Mutational analyses of restriction endonuclease-HindIII mutant E86K with higher activity and altered specificity

We have performed mutational analyses of restriction endonuclease HindIII in order to identify the amino acid residues responsible for enzyme activity. Four of the seven HindIII mutants, which had His-tag sequences at the N-termini, were expressed in Escherichia coli, and purified to homogeneity. The His-tag sequence did not affect enzyme activity, whereas it hindered binding of the DNA probe in gel retardation assays. A mutant E86K in which Lys was substituted for Glu at residue 86 exhibited high endonuclease activity. Gel retardation assays showed high affinity of this mutant to the DNA probe. Surprisingly, in the presence of a transition metal, Mo(2+) or Mn(2+), the E86K mutant cleaved substrate DNA at a site other than HindIII. Substitution of Glu for Val at residue 106 (V106E), and Asn for Lys at residue 125 (K125N) resulted in a decrease in both endonucleolytic and DNA binding activities of the enzyme. Furthermore, substitution of Leu for Asp at residue 108 (D108L) abolished both HindIII endonuclease and DNA binding activities. CD spectra of the wild type and the two mutants, E86K and D108L, were similar to each other, suggesting that there was little change in conformation as a result of the mutations. These results account for the notion that Asp108 could be directly involved in HindIII catalytic function, and that the substitution at residue 86 may bring about new interactions between DNA and cations.     

A mutant of Escherichia coli with altered inducer specificity for the fad regulon

A novel regulatory mutant of the fatty acid degradation ($\text{fad}$) regulon of $\text{Escherichia coli}$ was isolated. This mutant, D-2, was induced to synthesize the fatty acid β-oxidation enzymes during growth on decanoate and laurate whereas the wild type strain was induced only when fatty acids with a chain length greater than 12 carbon atoms were present in the growth medium. The fatty acid specificity of the acyl CoA synthetase was also changed in strain D-2. The data are consistant with the hypothesis that acyl CoA's themselves are the inducers of the $\text{fad}$ regulon and suggest that strain D-2 may synthesize an altered $\text{fad}$ regulatory protein. The results also suggest that the acyl CoA synthetase may possess regulatory as well as enzymatic activity.     

Novel splice variants of cyclin E with altered substrate specificity

Cyclin E, a G₁ cyclin, is overexpressed and present in low molecular weight (LMW) isoforms in breast cancer cells and tumor tissues. In this study we have examined the possibility that the shortened mRNA splice variants could give rise to tumor-specific cyclin E LMW proteins. We used the Splice Capture method to identify, enumerate and isolate known spliced mRNAs and to look for previously undetected mRNA forms of cyclin E that might be translated into the LMW proteins. We show that a new splice variant of cyclin E found in tumor cells isolated by the Splice Capture strategy, named Δ48, activates CDK2 more robustly than full-length cyclin E when assayed from transiently transfected cells with the natural substrate GST-Rb. We also found the Splice Capture method to be superior to the conventional RNase protection assay in analyzing the cyclin E mRNA present in normal and tumor cells. Splice Capture enumerated the relative abundance of known forms of cyclin E mRNA and easily discovered new splice variants in both normal and tumor cells. We conclude that the abundance of cyclin E splice variants in cells may represent a novel form of regulation of cyclin E, and if translated they show altered substrate specificity compared to the full length form of cyclin E.     

Tritium planigraphy comparative structural study of tobacco mosaic virus and its mutant with altered host specificity.

