Use of inteins for the in vivo production of stable cyclic peptide libraries in E. coli.
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Base Sequence↗
Biomedical subjects
Stephen J Benkovic
Publications and source records attributed to Stephen J Benkovic.
Using transcriptional-based systems for in vivo enzyme screening.
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Cloning, Molecular↗
Protein-protein interactions in the bacteriophage T4 replisome. The leading strand holoenzyme is physically linked to the lagging strand holoenzyme and the primosome.
The bacteriophage T4 replication complex is composed of eight proteins that function together to replicate DNA. This replisome can be broken down into four basic units: a primosome composed of gp41, gp61, and gp59; a leading strand holoenzyme composed of gp43, gp44/62, and gp45; a lagging strand holoenzyme; and a single strand binding protein polymer. These units interact further to form the complete replisome. The leading and lagging strand polymerases are physically linked in the presence of DNA or an active replisome. The region of interaction was mapped to an extension of the finger domain, such that Cys-507 of one subunit is in close proximity to Cys-507 of a second subunit. The leading strand polymerase and the primosome also associate, such that gp59 mediates the contact between the two complexes. Binding of gp43 to the primosome complex causes displacement of gp32 from the gp59.gp61.gp41 primosome complex. The resultant species is a complex of proteins that may allow coordinated leading and lagging strand synthesis, helicase DNA unwinding activity, and polymerase nucleotide incorporation.
Bacteriophage T4↗
Coupling interactions of distal residues enhance dihydrofolate reductase catalysis: mutational effects on hydride transfer rates.
Recently, the participation in catalysis of residues spatially removed from the enzyme's active site has received considerable attention. The influence of the distal Gly-121 residue on the chemical step of hydride transfer in dihydrofolate reductase (DHFR) catalysis had been demonstrated previously [Cameron, C. E., and Benkovic, S. J. (1997) Biochemistry 36, 15792-15800]. In our continuing effort to identify catalytically important residues that are distal from the active site, we used sequence conservation information, kinetic data on site-directed mutants, dynamic motion information from NMR methods, and correlated motions from MD simulations to identify a subset of residues. Among them, the region spanning positions 41-45 is distal to the active site and was chosen as the focus for the mutagenesis and kinetic studies reported here. Specifically, the highly conserved Met-42 was selected for site-directed mutagenesis. While the reaction kinetics for the M42F mutant enzyme did not deviate from wild-type behavior, a 41-fold reduction in the forward hydride transfer rate was found for the M42W mutant. Given the established role of Gly-121 in the hydride transfer process, double mutant enzymes involving positions 42 and 121 were constructed and characterized. These double mutant enzymes generally showed little changes in substrate and cofactor binding but synergistic decreases in forward hydride transfer rates, while the decreases in reverse rates were additive. Along with supporting information from mixed quantum/classical MD simulations [Agarwal, P. K., Billeter, S. R., Rajagopalan, P. T., Benkovic, S. J., and Hammes-Schiffer, S. (2002) Proc. Natl. Acad. Sci. U.S.A. 99, 2794-2799], the data suggest that a coupled dynamic motion of these distal residues enhances crossing of the chemical reaction barrier and imply an expanded nonstatic role for the protein fold in catalysis.
Bacterial Proteins↗
Interaction of dihydrofolate reductase with methotrexate: ensemble and single-molecule kinetics.
The thermodynamics and kinetics of the interaction of dihydrofolate reductase (DHFR) with methotrexate have been studied by using fluorescence, stopped-flow, and single-molecule methods. DHFR was modified to permit the covalent addition of a fluorescent molecule, Alexa 488, and a biotin at the N terminus of the molecule. The fluorescent molecule was placed on a protein loop that closes over methotrexate when binding occurs, thus causing a quenching of the fluorescence. The biotin was used to attach the enzyme in an active form to a glass surface for single-molecule studies. The equilibrium dissociation constant for the binding of methotrexate to the enzyme is 9.5 nM. The stopped-flow studies revealed that methotrexate binds to two different conformations of the enzyme, and the association and dissociation rate constants were determined. The single-molecule investigation revealed a conformational change in the enzyme-methotrexate complex that was not observed in the stopped-flow studies. The ensemble averaged rate constants for this conformation change in both directions is about 2-4 s(-1) and is attributed to the opening and closing of the enzyme loop over the bound methotrexate. Thus the mechanism of methotrexate binding to DHFR involves multiple steps and protein conformational changes.
