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R N Perham

Publications and source records attributed to R N Perham.

At least 91 records · Page 5Linked to original sources
PubMed1991

Engineering the substrate specificity of glutathione reductase toward that of trypanothione reduction.

Glutathione reductase (EC 1.6.4.2; CAS registry number 9001-48-3) and trypanothione reductase (CAS registry number 102210-35-5), which are related flavoprotein disulfide oxidoreductases, have marked specificities for glutathione and trypanothione, respectively. A combination of primary sequence alignments and molecular modeling, together with the high-resolution crystal structure of human glutathione reductase, identified certain residues as potentially being responsible for substrate discrimination. Site-directed mutagenesis of Escherichia coli glutathione reductase was used to test these predictions. The mutation of Asn-21 to Arg demonstrated that this single change was insufficient to generate the greater discrimination against trypanothione shown by human glutathione reductase compared with the E. coli enzyme. However, the mutation of Ala-18, Asn-21, and Arg-22 to the amino acid residues (Glu, Trp, and Asn, respectively) in corresponding positions in Trypanosoma congolense trypanothione reductase confirmed that this region of polypeptide chain is intimately involved in substrate recognition. It led to a mutant form of E. coli glutathione reductase that possessed essentially no activity with glutathione but that was able to catalyze trypanothione reduction with a kcat/Km value that was 10% of that measured for natural trypanothione reductases. These results should be of considerable importance in the design of trypanocidal drugs targeted at the differences between glutathione and trypanothione metabolism in trypanosomatids and their hosts.

Amino Acid Sequence↗
PubMed1991

Sequence-specific 1H-NMR assignments and secondary structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase multienzyme complex.

The lipoyl domain (residues 1-85) of the lipoate-acetyltransferase polypeptide chain of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been subjected to detailed structural analysis by means of two-dimensional (2D) 1H-NMR spectroscopy at 400 MHz. Sequence-specific proton resonance assignments were made, but at this field strength not all of the side-chain protons could be assigned, especially from complex spin systems like those of leucine, proline and lysine residues. Measurement of short-range interproton distances identified two extensive regions of beta-sheet, each containing four anti-parallel peptide strands. The lipoyl-lysine residue (Lys42) is located in a tight turn at a corner of one sheet, the N-terminal and C-terminal residues of the domain are close together in two adjacent beta-strands in the other. The lipoylated and unlipoylated forms of the domain have almost identical spectra, indicating that there is little, if any, conformational change in the protein as a result of the post-translational modification.

Acetyltransferases↗
PubMed1991

Multiple display of foreign peptides on a filamentous bacteriophage. Peptides from Plasmodium falciparum circumsporozoite protein as antigens.

We describe here two systems for encoding foreign amino acid sequences in the exposed N-terminal segment of the major coat protein of bacteriophage fd. Small peptides can be encoded directly; larger peptides are encoded in hybrid bacteriophage particles, in which the capsid is formed from a mixture of wild-type and modified coat proteins. In both cases, the peptides are present in multiple copies per phage particle. Peptides that represent the circumsporozoite protein, the major surface antigen of the sporozoites of the malaria parasite, Plasmodium falciparum, were inserted in this way and found to be highly immunogenic. These systems should prove to be valuable in displaying specific or random peptides as antigens, and could lead to cheap and effective vaccines. They will also allow rapid screening of peptides as potential agents of other biological effects, with important applications in biomolecular design.

Amino Acid Sequence↗
PubMed1991

Two lipoyl domains in the dihydrolipoamide acetyltransferase chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis.

A fragment of DNA incorporating the gene, pdhC, that encodes the dihydrolipoamide acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex of Streptococcus faecalis was cloned and a DNA sequence of 2360 bp was determined. The pdhC gene (1620 bp) corresponds to an E2 chain of 539 amino acid residues, Mr 56,466, comprising two lipoyl domains, a peripheral subunit-binding domain and an acetyltransferase domain, linked together by regions of polypeptide chain rich in alanine, proline and charged amino acids. The S. faecalis E2 chain differs in the number of its lipoyl domains from the E2 chains of all bacterial pyruvate dehydrogenase complexes hitherto described.

