Resolution of branched-chain oxo acid dehydrogenase complex of <i>Pseudomonas aeruginosa</i> PAO.

McCully, V; Burns, Gayle; Sokatch, John R.

doi:10.1042/bj2330737

Cited by 45 publications

(36 citation statements)

References 37 publications

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“…The start of bkdB was identified with the aid of the Nterminal sequence of E2b purified by HPLC. The source of E2b was P. aeruginosa since we have not yet been able to isolate E2b from P. putida [2]. The N-terminal sequence of E2b from P. aeruginosa closely matched the translated sequence beginning at base 2333 with the exception of the absence of Nterminal methionine and glutamic acid in place of glutamine at residue 17 of the translated sequence (Fig.…”

Section: Nucleotide Sequence Of Bkdbmentioning

confidence: 85%

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli

Burns

Brown

Hatter

et al. 1988

European Journal of Biochemistry

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The nucleotide sequence of bkdB, the structural gene for E2b, the transacylase component of branched-chainoxoacid dehydrogenase of Pseudornonas putida has been determined and translated into its amino acid sequence. The start of bkdB was identified from the N-terminal sequence of E2b isolated from branched-chain-oxoacid dehydrogenase of the closely related species, P. aeruginosa. The reading frame was composed of 65.5% G + C with 82.3% of the codons ending in G or C. There was no intergenic space between bkdA2 and bkdB. No codons requiring minor tRNAs were utilized and the codon bias index indicated a preferential codon usage. The hkdB gene encoded 423 amino acids although the N-terminal methionine was absent from E2b prepared from P. ueruginosa. The relative molecular mass of the encoded protein was 45134 (45003 minus methionine) vs 47000 obtained by SDS/polyacrylamide gel electrophoresis. There was a single lipoyl domain in E2b compared to three lipoyl domains in E2p, and one domain in E20, the transacylases of pyruvate and 2-oxoglutarate dehydrogenases of Escherichiu coli respectively. There was significant similarity between the lipoyl domain of E2b and of E2p and E20 as well as between the EI-E2 binding domains of E2b, E2p and E20. There was no similarity between the E3 binding domain of E2b to E2p and E20 which may reflect the uniqueness of the E3 component of branchedchain-oxoacid dehydrogenase of P. putida. The conclusions drawn from these comparisons are that the transacylases of prokaryotic pyruvate, 2-oxoglutarate and branched-chain-oxoacid dehydrogenases descended from a common ancestral protein probably at about the same time.Branched-chain-oxoacid dehydrogenase is a multienzyme complex which is common to the metabolism of the branched chain oxoacids which are formed from valine, isoleucine and leucine by transamination. Branched-chain-oxoacid dehydrogenase complex performs three catalytic activities. The El component is the dehydrogenase and catalyzes the decarboxylation of the oxoacid substrate and transfer of the branched-chain fatty acyl residue to E2 reducing the lipoyl prosthetic group of the latter subunit. E2 catalyzes transacylation of the fatty acyl group from the lipoyl residue of E2 to coenzyme A. E2 is the core of all of the oxoacid multienzyme complexes and binds the El and E3 components. The E3 component is a lipoamide dehydrogenase which catalyzes the oxidation of the dihydrolipoyl residue of the E2 subunit and the reduction of NAD+. The overall reaction catalyzed by Correspondence to J. R. Sokatch, Department of Biochemistry and Molecular Biology, University of Oklahoma, Health Sciences Center, P.O. Box 26901, Oklahoma City, Oklahoma, USA-73190Abhreviutions. E2b, E2p and E20 are the transacylase components of branched-chain-oxoacid, pyruvate and 2-oxoglu'tarate dehydrogenase respectively. The structural genes for the components of branched-chain-oxoacid dehydrogenase from Pseudomonas putida are hkdA1, E l i , hkdA2, EIP, hkdB, E2 and lpdV, E3 or LPD-Val. The structural genes for the...

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Section: Nucleotide Sequence Of Bkdbmentioning

confidence: 85%

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli

Burns

Brown

Hatter

et al. 1988

European Journal of Biochemistry

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“…Lpd is the second Factor H, FHL-1, CFHR1, and plasminogenbinding immune evasion protein identified from P. aeruginosa. Lpd, which has a predicted mass of 51.6 kDa, was initially identified as a cytoplasmic protein and as a component of the enzymatic pyruvate dehydrogenase complex, which catalyzes the electron transfer between pyridine nucleotides and disulfide components (32,38,39). Sequence analysis of the 5 laboratory strains and 13 clinical A fraction containing outer membrane proteins was prepared from the 13 clinical P. aeruginosa isolates, separated by SDS-PAGE, transferred to a membrane, and analyzed by Western blotting using the Lpd antiserum.…”