Base Sequence↗
PurT-encoded glycinamide ribonucleotide transformylase. Accommodation of adenosine nucleotide analogs within the active site.
PurT-encoded glycinamide ribonucleotide transformylase, or PurT transformylase, functions in purine biosynthesis by catalyzing the formylation of glycinamide ribonucleotide through a catalytic mechanism requiring Mg(2+)ATP and formate. From previous x-ray diffraction analyses, it has been demonstrated that PurT transformylase from Escherichia coli belongs to the ATP-grasp superfamily of enzymes, which are characterized by three structural motifs referred to as the A-, B-, and C-domains. In all of the ATP-grasp enzymes studied to date, the adenosine nucleotide ligands are invariably wedged between the B- and C-domains, and in some cases, such as biotin carboxylase and carbamoyl phosphate synthetase, the B-domains move significantly upon nucleotide binding. Here we present a systematic and high-resolution structural investigation of PurT transformylase complexed with various adenosine nucleotides or nucleotide analogs including Mg(2+)ATP, Mg(2+)-5'-adenylylimidodiphosphate, Mg(2+)-beta,gamma-methyleneadenosine 5'-triphosphate, Mg(2+)ATPgammaS, or Mg(2+)ADP. Taken together, these studies indicate that the conformation of the so-called "T-loop," delineated by Lys-155 to Gln-165, is highly sensitive to the chemical identity of the nucleotide situated in the binding pocket. This sensitivity to nucleotide identity is in sharp contrast to that observed for the "P-loop"-containing enzymes, in which the conformation of the binding motif is virtually unchanged in the presence or absence of nucleotides.
Adenosine Diphosphate↗
Assembly of the bacteriophage T4 helicase: architecture and stoichiometry of the gp41-gp59 complex.
The bacteriophage T4 59 protein (gp59) plays an essential role in recombination and replication by mediating the assembly of the gene 41 helicase (gp41) onto DNA. gp59 is required to displace the gp32 single-stranded binding protein on the lagging strand to expose a site for helicase binding. To gain a better understanding of the mechanism of helicase assembly, the architecture and stoichiometry of the gp41-gp59 complex were investigated. Both the N and C termini of gp41 were found to lie close to or in the gp41-gp41 subunit interface and interact with gp59. The site of interaction of gp41 on gp59 is proximal to Cys-215 of gp59. Binding of gp41 to gp59 stimulates a conformational change in the protein resulting in hexamer formation of gp59, and gp59 likewise stimulates oligomer formation of gp41. The gp59 subunits in this complex are arranged in a head to head orientation, such that Cys-42 of one subunit is in close proximity to Cys-42 on an adjacent subunit, and Cys-215 on one subunit is close to Cys-215 on a neighboring subunit. As the helicase is loaded onto DNA, a conformational change in the gp41-gp59 complex occurs, which may serve to displace gp32 from the lagging strand and load the hexameric helicase in its place.
Base Sequence↗
Network of coupled promoting motions in enzyme catalysis.
A network of coupled promoting motions in the enzyme dihydrofolate reductase is identified and characterized. The present identification is based on genomic analysis for sequence conservation, kinetic measurements of multiple mutations, and mixed quantum/classical molecular dynamics simulations of hydride transfer. The motions in this network span time scales of femtoseconds to milliseconds and are found on the exterior of the enzyme as well as in the active site. This type of network has broad implications for an expanded role of the protein fold in catalysis as well as ancillaries such as the engineering of altered protein function and the action of drugs distal to the active site.
Animals↗
Preorganization and protein dynamics in enzyme catalysis.