Acetyltransferases↗
PubMed1991

Lipoylation of the E2 components of the 2-oxo acid dehydrogenase multienzyme complexes of Escherichia coli.

The number of functional lipoyl groups in the dihydrolipoyl acetyltransferase (E2) chain of the pyruvate dehydrogenase multienzyme complex from Escherichia coli has been re-assessed by means of a combination of protein-chemical and mass-spectrometric techniques. (1) After the complex had been treated with N-ethyl[2,3-14C]maleimide in the presence of pyruvate, the lipoyl domains were excised from the complex, treated with NaBH4 and re-exposed to N-ethyl[2,3-14C]maleimide. All the chemically reactive lipoyl groups in the native complex were found to be catalytically active. (2) Proteolytic digests of the separated lipoyl domains were examined for the presence of the lipoylation-site peptide, GDKASME, with and without the lipoyl group in N6-linkage to the lysine residue. Only the lipoylated form of the peptide was detected, suggesting that all three lipoyl domains are fully substituted at this site. (3) The behaviour of each lipoyl domain was examined on ion-exchange chromatography in response to alkylation with 4-vinylpyridine after either chemical reduction of the lipoyl group with dithiothreitol or reductive acetylation by the pyruvate dehydrogenase complex in the presence of pyruvate. All three domains exhibited a quantitative shift in retention time, confirming that each domain was fully substituted by an enzymically reactive lipoyl group. (4) When subjected to electrospray mass spectrometry, each domain gave a mass consistent with a fully lipoylated domain, and no aberrant substitution of the target lysine residue was detected. The same result was obtained for the lipoyl domain from the E. coli 2-oxoglutarate dehydrogenase complex. (5) Previous widespread attempts to assess the number of functional lipoyl groups in the pyruvate dehydrogenase multienzyme complex, which have led to the view that a maximum of two lipoyl groups per E2 chain may be involved in the catalytic mechanism, are in error.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1991

DNA sequence of the filamentous bacteriophage Pf1.

The genome of the class II filamentous bacteriophage Pf1 has been sequenced by a combination of the chain termination and chemical degradation methods. It consists of 7349 nucleotides in a closed, circular loop of single-stranded DNA. The size and position of its open reading frames (ORFs) in general resemble those of other filamentous bacteriophage genomes. The size and position of the spaces between the ORFs have not been conserved, however, and six short reading frames (2 of which overlap) occupy a region corresponding to that filled by genes 2 and 10 in the Ff genome. Most of the ORFs are preceded by sequences resembling ribosome binding sites from the phage's host. Pseudomonas aeruginosa, that appear to differ somewhat from their counterparts in Escherichia coli. A search for sequences related to known pseudomonad promoters suggests that the promoters in this bacteriophage may well be ntr-dependent, with the two strongest preceding the gene for the major coat protein (gene 8) and another ORF (430). Gene 8 is followed by a sequence with the properties of a rho-independent terminator of transcription, like that at the same position in the genome of Ff. The Pf1 genome contains no collection of potential stem-and-loop structures corresponding to those that initiate replication of Ff DNA and assembly of the Ff virion, although isolated structures of this kind are present. The available evidence suggests that at least 13 of the 14 major ORFs are expressed. Overall, the organization of the Pf1 genome differs from that of the other class II filamentous phage whose genome has been sequenced, Pf3, as much as it does from that of the class I phages Ff and IKe.

Amino Acid Sequence↗
PubMed1991

Regulation of filamentous bacteriophage length by modification of electrostatic interactions between coat protein and DNA.

Bacteriophage fd gene VIII, which encodes the major capsid protein, was mutated to convert the serine residue at position 47 to a lysine residue (S47K), thereby increasing the number of positively charged residues in the C-terminal region of the protein from four to five. The S47K coat protein underwent correct membrane insertion and processing but could not encapsidate the viral DNA, nor was it incorporated detectably with wild-type coat proteins into hybrid bacteriophage particles. However, hybrid virions could be constructed from the S47K coat protein and a second mutant coat protein, K48Q, the latter containing only three lysine residues in its C-terminal region. K48Q phage particles are approximately 35% longer than wild-type. Introducing the S47K protein shortened these particles, the S47K/K48Q hybrids exhibiting a range of lengths between those of K48Q and wild-type. These results indicate that filamentous bacteriophage length (and the DNA packaging underlying it) are regulated by unusually flexible electrostatic interactions between the C-terminal domain of the coat protein and the DNA. They strongly suggest that wild-type bacteriophage fd makes optimal use of the minimum number of coat protein subunits to package the DNA compactly.

