Defining Electron Bifurcation in the Electron-Transferring Flavoprotein Family

ABSTRACT Electron bifurcation is the coupling of exergonic and endergonic redox reactions to simultaneously generate (or utilize) low- and high-potential electrons. It is the third recognized form of energy conservation in biology and was recently described for select electron-transferring flavoproteins (Etfs). Etfs are flavin-containing heterodimers best known for donating electrons derived from fatty acid and amino acid oxidation to an electron transfer respiratory chain via Etf-quinone oxidoreductase. Canonical examples contain a flavin adenine dinucleotide (FAD) that is involved in electron transfer, as well as a non-redox-active AMP. However, Etfs demonstrated to bifurcate electrons contain a second FAD in place of the AMP. To expand our understanding of the functional variety and metabolic significance of Etfs and to identify amino acid sequence motifs that potentially enable electron bifurcation, we compiled 1,314 Etf protein sequences from genome sequence databases and subjected them to informatic and structural analyses. Etfs were identified in diverse archaea and bacteria, and they clustered into five distinct well-supported groups, based on their amino acid sequences. Gene neighborhood analyses indicated that these Etf group designations largely correspond to putative differences in functionality. Etfs with the demonstrated ability to bifurcate were found to form one group, suggesting that distinct conserved amino acid sequence motifs enable this capability. Indeed, structural modeling and sequence alignments revealed that identifying residues occur in the NADH- and FAD-binding regions of bifurcating Etfs. Collectively, a new classification scheme for Etf proteins that delineates putative bifurcating versus nonbifurcating members is presented and suggests that Etf-mediated bifurcation is associated with surprisingly diverse enzymes. IMPORTANCE Electron bifurcation has recently been recognized as an electron transfer mechanism used by microorganisms to maximize energy conservation. Bifurcating enzymes couple thermodynamically unfavorable reactions with thermodynamically favorable reactions in an overall spontaneous process. Here we show that the electron-transferring flavoprotein (Etf) enzyme family exhibits far greater diversity than previously recognized, and we provide a phylogenetic analysis that clearly delineates bifurcating versus nonbifurcating members of this family. Structural modeling of proteins within these groups reveals key differences between the bifurcating and nonbifurcating Etfs.

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Rhodococcus sp. Strain CR-53 LipR, the First Member of a New Bacterial Lipase Family (Family X) Displaying an Unusual Y-Type Oxyanion Hole, Similar to the Candida antarctica Lipase Clan

Applied and Environmental Microbiology ◽

10.1128/aem.06332-11 ◽

2012 ◽

Vol 78 (6) ◽

pp. 1724-1732 ◽

Cited By ~ 30

Author(s):

Arnau Bassegoda ◽

F. I. Javier Pastor ◽

Pilar Diaz

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Candida Antarctica ◽

Burkholderia Cenocepacia ◽

Sequence Motifs ◽

Oxyanion Hole ◽

Content Type ◽

New Family ◽

Consensus Amino Acid Sequence ◽

Four Blocks

ABSTRACTBacterial lipases constitute the most important group of biocatalysts for synthetic organic chemistry. Accordingly, there is substantial interest in developing new valuable lipases. Considering the lack of information concerning the lipases of the genusRhodococcusand taking into account the interest raised by the enzymes produced by actinomycetes, a search for putative lipase-encoding genes fromRhodococcussp. strain CR-53 was performed. We isolated, cloned, purified, and characterized LipR, the first lipase described from the genusRhodococcus. LipR is a mesophilic enzyme showing preference for medium-chain-length acyl groups without showing interfacial activation. It displays good long-term stability and high tolerance for the presence of ions and chemical agents in the reaction mixture. Amino acid sequence analysis of LipR revealed that it displays four unique amino acid sequence motifs that clearly separate it from any other previously described family of bacterial lipases. Using bioinformatics tools, LipR could be related only to several uncharacterized putative lipases from different bacterial origins, all of which display the four blocks of consensus amino acid sequence motifs that contribute to define a new family of bacterial lipases, namely, family X. Therefore, LipR is the first characterized member of the new bacterial lipase family X. Further confirmation of this new family of lipases was performed after cloningBurkholderia cenocepaciaputative lipase, bearing the same conserved motifs and clustering in family X. Interestingly, all lipases grouping in the new bacterial lipase family X display a Y-type oxyanion hole, a motif conserved in theCandida antarcticalipase clan but never found among bacterial lipases. This observation contributes to confirm that LipR and its homologs belong to a new family of bacterial lipases.