Recently, an alternative has been offered to the concept of transition state (TS) stabilization as an explanation for rate enhancements in enzyme-catalyzed reactions. Instead, most of the rate increase has been ascribed to preorganization of the enzyme active site to bind substrates in a geometry close to that of the TS, which then transit the activation barrier impelled by motions along the reaction coordinate. The question as to how an enzyme achieves such preorganization and concomitant TS stabilization as well as potential coupled motions along the reaction coordinate leads directly to the role of protein dynamic motion. Dihydrofolate reductase (DHFR) is a paradigm in which the role of dynamics in catalysis continues to be unraveled by a wealth of kinetic, structural, and computational studies. DHFR has flexible loop regions adjacent to the active site whose motions modulate passage through the kinetically preferred pathway. The participation of residues distant from the DHFR active site in enhancing the rate of hydride transfer, however, is unanticipated and may signify the importance of long range protein motions. The general significance of protein dynamics in understanding other biological processes is briefly discussed.
Alcohol Oxidoreductases↗
10-Formyl-5,10-dideaza-acyclic-5,6,7,8-tetrahydrofolic acid (10-formyl-DDACTHF): a potent cytotoxic agent acting by selective inhibition of human GAR Tfase and the de novo purine biosynthetic pathway.
The synthesis of 10-formyl-DDACTHF (3) as a potential inhibitor of glycinamide ribonucleotide transformylase (GAR Tfase) and aminoimidazole carboxamide ribonucleotide transformylase (AICAR Tfase) is reported. Aldehyde 3, the corresponding gamma- and alpha-pentaglutamates 21 and 25 and related agents were evaluated for inhibition of folate-dependent enzymes including GAR Tfase and AICAR Tfase. The inhibitors were found to exhibit potent cytotoxic activity (CCRF-CEM IC(50) for 3=60nM) that exceeded their enzyme inhibition potency [K(i) (3)=6 and 1 microM for Escherichia coli GAR and human AICAR Tfase, respectively]. Cytotoxicity rescue by medium purines, but not pyrimidines, indicated that the potent cytotoxic activity is derived from selective purine biosynthesis inhibition and rescue by AICAR monophosphate established that the activity is derived preferentially from GAR versus AICAR Tfase inhibition. The potent cytotoxic compounds including aldehyde 3 lost activity against CCRF-CEM cell lines deficient in the reduced folate carrier (CCRF-CEM/MTX) or folylpolyglutamate synthase (CCRF-CEM/FPGS(-)) establishing that their potent activity requires both reduced folate carrier transport and polyglutamation. Unexpectedly, the pentaglutamates displayed surprisingly similar K(i)'s versus E. coli GAR Tfase and only modestly enhanced K(i)'s versus human AICAR Tfase. On the surface this initially suggested that the potent cytotoxic activity of 3 and related compounds might be due simply to preferential intracellular accumulation of the inhibitors derived from effective transport and polyglutamation (i.e., ca. 100-fold higher intracellular concentrations). However, a subsequent examination of the inhibitors against recombinant human GAR Tfase revealed they and the corresponding gamma-pentaglutamates were unexpectedly much more potent against the human versus E. coli enzyme (K(i) for 3, 14nM against rhGAR Tfase versus 6 microM against E. coli GAR Tfase) which also accounts for their exceptional cytotoxic potency.
Antineoplastic Agents↗
A universal, vector-based system for nucleic acid reading-frame selection.
The identification of a nucleic acid sequence's correct reading frame has important implications for homology-independent protein engineering techniques such as incremental truncation and SCRATCHY. We report the development and experimental implementation of a general in-frame selection system, pSALect, a plasmid vector that utilizes two marker sequences flanking the DNA of interest. This dual selection approach overcomes inconsistencies observed with traditional C-terminally fused reporter proteins. In the pSALect vector, sequences of interest are positioned between an N-terminal Tat-signal sequence and a C-terminal beta-lactamase reporter. In-frame selection of the resulting three-domain protein is performed by growing colonies on ampicillin-containing plates, requiring full-length translation in order to link covalently the signal sequence to the lactamase for export into the periplasm. This dual selection scheme has been validated successfully using defined sequences of glycinamide ribonucleotide formyltransferases (GARTs) from Escherichia coli and human and, in contrast to C-terminal fusion systems, proved effective when applied towards the selection of in-frame constructs in an incremental truncation library.
Ampicillin Resistance↗