Amino Acid Sequence↗
PubMed1991

New enzymes for old: redesigning the coenzyme and substrate specificities of glutathione reductase.

A set of amino acid side chains that confer specificity for the coenzyme NADPH and the substrate glutathione in the flavoprotein disulphide oxidoreductase, glutathione reductase, has been identified. Systematic replacement of these amino acid residues in the coenzyme-binding site switches the specificity of the enzyme from its natural strong preference for NADPH to a marked preference for NADH. The amino acids replaced all lie in a structural motif within the dinucleotide-binding domain of the protein. Since this domain is a feature common to most dehydrogenases (reductases) that use nicotinamide coenzymes, it may be that the coenzyme specificities of all such enzymes can be manipulated in this way. Similarly, amino acid residues involved in the selective recognition of trypanothione by trypanothione reductase, an enzyme related to glutathione reductase and exclusive to trypanosomatids, were identified. Suitable mutation of the corresponding residues in E. coli glutathione reductase switched its substrate specificity towards trypanothione. A better understanding of the substrate specificity of these enzymes could open up a route to the chemotherapy of trypanosomal infections.

Amino Acid Sequence↗
PubMed1991

A synthesis of acylphosphonic acids and of 1-aminoalkylphosphonic acids: the action of pyruvate dehydrogenase and lactate dehydrogenase on acetylphosphonic acid.

Acylphosphonic acids, R-CO-PO(OH)2, have been synthesized by the steps [formula: see text] of which the last is new and provides a mild method for de-esterifying acylphosphonic acids. Their reductive amination gives a simple way of making 1-aminoalkylphosphonic acids. Acetylphosphonic acid inhibited NAD+ reduction by pyruvate with the pyruvate dehydrogenases from Escherichia coli and Bacillus stearothermophilus. The inhibition was competitive with pyruvate, with Ki of 6 microM for the E. coli enzyme (pyruvate Km 0.5 mM) and one of 0.4 mM of the B. stearothermophilus enzyme (pyruvate Km 0.1 mM). Acetylphosphonate and its monomethyl ester are substates for pig heart lactate dehydrogenase, with Km values of 15 mM and 10 mM respectively (pyruvate Km 0.05 mM) and specificity constants one thousandth that for pyruvate.

Acetaldehyde↗
PubMed1991

Autoantibodies in primary biliary cirrhosis: analysis of reactivity against eukaryotic and prokaryotic 2-oxo acid dehydrogenase complexes.

Six components of the mammalian 2-oxo acid dehydrogenase complexes have previously been identified as M2 autoantigens in primary biliary cirrhosis. In this report, we present data showing that both polypeptide-specific and cross-reacting antibodies are present in patients' sera. Antibodies reacting with E2 of the pyruvate dehydrogenase complex cross-react with protein X but not with any other mammalian antigen. The main immunogenic region on protein X has been localized to within its single lipoyl domain. Polypeptide-specific antibodies bind to E1 alpha and E1 beta of the pyruvate dehydrogenase complex. Antibodies reacting with the E2 polypeptides of the 2-oxoglutarate dehydrogenase complex and branched-chain 2-oxo acid dehydrogenase complex show some cross-reactivity but do not recognize any of the antigens of the pyruvate dehydrogenase complex. Antibodies against the E2 component of the mammalian pyruvate dehydrogenase complex cross-react effectively with the corresponding protein from yeast but not with E2 from Escherichia coli. Antibody titer against mammalian antigens is significantly higher than against the bacterial antigens, arguing against a bacterial origin for primary biliary cirrhosis.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1990

Active site complementation in engineered heterodimers of Escherichia coli glutathione reductase created in vivo.