Spatial organization of wild-type (strain U1) tobacco mosaic virus (TMV) and of the temperature-sensitive TMV ts21-66 mutant was compared by tritium planigraphy. The ts21-66 mutant contains two substitutions in the coat protein (Ile21-->Thr and Asp66-->Gly) and, in contrast with U1, induces a hypersensitive response (formation of necroses) on the leaves of plants bearing a host resistance gene N' (for example Nicotiana sylvestris); TMV U1 induces systemic infection (mosaic) on the leaves of such plants. Tritium distribution along the coat protein (CP) polypeptide chain was determined after labelling of both isolated CP preparations and intact virions. In the case of the isolated low-order (3-4S) CP aggregates no reliable differences in tritium distribution between U1 and ts21-66 were found. But in labelling of the intact virions a significant difference between the wild-type and mutant CPs was observed: the N-terminal region of ts21-66 CP incorporated half the amount of tritium than the corresponding region of U1 CP. This means that in U1 virions the CP N-terminal segment is more exposed on the virion surface than in ts21-66 virions. The possibility of direct participation of the N-terminal tail of U1 CP subunits in the process of the N' hypersensitive response suppression is discussed.     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

Mapping the pro-region of carboxypeptidase B by protein engineering. Cloning, overexpression, and mutagenesis of the porcine proenzyme.

The proteolytic processing of pancreatic procarboxypeptidase B to a mature and functional enzyme is much faster than that of procarboxypeptidase A1. This different behavior has been proposed to depend on specific conformational features at the region that connects the globular domain of the pro-segment to the enzyme and at the contacting surfaces on both moieties. A cDNA coding for porcine procarboxypeptidase B was cloned, sequenced, and expressed at high yield (250 mg/liter) in the methylotrophic yeast Pichia pastoris. To test the previous hypothesis, different mutants of the pro-segment at the putative tryptic targets in its connecting region and at some of the residues contacting the active enzyme were obtained. Moreover, the complete connecting region was replaced by the homologous sequence in procarboxypeptidase A1. The detailed study of the tryptic processing of the mutants shows that limited proteolysis of procarboxypeptidase B is a very specific process, as Arg-95 is the only residue accessible to tryptic attack in the proenzyme. A fast destabilization of the connecting region after the first tryptic cut allows subsequent proteolytic processing and the expression of carboxypeptidase B activity. Although all pancreatic procarboxypeptidases have a preformed active site, only the A forms show intrinsic activity. Mutational substitution of Asp-41 in the globular activation domain, located at the interface with the enzyme moiety, as well as removal of the adjacent 310 helix allow the appearance of residual activity in the mutated procarboxypeptidase B, indicating that the interaction of both structural elements with the enzyme moiety prevents the binding of substrates and promotes enzyme inhibition. In addition, the poor heterologous expression of such mutants indicates that the mutated region is important for the folding of the whole proenzyme.     

Interaction of subtilisins with serpins.

Serpins are well-characterized inhibitors of the chymotrypsin family serine proteinases. We have investigated the interaction of two serpins with members of the subtilisin family, proteinases that possess a similar catalytic mechanism to the chymotrypsins, but a totally different scaffold. We demonstrate that alpha 1 proteinase inhibitor inhibits subtilisin Carlsberg and proteinase K, and alpha 1 antichymotrypsin inhibits proteinase K, but not subtilisin Carlsberg. When inhibition occurs, the rate of formation and stability of the complexes are similar to those formed between serpins and chymotrypsin family members. However, inhibition of subtilisins is characterized by large partition ratios where more than four molecules of each serpin are required to inhibit one subtilisin molecule. The partition ratio is caused by the serpins acting as substrates or inhibitors. The ratio decreases as temperature is elevated in the range 0-45 degrees C, indicating that the serpins are more efficient inhibitors at high temperature. These aspects of the subtilisin interaction are all observed during inhibition of chymotrypsin family members by serpins, indicating that serpins accomplish inhibition of these two distinct proteinase families by the same mechanism.     

Crystallographic analysis of trypsin-G226A. A specificity pocket mutant of rat trypsin with altered binding and catalysis