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Identification and characterization of tRNA (Gm18) methyltransferase from Thermus thermophilus HB8: domain structure and conserved amino acid sequence motifs

Genes to Cells ◽

10.1046/j.1365-2443.2002.00520.x ◽

2002 ◽

Vol 7 (3) ◽

pp. 259-272 ◽

Cited By ~ 43

Author(s):

Hiroyuki Hori ◽

Tsutomu Suzuki ◽

Kazumasa Sugawara ◽

Yorinao Inoue ◽

Takehiko Shibata ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Domain Structure ◽

Thermus Thermophilus ◽

Sequence Motifs ◽

Thermus Thermophilus Hb8 ◽

Identification And Characterization

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Searching for amino acid sequence motifs among enzymes: the Enzyme–Reaction Database

Bioinformatics ◽

10.1093/bioinformatics/9.1.9 ◽

1993 ◽

Vol 9 (1) ◽

pp. 9-15 ◽

Cited By ~ 3

Author(s):

Mikita Suyama ◽

Atsushi Ogiwara ◽

Takaaki Nishioka ◽

Jun'ichi Oda

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Enzyme Reaction ◽

Sequence Motifs

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Broad-spectrum detection of papillomaviruses in bovine teat papillomas and healthy teat skin

Journal of General Virology ◽

10.1099/vir.0.80086-0 ◽

2004 ◽

Vol 85 (8) ◽

pp. 2191-2197 ◽

Cited By ~ 77

Author(s):

Tomoko Ogawa ◽

Yoshimi Tomita ◽

Mineyuki Okada ◽

Kuniko Shinozaki ◽

Hiroko Kubonoya ◽

...

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Broad Spectrum ◽

Phylogenetic Analyses ◽

Genomic Diversity ◽

Bovine Papillomavirus ◽

Subclinical Infection ◽

Healthy Skin ◽

Sequence Alignments ◽

Spectrum Detection

To investigate the prevalence of bovine papillomavirus (BPV) in bovine papilloma and healthy skin, DNA extracted from teat papillomas and healthy teat skin swabs was analysed by PCR using the primer pairs FAP59/FAP64 and MY09/MY11. Papillomavirus (PV) DNA was detected in all 15 papilloma specimens using FAP59/FAP64 and in 8 of the 15 papilloma specimens using MY09/MY11. In swab samples, 21 and 8 of the 122 samples were PV DNA positive using FAP59/FAP64 and MY09/MY11, respectively. Four BPV types (BPV-1, -3, -5 and -6), two previously identified putative BPV types (BAA1 and -5) and 11 putative new PV types (designated BAPV1 to -10 and BAPV11MY) were found in the 39 PV DNA-positive samples. Amino acid sequence alignments of the putative new PV types with reported BPVs and phylogenetic analyses of the putative new PV types with human and animal PV types showed that BAPV1 to -10 and BAPV11MY are putative new BPV types. These results also showed the genomic diversity and extent of subclinical infection of BPV.

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Short translational ramp determines efficiency of protein synthesis

10.1101/571059 ◽

2019 ◽

Cited By ~ 2

Author(s):

Manasvi Verma ◽

Junhong Choi ◽

Kyle A. Cottrell ◽

Zeno Lavagnino ◽

Erica N. Thomas ◽

...

Keyword(s):