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the dimeric flavoprotein glutathione reductase, Cys-47 (a cysteine residue forming an essential charge-transfer complex with enzyme-bound FAD) was converted to serine (C47S) and His-439 (required to facilitate protonation of the reduced glutathione) was converted to glutamine (H439Q). Both mutant genes were placed in the same plasmid, pHD, where each of them came under the control of a strong tac promoter. This was designed to achieve equal over-expression of both genes in the same E. coli cell. The parental homo-dimers show no (C47S) or very little (H439Q) activity as glutathione reductases. The formation in vivo of heterodimers, carrying one crippled and one fully functional active site, was detected by absorbance spectroscopy and fluorescence emission spectrometry of enzyme-bound FAD and by active site complementation. The fractional distribution of homo- and hetero-dimers was in accord with that expected for a random association of enzyme subunits. In a homo-dimer, the H439Q mutation leads to a big fall in the value of Km for NADPH which binds some 1.8 nm from the point of mutation (Berry, A., Scrutton, N.S. & Perham, R. N. Biochemistry 28, 1264-1269 (1989)). However, the one active site in the H439Q/C47S hetero-dimer exhibited kinetic parameters similar to those of the wild-type enzyme. Thus, the effect of the H439Q mutation must be retained within the active site that accommodates it and is not transmitted through the protein to the second active site across the subunit interface.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1990

Cloning and sequence analysis of the genes encoding the dihydrolipoamide acetyltransferase and dihydrolipoamide dehydrogenase components of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 2641-bp EcoRI fragment of DNA that encodes the C-terminal part of the dihydrolipoyl acetyltransferase (E2) component and the dihydrolipoamide dehydrogenase (E3) component of the pyruvate dehydrogenase complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. A 705-bp truncated open reading frame was located at the 5'end of the insert which, together with the 588-bp truncated open reading frame at the 3' end of another EcoRI fragment of B. stearothermophilus DNA previously cloned and sequenced [Hawkins, C. F., Borges, A. & Perham, R. N. (1990) Eur. J. Biochem. 191, 337-446], was identified as the gene, pdhC, encoding the E2 polypeptide chain. Direct sequence analysis of the purified E2 chain confirmed that the two EcoRI fragments are adjoining in the B. stearothermophilus genome. The E3 gene, pdhD, begins just 4 bp downstream from the stop codon of the pdhC gene. The amino acid sequences deduced from the pdhC and pdhD genes correspond to proteins of 427 amino acids (E2, Mr 46,265) and 469 amino acids (E3, Mr 49,193), respectively. Both genes are preceded by potential ribosome-binding sites and the E3 gene is followed by a stemloop structure characteristic of rho-independent transcription terminators. The B. stearothermophilus E2 and E3 chains exhibit substantial sequence similarity with the corresponding subunits of other 2-oxo-acid dehydrogenase multienzyme complexes. The cloning and sequence analysis described here complete the description of the gene cluster (pdhA, B, C and D) which encodes the B. stearothermophilus pyruvate dehydrogenase multienzyme complex.

Acetyltransferases↗
PubMed1990

Directed mutagenesis of the redox-active disulphide bridge in glutathione reductase from Escherichia coli.

Directed mutagenesis of the gor gene from Escherichia coli encoding the flavoprotein glutathione reductase was used to convert the two cysteine residues that comprise its redox-active disulphide bridge to alanine (C42A) and serine (C47S) residues. A double mutant (C42AH439A) was also created in which His-439, the proton donor/acceptor in the glutathione-binding site, was additionally converted into an alanine residue. The C42A and C47S mutants were both unable to catalyse the reduction of glutathione by NADPH. The C42A mutant retained the transhydrogenase activity of the wild-type enzyme, whereas the C47S mutant was also inhibited in this reaction. These results support the view that in the catalytic mechanism of E. coli glutathione reductase, the thiolate form of Cys-42 acts as a nucleophile to initiate disulphide exchange with enzyme-bound glutathione and that the thiolate form of Cys-47 generates an essential charge-transfer complex with enzyme-bound FAD. Titration of the C42A and C42AH439A mutants indicated that the imidazole side-chain of His-439 lowered the pKa of the charge-transfer thiol (Cys-47) from 7.7 to 5.7, enhancing its ability to act as an anion at neutral pH. Several important differences between these mutants of E. coli glutathione reductase and similar mutants (or chemically modified forms) of other members of the flavoprotein disulphide oxidoreductase family were noted, but these could be explained in terms of the different redox chemistries of the enzymes concerned.

