Lipoate Acetyltransferase Research Articles

Salt-Induced Changes in the Subunit Structure of theBacillus stearothermophilusLipoate Acetyltransferase

The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.

Cloning, Sequencing, and Overexpression of Gene Encoding the Bacillus stearothermophilus Lipoate Acetyltransferase

Cloning, Sequencing, and Overexpression of Gene Encoding the Bacillus stearothermophilus Lipoate Acetyltransferase

Thermally induced changes of lipoate acetyltransferase inner core isolated from the Bacillus stearothermophilus pyruvate dehydrogenase complex.

Incubation at 70 degrees C converted the Bacillus stearothermophilus lipoate acetyltransferase inner core into an unidentified active molecular form, X, yielding an inactive aggregate. The core and X showed similar thermostabilities, but they were different in the recovery of enzyme activity after incubation with 1.2-2.0 M guanidine hydrochloride and its subsequent removal; the core was hardly recovered, but X was well recovered.

Guanidine Hydrochloride-Induced Changes of the E2 Inner Core of the Bacillus stearothermophilus Pyruvate Dehydrogenase Complex

The limited proteolysis of the Bacillus stearothermophilus pyruvate dehydrogenase complex by V8 protease yields its core structure solely composed of lipoate acetyltransferase (E2) fragments. The changes in the core with guanidine hydrochloride (GdnHCl) were biphasic: below 0.8 M (first) and above 1.0 M (second) GdnHCl. The changes in the first phase were slight but significant: decreases in ellipticity and light scattering, and an increase in E2 activity. Insignificant changes in the molecular shape and size of the core were detected on fluorescence spectroscopy, ultracentrifugation, gel filtration, and electron microscopy. On the other hand, the changes in the second phase were drastic; the core was disassembled and denatured.

Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli

The effects of changing environmental conditions on expression of the pdh operon were studied in strains containing pyruvate dehydrogenase (PDH) complexes having either one or three lipoyl domains per lipoate acetyltransferase chain. The expression of the pdh operon was lowered during growth on reduced carbon sources and when the mode of energy generation was changed from aerobic respiration to anaerobic respiration and fermentation. In contrast, growth at non-optimal pH increased expression. Operon expression was generally higher in the 1 lip strain compared to the 3 lip strain. Expression of the pdh operon was shown to be tightly controlled in response to environmental stimuli, consistent with its importance in defining metabolic flux.

Environmental control of pyruvate dehydrogenase complex expression in Escherichia coli.

The effects of changing environmental conditions on expression of the pdh operon were studied in strains containing pyruvate dehydrogenase (PDH) complexes having either one or three lipoyl domains per lipoate acetyltransferase chain. The expression of the pdh operon was lowered during growth on reduced carbon sources and when the mode of energy generation was changed from aerobic respiration to anaerobic respiration and fermentation. In contrast, growth at non-optimal pH increased expression. Operon expression was generally higher in the 1 lip strain compared to the 3 lip strain. Expression of the pdh operon was shown to be tightly controlled in response to environmental stimuli, consistent with its importance in defining metabolic flux.

Potassium Iodide-Induced Changes in Pyruvate Dehydrogenase Complex from Bacillus stearothermophilus.

Treatment with KI and its subsequent removal induced disassembly of Bacillus stearothermophilus pyruvate dehydrogenase complex (PDC) and association of disassembly products, respectively. The disassembly yielded neither completely dissociated components nor aggregate, but did yield a few molecular forms smaller than PDC. Depending on the KI concentration, these changes were of three phases: K-1, below 0.6 M; K-2, 1.0-1.5 M; K-3, above 1.8 M. PDC was disassembled in K-1 to C1 comprising pyruvate decarboxylase and lipoate acetyltransferase mainly and C2 comprising the decarboxylase and dihydrolipoamide dehydrogenase. In K-1, the removal of KI resulted in an apparent reconstitution of PDC. The mixing of C1 with an excess of C2 yielded an assembly larger than PDC and restored enzyme activities, but specific activities were different from those of PDC. In K-2 and K-3 phases, complexes smaller than PDC were yielded from disassembly products, and activities except for that of the acetyltransferase were not restored.

Mutations in PDX1, the Human Lipoyl-Containing Component X of the Pyruvate Dehydrogenase–Complex Gene on Chromosome 11p1, in Congenital Lactic Acidosis

We have identified and sequenced a cDNA that encodes an apparent human orthologue of a yeast protein-X component (ScPDX1) of pyruvate dehydrogenase multienzyme complexes. The new human cDNA that has been referred to as "HsPDX1" cDNA was cloned by use of the "database cloning" strategy and had a 1,506-bp open reading frame. The amino acid sequence of the protein encoded by the cDNA was 20% identical with that encoded by the yeast PDX1 gene and 40% identical with that encoded by the lipoate acetyltransferase component of the pyruvate dehydrogenase and included a lipoyl-bearing domain that is conserved in some dehydrogenase enzyme complexes. Northern blot analysis demonstrated that the major HsPDX1 mRNA was 2.5 kb in length and was expressed mainly in human skeletal and cardiac muscles but was also present, at low levels, in other tissues. FISH analysis performed with a P1-derived artificial chromosome (PAC)-containing HsPDX1 gene sublocalized the gene to 11p1.3. Molecular investigation of PDX1 deficiency in four patients with neonatal lactic acidemias revealed mutations 78del85 and 965del59 in a homozygous state, and one other patient had no PDX1 mRNA expression.