Protein Synthesis ◽

Amino Acid ◽

Amino Acid Sequence ◽

Single Molecule ◽

Translation Initiation ◽

Protein Translation ◽

Translational Efficiency ◽

Translation Efficiency ◽

Sequence Motifs ◽

Peptide Bond Formation

AbstractIt is generally assumed that translation efficiency is governed by translation initiation. However, the efficiency of protein synthesis is regulated by multiple factors including tRNA abundance, codon composition, mRNA motifs and amino-acid sequence1–4. These factors influence the rate of protein synthesis beyond the initiation phase of translation, typically by modulating the rate of peptide-bond formation and to a lesser extent that of translocation. The slowdown in translation during the early elongation phase, known as the 5’ translational ramp, likely contributes to the efficiency of protein synthesis 5–9. Multiple mechanisms, which could explain the molecular basis for this translational ramp, have been proposed that include tRNA abundance bias6,9, the rate of translation initiation10–15, mRNA and ribosome structure 11,12,14,16–18, or retention of initiation factors during early elongation events 19. Here, we show that the amount of synthesized protein (translation efficiency) depends on a short translational ramp that comprises the first 5 codons in mRNA. Using a library of more than 250,000 reporter sequences combined with in vitro and in vivo protein expression assays, we show that differences in the short ramp can lead to 3 to 4 orders of magnitude changes in protein abundance. The observed difference is not dependent on tRNA abundance, efficiency of translation initiation, or overall mRNA structure. Instead, we show that translation is regulated by amino-acid-sequence composition and local mRNA sequence. Single-molecule measurements of translation kinetics indicate substantial pausing of ribosome and abortion of protein synthesis on the 4th or 5th codon for distinct amino acid or nucleotide compositions. Introduction of preferred sequence motifs, only at the exact positions within the mRNA, improves protein synthesis for recombinant proteins, indicating an evolutionarily conserved mechanism for controlling translational efficiency.

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The Use of Amino Acid Sequence Alignments to Assess Potential Allergenicity of Proteins Used in Genetically Modified Foods

Advances in Food and Nutrition Research ◽

10.1016/s1043-4526(08)60092-3 ◽

1998 ◽

pp. 45-62 ◽

Cited By ~ 37

Author(s):

Steven M. Gendel

Keyword(s):

Amino Acid ◽

Amino Acid Sequence ◽

Genetically Modified ◽

Genetically Modified Foods ◽

Sequence Alignments

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A Novel Lipolytic Enzyme Located in the Outer Membrane of Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.181.22.6977-6986.1999 ◽

1999 ◽

Vol 181 (22) ◽

pp. 6977-6986 ◽

Cited By ~ 112

Author(s):

Susanne Wilhelm ◽

Jan Tommassen ◽

Karl-Erich Jaeger

Keyword(s):

Pseudomonas Aeruginosa ◽

Amino Acid ◽

Amino Acid Sequence ◽

Outer Membrane ◽

Open Reading Frame ◽

Lipolytic Enzyme ◽

Cell Fractionation ◽

Sequence Alignments ◽

Reading Frame ◽

Amino Terminal

ABSTRACT A lipase-negative deletion mutant of Pseudomonas aeruginosa PAO1 still showed extracellular lipolytic activity toward short-chain p-nitrophenylesters. By screening a genomic DNA library of P. aeruginosa PAO1, an esterase gene, estA, was identified, cloned, and sequenced, revealing an open reading frame of 1,941 bp. The product ofestA is a 69.5-kDa protein, which is probably processed by removal of an N-terminal signal peptide to yield a 67-kDa mature protein. A molecular mass of 66 kDa was determined for35S-labeled EstA by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The amino acid sequence of EstA indicated that the esterase is a member of a novel GDSL family of lipolytic enzymes. The estA gene showed high similarity to an open reading frame of unknown function located in thetrpE-trpG region of P. putida and to a gene encoding an outer membrane esterase of Salmonella typhimurium. Amino acid sequence alignments led us to predict that this esterase is an autotransporter protein which possesses a carboxy-terminal β-barrel domain, allowing the secretion of the amino-terminal passenger domain harboring the catalytic activity. Expression of estA in P. aeruginosa andEscherichia coli and subsequent cell fractionation revealed that the enzyme was associated with the cellular membranes. Trypsin treatment of whole cells released a significant amount of esterase, indicating that the enzyme was located in the outer membrane with the catalytic domain exposed to the surface. To our knowledge, this esterase is unique in that it exemplifies in P. aeruginosa(i) the first enzyme identified in the outer membrane and (ii) the first example of a type IV secretion mechanism.

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Photosynthetic Reaction Center Variants Made via Genetic Code Expansion Show Tyr at M210 Tunes the Initial Electron Transfer Mechanism

10.26434/chemrxiv.14691339 ◽

2021 ◽

Author(s):

Jared Weaver ◽

Chi-Yun Lin ◽

Kaitlyn M. Faries ◽

Irimpan Mathews ◽

Silvia Russi ◽

...