Section: Discussionmentioning

confidence: 99%

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

Hallström

Mörgelin

Barthel

et al. 2012

The Journal of Immunology

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The opportunistic human pathogen Pseudomonas aeruginosa causes a wide range of diseases. To cross host innate immune barriers, P. aeruginosa has developed efficient strategies to escape host complement attack. In this study, we identify the 57-kDa dihydrolipoamide dehydrogenase (Lpd) as a surface-exposed protein of P. aeruginosa that binds the four human plasma proteins, Factor H, Factor H-like protein-1 (FHL-1), complement Factor H-related protein 1 (CFHR1), and plasminogen. Factor H contacts Lpd via short consensus repeats 7 and 18–20. Factor H, FHL-1, and plasminogen when bound to Lpd were functionally active. Factor H and FHL-1 displayed complement-regulatory activity, and bound plasminogen, when converted to the active protease plasmin, cleaved the chromogenic substrate S-2251 and the natural substrate fibrinogen. The lpd of P. aeruginosa is a rather conserved gene; a total of 22 synonymous and 3 nonsynonymous mutations was identified in the lpd gene of the 5 laboratory strains and 13 clinical isolates. Lpd is surface exposed and contributes to survival of P. aeruginosa in human serum. Bacterial survival was reduced when Lpd was blocked on the surface prior to challenge with human serum. Similarly, bacterial survival was reduced up to 84% when the bacteria was challenged with complement active serum depleted of Factor H, FHL-1, and CFHR1, demonstrating a protective role of the attached human regulators from complement attack. In summary, Lpd is a novel surface-exposed virulence factor of P. aeruginosa that binds Factor H, FHL-1, CFHR1, and plasminogen, and the Lpd-attached regulators are relevant for innate immune escape and most likely contribute to tissue invasion.

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“…3. The sequences of cyanogen bromide peptides including the sequence of the C-terminal peptide previously reported [5], are underlined and designated (3), (4) and ( 5 ) in Fig. 3.…”

Section: Analysis Of Nucleotide Sequencesmentioning

confidence: 99%

“…Nuckotide sequence and amino acid trunslution cf IpdV. The underlined amino acids correspond to (1) the N-terminal amino acids identified by use o f the gas-phase amino acid sequencer, (2) the redox-active disulfide region, (3), (4) and (5) are cyanogen bromide peptides, ( 5 ) being the previously-reported C-terminal peptide [5]. The underlined sequence marked SD is the Shine-Dalgarno ribosome-binding site Table 2 is 48 164.…”

Section: Amino Acid Composition and Mmentioning

confidence: 99%

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Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida

Burns

Brown

Hatter

et al. 1989

European Journal of Biochemistry

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The production of two lipoamide dehydrogenases by Pseudomonus is so far unique. One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins. Northern blots using mRNA isolated from P. putidu grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD-Val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C . Codon usagc is characteristic of moderately strongly expressed genes. There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame. The M , of the protein encoded is 48164 and when the M , of FAD is added, the total M , is 48949, which is very close to the value of 49 000 obtained by SDS-polyacrylamide gel electrophoresis.Similarity comparisons of LPD-Val with sequences of three other lipoamide dehydrogenases showed that LPDval was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.Branched-chain-oxoacid dehydrogenase, the second enzyme in the catabolism of valine, leucine and isoleucine [I], is a multienzyme complex composed of three subunits: El, the dehydrogenase-decarboxylase, E2, the transacylase and E3, lipoamide dehydrogenase. The complex has been purified from bovine kidney [2], Pseudomonas putidu [3], rabbit liver [4] and Pseudomonus ueruginosu [5]. In addition, an oxoacid dehydrogenase has been purified from Bacillus subtilis [6] which has activity with pyruvate and branched-chain oxoacids. All of these complexes are composed of four polypeptides including Elcl and Elfl subunits. The mammalian complexes are regulated by phosphorylation [4, 71, while the P.~eudomonus complex is allosterically regulated by L-valine I3, 51.P. putidu and P. ueruginosu contain two lipoamide dehydrogenases, LPD-Val and LPD-glc. LPD-val was so named because it is induced in media containing branched-chain amino acids as carbon sources and is the specific E3 subunit of branched-chain-oxoacid dehydrogenase, which is its only known function [5, 81. LPD-glc is found in cells grown in glucose synthetic medium and is the E3 subunit of 2-oxoglutarate dehydrogenase [9] and the L-factor of the glycine-oxidation system in P. putidu [lo]. LPD-val and LPDglc are specific for their functions and are not interchangeable. Mutants affected in IpdV, the structural gene for LPD-val, lack b...

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Resolution of branched-chain oxo acid dehydrogenase complex of Pseudomonas aeruginosa PAO.

Cited by 45 publications

References 37 publications

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli

Comparison of the amino acid sequences of the transacylase components of branched chain oxoacid dehydrogenase of Pseudomonas putida, and the pyruvate and 2‐oxoglutarate dehydrogenases of Escherichia coli

Dihydrolipoamide Dehydrogenase of Pseudomonas aeruginosa Is a Surface-Exposed Immune Evasion Protein That Binds Three Members of the Factor H Family and Plasminogen

Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched‐chain‐oxoacid dehydrogenase of Pseudomonas putida

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