Enzymological and physiological consequences of restructuring the lipoyl domain content of the pyruvate dehydrogenase complex of Escherichia coli

The core-forming lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli contain three tandemly repeated lipoyl domains although one lipoyl domain is apparently sufficient for full catalytic activity in vitro. Plasmids containing IPTG-inducible aceEF-IpdA operons which express multilip-PDH complexes bearing one N-terminal lipoyl domain and up to seven unlipoylated (mutant) domains per E2p chain, were constructed. Each plasmid restored the nutritional lesion of a strain lacking the PDH complex and expressed a sedimentable PDH complex, although the catalytic activities declined significantly as the number of unlipoylated domains increased above four per E2p chain. It was concluded that the extra domains protrude from the 24-meric E2p core without affecting assembly of the E1p and E3 subunits, and that the lipoyl cofactor bound to the outermost domain can participate successfully at each of the three types of active site in the assembled complex. Physiological studies with two series of isogenic strains expressing multilip-PDH complexes from modified chromosomal pdh operons (pdhR-aceEF-IpdA) showed that three lipoyl domains per E2p chain is optimal and that only the outermost domain need be lipoylated for optimal activity. It is concluded that the reason for retaining three lipoyl domains is to extend the reach of the outermost lipoyl cofactor rather than to provide extra cofactors for catalysis.

Further studies on thermal denaturation of pyruvate dehydrogenase complex from Bacillus stearothermophilus.

Thermally induced changes in pyruvate dehydrogenase complex (PDC) from B. stearothermophilus were examined mainly at temperatures from 60 degrees to 70 degrees C. Accompanied by inactivation of pyruvate decarboxylase, light scattering decreased, and ANS fluorescence increased. These changes including the inactivation were approximately first-order reactions, and the values of rate constants were greatly dependent on temperature. Chromatographic studies showed that any polypeptides were in associated forms and that final products were aggregates (> 230S) and an assembly (48S) smaller than PDC. The aggregates and assembly were rich in decarboxylase and lipoate acetyltransferase, respectively. It was suggested that, during the thermal denaturation, a decarboxylase was dissociated from PDC and immediately involved in aggregates.

Purification and properties of the lipoate protein ligase of Escherichia coli

Lipoate is an essential component of the 2-oxoacid dehydrogenase complexes and the glycine-cleavage system of Escherichia coli. It is attached to specific lysine residues in the lipoyl domains of the E2p (lipoate acetyltransferase) subunit of the pyruvate dehydrogenase complex by a Mg(2+)- and ATP-dependent lipoate protein ligase (LPL). LPL was purified from wild-type E. coli, where its abundance is extremely low (< 10 molecules per cell) and from a genetically amplified source. The purified enzyme is a monomeric protein (M(r) 38,000) which forms irregular clusters of needle-like crystals. It is stable at -20 degrees C, but slowly oxidizes to an inactive form containing at least one intramolecular disulphide bond at 4 degrees C. The inactive form could be re-activated by reducing agents or by an as-yet unidentified component (reactivation factor) which is resolved from LPL at the final stage of purification. The pI is 5.80, and the Km values for ATP, Mg2+ and DL-lipoate were determined. Selenolipoate and 6-thio-octanoate were alternative but poorer substrates. Lipoylation was reversibly inhibited by the 6- and 8-seleno-octanoates and 8-thio-octanoate, which reacted with the six cysteine thiol groups of LPL. LPL was inactivated by Cu2+ ions in a process that involved the formation of inter- and intra-molecular disulphide bonds. Studies with lplA mutants lacking LPL activity indicated that E. coli possesses another distinct lipoylation system, although no such activity could be detected in vitro.

Metabolic engineering in Escherichia coli: lowering the lipoyl domain content of the pyruvate dehydrogenase complex adversely affects the growth rate and yield.