Alanine↗
PubMed1990

Cloning and sequence analysis of the genes encoding the alpha and beta subunits of the E1 component of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus.

A 4175-bp EcoRI fragment of DNA that encodes the alpha and beta chains of the pyruvate dehydrogenase (lipoamide) component (E1) of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus has been cloned in Escherichia coli. Its nucleotide sequence was determined. Open reading frames (pdhA, pdhB) corresponding to the E1 alpha subunit (368 amino acids, Mr 41,312, without the initiating methionine residue) and E1 beta subunit (324 amino acids, Mr 35,306, without the initiating methionine residue) were identified and confirmed with the aid of amino acid sequences determined directly from the purified polypeptide chains. The E1 beta gene begins just 3 bp downstream from the E1 alpha stop codon. It is followed, after a longer gap of 73 bp, by the start of another but incomplete open reading frame that, on the basis of its known amino acid sequence, encodes the dihydrolipoyl acetyltransferase (E2) component of the complex. All three genes are preceded by potential ribosome-binding sites and the gene cluster is located immediately downstream from a region of DNA showing numerous possible promoter sequences. The E1 alpha and E1 beta subunits of the B. stearothermophilus pyruvate dehydrogenase complex exhibit substantial sequence similarity with the E1 alpha and E1 beta subunits of pyruvate and branched-chain 2-oxo-acid dehydrogenase complexes from mammalian mitochondria and Pseudomonas putida. In particular, the E1 alpha chain contains the highly conserved sequence motif that has been found in all enzymes utilizing thiamin diphosphate as cofactor.

Acetyltransferases↗
PubMed1990

Expression in Escherichia coli of a sub-gene encoding the lipoyl domain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus.

A sub-gene encoding the lipoyl domain (residues 1-85) of the lipoate acetyltransferase chain of the pyruvate dehydrogenase complex of Bacillus stearothermophilus was over-expressed in Escherichia coli. Approx. 80% of the domain was unlipoylated but most of the remainder was correctly lipoylated on Lys-42 and could be reductively acetylated by the B stearothermophilus enzyme complex. A small proportion (approx. 4%) of the domain carried an aberrant substituent, possibly an octanoyl group, on Lys-42. The 400 MHz 1H NMR spectra of the lipoylated and unlipoylated domains were essentially identical and closely resembled that of the native lipoyl domain.

Acetyltransferases↗
PubMed1990

Interactions of lipoyl domains with the E1p subunits of the pyruvate dehydrogenase multienzyme complex from Escherichia coli.

Equilibrium binding experiments were carried out with lipoyl domains and the pyruvate decarboxylase [pyruvate dehydrogenase (lipoamide), E1p, EC 1.2.4.1)] component of the pyruvate dehydrogenase multienzyme complex of Escherichia coli. The dissociation constant (Ks) was estimated to be not less than 0.3 mM, exceeding the Km value (33 microM) for reductive acetylation of the domains by an order of magnitude. Thus, the lipoyl domain, which is required to promote reductive acetylation of the lipoyl group, does not appear to do this simply by enhancing the binding to E1p. The difference between Ks and Km suggests that the formation and release of reductively acetylated lipoyl domains from the enzyme may be a relatively rapid step in the mechanism.

Escherichia coli↗
PubMed1990

Redesign of the coenzyme specificity of a dehydrogenase by protein engineering.

Directed mutagenesis and molecular modelling have been used to identify a set of amino-acid side chains in glutathione reductase that confer specificity for the coenzyme NADP+. Systematic replacement of these amino acids, all of which occur in a 'fingerprint' structural motif in the NADP+-binding domain, leaves the substrate specificity unchanged but converts the enzyme into one displaying a marked preference for the coenzyme NAD+.

Amino Acid Sequence↗