These data show that the mechanism is random, but there is a high level of synergism in the binding of sugar and MgATP, and the path in which the sugar adds first is strongly preferred.     

Subunit and amino acid interactions in the Escherichia coli mannitol permease: a functional complementation study of coexpressed mutant permease proteins.

Mannitol-specific enzyme II, or mannitol permease, of the phosphoenolpyruvate-dependent carbohydrate phosphotransferase system of Escherichia coli carries out the transport and phosphorylation of D-mannitol and is most active as a dimer in the membrane. We recently reported the importance of a glutamate residue at position 257 in the binding and transport of mannitol by this protein (C. Saraceni-Richards and G. R. Jacobson, J. Bacteriol. 179:1135-1142, 1997). Replacing Glu-257 with alanine (E257A) or glutamine (E257Q) eliminated detectable mannitol binding and transport by the permease. In contrast, an E257D mutant protein was able to bind and phosphorylate mannitol in a manner similar to that of the wild-type protein but was severely defective in mannitol uptake. In this study, we have coexpressed proteins containing mutations at position 257 with other inactive permeases containing mutations in each of the three domains of this protein. Activities of any active heterodimers resulting from this coexpression were measured. The results show that various inactive mutant permease proteins can complement proteins containing mutations at position 257. In addition, we show that both Glu at position 257 and His at position 195, both of which are in the membrane-bound C domain of the protein, must be on the same subunit of a permease dimer in order for efficient mannitol phosphorylation and uptake to occur. The results also suggest that mannitol bound to the opposite subunit within a permease heterodimer can be phosphorylated by the subunit containing the E257A mutation (which cannot bind mannitol) and support a model in which there are separate binding sites on each subunit within a permease dimer. Finally, we provide evidence from these studies that high-affinity mannitol binding is necessary for efficient transport by mannitol permease.     

Substrate specificity engineering of Escherichia coli derived fructosamine 6-kinase

A three-dimensional structural model of Escherichia coli fructosamine 6-kinase (FN6K), an enzyme that phosphorylates fructosamines at C6 and catalyzes the production of the fructosamine 6-phosphate stable intermediate, was generated using the crystal structure of 2-keto-3-deoxygluconate kinase isolated from Thermus thermophilus as template. The putative active site region was then investigated by site-directed mutagenesis to reveal several amino acid residues that likely play important roles in the enzyme reaction. Met220 was identified as a residue that plays a role in substrate recognition when compared to Bacillus subtilis derived FN6K, which shows different substrate specificity from the E. coli FN6K. Among the various Met220-substituted mutant enzymes, Met220Leu, which corresponded to the B. subtilis residue, resulted in an increased activity of fructosyl-valine and decreased activity of fructosyl-lysine, thus increasing the specificity for fructosyl-valine by 40-fold.     

Membrane insertion of the mannitol permease of Escherichia coli occurs under conditions of impaired SecA function.

The membrane insertion of the mannitol permease (MtlA protein) of Escherichia coli, a polytopic cytoplasmic membrane protein possessing an uncleaved amphiphilic signal sequence, was studied using a cell-free protein synthesis system. The MtlA protein synthesized in the presence of inside-out cytoplasmic membrane vesicles was shown to insert into the membranes based on the following criteria: (a) co-sedimentation of the majority of the MtlA protein with the vesicles; (b) selective extraction of the membrane-associated MtlA by doxycholate but not by urea treatment; and (c) protease resistance of a defined MtlA fragment observed exclusively in the presence of membranes. Post-translational addition of membrane vesicles allowed membrane association of MtlA but did not allow efficient integration. In cell-free systems having reduced levels of the export factors SecA and SecB and exhibiting defective translocation of preOmpA and preLamB, insertion of the in vitro synthesized MtlA apparently occurred normally. In contrast, when membranes from the secY24ts mutant or trypsin-treated membranes were used, insertion of MtlA was reduced. In vivo experiments monitoring the permease activity of MtlA in the secA and secY mutants supported the conclusions of the in vitro results. Thus, the insertion of MtlA is essentially SecA- and SecB-independent but may be dependent on SecY and/or an as yet unidentified membrane protein. The requirements for the insertion of the mannitol permease are therefore clearly different from those for the translocation of most proteins having a cleavable hydrophobic signal sequence.     

Characterization of purified, reconstituted site-directed cysteine mutants of the lactose permease of Escherichia coli

lac permease mutated at each of the 8 cysteinyl residues in the molecule was solubilized from the membrane, purified, and reconstituted into proteoliposomes. The transport activity of proteoliposomes reconstituted with each mutant permease relative to the wild-type is virtually identical with that reported for intact cells and/or right-side-out membrane vesicles. Moreover, a double mutant containing Ser in place of both Cys148 and Cys154 exhibits significant ability to catalyze active lactose transport. The results provide strong confirmation for the contention that cysteinyl residues in lac permease do not play an important role in the transport mechanism. The effect of sulfhydryl oxidant 5-hydroxy-2-methyl-1,4-naphthoquinone on lactose transport in proteoliposomes reconstituted with wild-type or mutant permeases was also investigated, and the results indicate that inactivation is probably due to formation of a covalent adduct with Cys148 and/or Cys154 rather than disulfide formation. Thus, it seems unlikely that sulfhydryl-disulfide interconversion functions to regulate permease activity.     

Substrate specificity and transport properties of the glycerol facilitator of Escherichia coli.

The specificity of the glycerol facilitator (glpF) of Escherichia coli was studied with an osmotic method. This transport system allowed the entry of polyols (glycerol and erythritol), pentitols, and hexitols. The analogous sugars were not transported. However, urea, glycine, and DL-glyceraldehyde could use this pathway to enter the cell. The glpF protein allowed the rapid efflux of preequilibrated xylitol. Glycerol surprisingly did not inhibit the uptake of xylitol, and xylitol only slightly reduced the uptake of glycerol. The observation and the insensitivity of the xylitol transport to low temperature suggest that the facilitator behaves as a membrane channel.     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.