Isogenic strains of Escherichia coli W3110 containing pyruvate dehydrogenase complexes with three (wild-type), two or one lipoyl domains per lipoate acetyltransferase (E2p) chain, were constructed. The maximum growth rates (mumax) for batch cultures growing in minimal medium containing different carbon sources showed that reducing the number of lipoyl domains adversely mumax value of the mutant containing one lipoyl domain per E2p chain was restored by the presence of compatible multicopy plasmids encoding PDH complexes with either one or three lipoyl domains per E2p chain. In glucose-limited chemostat cultures the protein contents of all strains were similar and substrate carbon was totally accounted for in the biomass and CO2 produced. However, the carbon efficiencies (percentage carbon conversion to biomass) were significantly lower when the lipoyl domain content of the E2p subunit was reduced from three to one. Similarly, the cellular maintenance energy (m(e)) and the maximum growth yield (Ymax) were lower in bacteria containing PDH complexes with fewer than three lipoyl domains per E2p chain. Wild-type values were restored by supplementing the medium with either casamino acids (0.01%) or acetate (up to 0.1 mM). The lower growth efficiencies of the mutants were further confirmed in competition experiments where equal numbers of genetically marked (NalR) mutant and wild-type bacteria were used to inoculate glucose-limited chemostat cultures (dilution rate 0.075 h-1). The mutants with one or two lipoyl domains per E2p chain were washed out, whereas in controls, the initial ratio of wild-type (Nals) to reconstructed wild-type (NalR) bacteria was maintained over 50 generations.

Thermal Disassembly of Pyruvate Dehydrogenase Multienzyme Complex fromBacillus stearothermophilus

Thermostabilities of component enzymes in the pyruvate dehydrogenase complex from Bacillus stearothermophilus decreased in the order lipoamide dehydrogenase, lipoate acetyltransferase, and pyruvate decarboxylase (E1). Fluorescence of an extrinsic 8-amino-1-naphthalenesulfonate (ANS) increased with inactivation of E1. The thermal denaturation of the enzymes resulted in disassembly of the complex. El was involved in a resulting aggregate of the complex. The interaction between ANS and denatured E1 accounted for an increase in fluorescence.

Mobility in pyruvate dehydrogenase complexes with multiple lipoyl domains

High-field NMR studies were carried out with genetically-reconstructed pyruvate dehydrogenase (PDH) complexes of Escherichia coli containing from zero to nine lipoyl domains per lipoate acetyltransferase (E2p) subunit. The only significant differences between the NMR spectra were the increasing intensities of the signals derived from the lipoyl domains and their associated linkers, and the much enhanced signal from the E3-binding domain and its linker in complexes that are devoid of lipoyl domains. The results suggest an explanation for the presence of three lipoyl domains per E2p subunit in the wild-type PDH complex, based on its greater inherent mobility, and potentially more efficient active-site coupling, than any of the other complexes.

Purification of Pyruvate Dehydrogenase Complex from an Extreme Thermophile, Bacillus caldolyticus, and Its Thermal Stability.

Pyruvate dehydrogenase multienzyme complex was purified from Bacillus caldolyticus. The complex was composed of four polypeptides with molecular masses of 39.8, 41.7, 53.7, and 57.5kDa estimated by SDS-PAGE and they were presumed to be pyruvate decarboxylase (E1, dimeric), lipoate acetyltransferase (E2), and lipoamide dehydrogenase (E3) on the analogy of those from Bacillus stearothermophilus. E1 and E3 were stable at pH 5.7-10.2 and 4.5-11.3, respectively. Halves of E1 and E3 activity were abolished by incubation for 30min at 65°C and 85°C, respectively. Loss of overall activity was principally due to inactivation of E1. Structural changes in the complex incubated at high temperature were studied by fluorescence spectroscopy. The results suggested that the thermal denaturation of the complex proceeded through at least two different steps: inactivations of E1 and E3, and the former process is accompanied by a reduction of the complex size.

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain.

The lipoate acetyltransferase (E2p) subunits of the pyruvate dehydrogenase (PDH) complex of Escherichia coli have three tandemly repeated lipoyl domains, although net deletions of one or two has no apparent effect on the activity of the purified complexes. Plasmids containing IPTG-inducible aceEF-lpd operons, which encode PDH complexes bearing from one to nine lipoyl domains per E2p chain (24-216 per complex), were constructed. They were all capable of restoring the nutritional lesion of a strain lacking PDH complex and they all expressed active sedimentable multienzyme complexes having a relatively normal range of subunit stoichiometries. The extra domains are presumed to protrude from the E2p core (24-mer) without significantly affecting the assembly of the E1p and E3 subunits on the respective edges and faces of the cubic core. However, the catalytic activities of the overproduced complexes containing four to nine lipoyl domains per E2p chain were lower than those with fewer lipoyl domains. This could be due to under-lipoylation of the domains participating in catalysis and interference from unlipoylated domains.

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain

Construction and properties of pyruvate dehydrogenase complexes with up to nine lipoyl domains per lipoate acetyltransferase chain

The human pyruvate dehydrogenase complex: a polymorphic region of the lipoate acetyl transferase (E2) subunit gene

A major issue in the study of the pathogenesis of primary biliary cirrhosis is whether the E2 subunit of the pyruvate dehydrogenase complex (PDH-E2), the major autoantigen in the disease, exists as a tissue-specific isoform. cDNA clones spanning a segment of the 3′-catalytic region of PDH-E2 (nt 1158–1361) have been isolated from human kidney, placenta and bile epithelium cells. Nucleotide sequence analysis of the clones showed differences consistent with the presence of normal variants of PDH-E2 in the human population. However, the existence of tissue-specific isoforms of PDH-E2 cannot yet be discounted.

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.