The crystal structure of trypsin-G226A has been determined, in the presence of benzamidine, to a resolution of 1.75 A with an R-factor of 14.6%. The mutation was designed to alter substrate specificity by disrupting arginine binding, but was previously found to disrupt catalysis to a greater extent than binding. The arginine analog, benzamidine, has rotated 40 degrees and 49 degrees and translated 1.1 A in the specificity pocket, relative to the position in wild-type trypsin. The salt-bridge between the amidinium group of benzamidine and the carboxylate of D189 as well as four other hydrogen bonds have been replaced by a set of six new hydrogen bonds. Based on these interactions, computer modeling of an arginine substrate demonstrates that arginine terminal nitrogen atoms can occupy the new benzamidine nitrogen positions with torsion angle adjustments and without short contacts. In the secondary orientation, arginine substrates appear to be forced out of alignment with the active site. This may account for the larger drop in kcat with arginine relative to lysine substrates. A second possible cause of the altered activity is a change of the enzyme structure with concomitant loss of activity. No evidence of such a change is seen in the co-ordinates or temperature factors of the trypsin-G226A-benzamidine complex. A226 disrupts mainly the co-ordinates of amino acids with which it has direct contacts such that the effects of the mutation are absorbed locally.     

Pediococcus cerevisiae mutant with altered transport of folates.

A Pediococcus cerevisiae mutant that actively accumulated folate (PteGlu), in contrast to the wild-type, was also found to exhibit changes in the pattern of uptake of 5-methyl-tetrahydrofolate (5-CH3-H4PteGlu) and amethopterin. Most of the 5-CH3-H4PteGlue accumulated through a glucose- and temperature-dependent process, and a concentrative uptake was also found in gluocse-starved cells and in cells incubated at OC. About 75% of the accumulated 5-CH3-H4PteGlu exchanged with amethopterin. In contrast to the wild type, the mutant accumulated both diastereoisomers of 5-CH3-H4PteGlue by glucose-dependent and glucose-independent processes. Amethopterin and PteGlue competitively inhibited the uptake in both processes, with an apparent lower affinity of the carrier for PteGlu than for the analogue. p-Chloromercuribenzoate strongly inhibited the uptake (75%). The p-chloromercuribenzoate-nonsusceptible and temperature-independent uptake was also competed by amethopterin. Metabolic poisons like sodium azide, potassium fluoride, iodoacetate, and 2,4-dimitrophenol inhibited the glucose-dependent process. Uptake, in the absence of glucose, was enhanced by sodium azide and potassium fluoride.     

FeMo cofactor synthesis by a nifH mutant with altered MgATP reactivity.

We have characterized a Nif- mutant of Azotobacter vinelandii, designated UW91 (Shah, V. K., Davis, L. C., Gordon, J. K., Orme-Johnson, W. H., and Brill, W. J. (1973) Biochim. Biophys. Acta 292, 246-255). The specific Fe protein mutation giving rise to the Nif- phenotype was shown by DNA sequencing and site-directed mutagenesis to be the substitution of a conserved alanine at position 157 by a serine. The UW91 Fe protein was purified and shown to have a normal [4Fe-4S] cluster and normal MgATP binding activity. The substitution of alanine 157 by serine, however, prevents the MgATP-induced conformational change that occurs for the wild-type Fe protein, prevents MgATP hydrolysis, and prevents productive electron transfer to the MoFe protein. The UW91 Fe protein does bind to the MoFe protein to give a normal cross-linking pattern; however, it does not compete very successfully with wild-type Fe protein in an activity assay. The UW91 MoFe protein was also purified and characterized and shown to be indistinguishable from the wild-type protein. Thus, the substitution of Fe protein residue alanine 157 by serine does not change the Fe protein's ability to function in FeMo cofactor biosynthesis or insertion. This demonstrates that these events do not require the MgATP-induced conformational change, MgATP hydrolysis, or productive electron transfer to the MoFe protein.     

AraC proteins with altered DNA sequence specificity which activate a mutant promoter in Escherichia coli

We examined the recognition of the araBAD promoter by the AraC protein in the Escherichia coli arabinose operon. A mutant promoter, with base substitutions at positions contacted by AraC, was used to isolate suppressor mutations in araC by direct selection. Two hydroxylamine-induced araC mutations were isolated repeatedly; each contained a single amino acid substitution. When tested against a set of base substitution promoter mutants, one revertant, an Arg to His substitution at residue 250, displayed altered base specificity for a single position within the araBAD promoter. The other revertant, a Cys to Tyr substitution at residue 204, did not show consistent base-specific suppression. Neither demonstrated a higher affinity than the wild type protein for the mutant promoter in vitro. Both proteins suppress mutant sequences by a mechanism that does not appear to involve the formation of new net favorable contacts with the mutant base pairs of the promoter.     

Isolation and mapping of a mutant penicillin G acylase with altered substrate specificity from Escherichia coli

Summary Penicillin G acylase of Escherichia coli ATCC 11105 catalyzes hydrolysis as wellas synthesis of penicillin G. In this work a recombinant penicillin G acylase genewas mutagenized in vivo. A mutant with altered penicillin G acylase was selectedby its ability to grow with phthalyl-L-leucine as sole source of leucine. Themutant enzyme obtained was deficient in hydrolyzing penicillin G. A mutation ofGly359 to aspartic acid was mapped first by construction of chimeric pac genescomposed of wild type and mutant DNA, followed by nucleotide sequencing.     

Phage-display evolution of tyrosine kinases with altered nucleotide specificity.

The problem of identifying downstream targets of kinase phosphorylation remains a challenge despite technological advances in genomics and proteomics. A recent approach involves the generation of kinase mutants that can uniquely use "orthogonal" ATP analogs to phosphorylate substrates in vivo. Using structure-based design, mutants of several protein kinase superfamily members have been found; robust and general methods are needed, however, for altering the nucleotide specificity of the remaining kinases in the genome. Here we demonstrate the application of a new phage display technique for direct functional selection to the identification of a tyrosine kinase mutant with the ability to use N6-benzyl-ATP. Our method produces, in five rounds of selection, a mutant identical to the best orthogonal Src kinase found to date. In addition, we isolate from a larger library of kinase mutants a promiscuous clone capable of using many different ATP analogs. This approach to engineering orthogonal kinases, combined with others, will facilitate the mapping of phosphorylation targets of any kinase in the genome.     

Computational method for the design of enzymes with altered substrate specificity.

A combination of enzyme kinetics and X-ray crystallographic analysis of site-specific mutants has been used to probe the determinants of substrate specificity for the enzyme alpha-lytic protease. We now present a generalized model for understanding the effects of mutagenesis on enzyme substrate specificity. This algorithm uses a library of side-chain rotamers to sample conformation space within the binding site for the enzyme-substrate complex. The free energy of each conformation is evaluated with a standard molecular mechanics force field, modified to include a solvation energy term. This rapid energy calculation based on coarse conformation sampling quite accurately predicts the relative catalytic efficiency of over 40 different alpha-lytic protease-substrate combinations. Unlike other computational approaches, with this method it is feasible to evaluate all possible mutations within the binding site. Using this algorithm, we have successfully designed a protease that is both highly active and selective for a non-natural substrate. These encouraging results indicate that it is possible to design altered enzymes solely on the basis of empirical energy calculations.     

The role of tryptophan residues in the autoprocessing of prosubtilisin E

Subtilisin E, a serine protease from Bacillus subtilis, requires an N-terminal propeptide for its correct folding. The propeptide is autocleaved and digested by the subtilisin domain upon proper folding. Here we investigated the individual roles of the three Trp residues within the subtilisin domain (Trp106, Trp113 and Trp241) on propeptide processing, enzymatic activity and stability of subtilisin. When the propeptide processing was examined by SDS-PAGE after refolding by rapid dilution, the mutation at either position Trp106 or Trp113 was found to significantly delay the propeptide processing, while the mutation at Trp241 had no effect. Far-UV circular dichroism (CD) spectra of the mutants revealed that the mutations at the three positions did not affect appreciably the alpha-helix content of subtilisin. Secondary structure thermal unfolding monitored by CD spectroscopy revealed that none of the tryptophan residues had any significant effect on the stability of mature subtilisin. The enzymatic activity measurements showed that only Trp106 plays a major role in the enzymatic activity of subtilisin E. These results demonstrate that both Trp106 and Trp113 play a specific role in propeptide processing and enzymatic activity, while Trp241 plays no considerable role on any of these activities.