Keyword(s):

Electron Transfer ◽

Free Energy ◽

Amino Acid ◽

Near Infrared ◽

Transient Absorption ◽

Global Analysis ◽

Primary Electron ◽

Wild Type ◽

Electron Transfer Mechanism ◽

Noncanonical Amino Acid

Photosynthetic reaction centers (RCs) from Rhodobacter sphaeroides were engineered to vary the electronic properties of a key tyrosine close to an essential electron transfer component (M210) via its replacement with site-specific genetically encoded noncanonical amino acid tyrosine analogs. High fidelity of noncanonical amino acid incorporation was verified with mass spectrometry and x-ray crystallography and demonstrated that RC variants exhibit no significant structural alterations relative to wild-type. Ultrafast transient absorption spectroscopy indicates the excited primary electron donor, P*, decays via an approximately 4 ps and 20 ps population to produce the charge-separated state P+HA- in all variants. Global analysis indicates that in the 4 ps population P+HA- forms through a 2-step process P* –> P+BA– –> P+HA-, while in the 20 ps population it forms via a 1-step P* –> P+HA– superexchange mechanism. The percentage of P* population that decays via the superexchange route varies from approximately 25% to 45% among variants while in wild-type this percentage is approximately 15%. Increases in the P* population which decays via superexchange correlates with increases in free energy of the P+BA– intermediate caused by a given M210 tyrosine analog. This was experimentally estimated through resonance Stark spectroscopy, redox titrations, and near-infrared absorption measurements. As the most energetically perturbative variant, 3-nitrotyrosine at M210 creates an approximately 110 meV increase in the free energy of P+BA– along with a dramatic diminution of the 1030 nm transient absorption band indicative of P+BA– formation. Collectively this work indicates the tyrosine at M210 tunes the mechanism of primary electron transfer in the RC.<br>

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Three-ComponentO-Demethylase System Essential for Catabolism of a Lignin-Derived Biphenyl Compound in Sphingobium sp. Strain SYK-6

Applied and Environmental Microbiology ◽

10.1128/aem.02236-14 ◽

2014 ◽

Vol 80 (23) ◽

pp. 7142-7153 ◽

Cited By ~ 22

Author(s):

Taichi Yoshikata ◽

Kazuya Suzuki ◽

Naofumi Kamimura ◽

Masahiro Namiki ◽

Shojiro Hishiyama ◽

...

Keyword(s):

Electron Transfer ◽

Amino Acid ◽

Amino Acid Sequence ◽

Reductase Activity ◽

Normal Growth ◽

Prosthetic Group ◽

Gene Encoding ◽

Content Type ◽

Sequence Identities ◽

Electron Transfer Component

ABSTRACTSphingobiumsp. strain SYK-6 is able to assimilate lignin-derived biaryls, including a biphenyl compound, 5,5′-dehydrodivanillate (DDVA). Previously,ligXa(SLG_07770), which is similar to the gene encoding oxygenase components of Rieske-type nonheme iron aromatic-ring-hydroxylating oxygenases, was identified to be essential for the conversion of DDVA; however, the genes encoding electron transfer components remained unknown. Disruption of putative electron transfer component genes scattered through the SYK-6 genome indicated that SLG_08500 and SLG_21200, which showed approximately 60% amino acid sequence identities with ferredoxin and ferredoxin reductase of dicambaO-demethylase, were essential for the normal growth of SYK-6 on DDVA. LigXa and the gene products of SLG_08500 (LigXc) and SLG_21200 (LigXd) were purified and were estimated to be a trimer, a monomer, and a monomer, respectively. LigXd contains FAD as the prosthetic group and showed much higher reductase activity toward 2,6-dichlorophenolindophenol with NADH than with NADPH. A mixture of purified LigXa, LigXc, and LigXd converted DDVA into 2,2′,3-trihydroxy-3′-methoxy-5,5′-dicarboxybiphenyl in the presence of NADH, indicating that DDVAO-demethylase is a three-component monooxygenase. This enzyme requires Fe(II) for its activity and is highly specific for DDVA, with aKmvalue of 63.5 μM andkcatof 6.1 s−1. Genome searches in six other sphingomonads revealed genes similar toligXcandligXd(>58% amino acid sequence identities) with a limited number of electron transfer component genes, yet a number of diverse oxygenase component genes were found. This fact implies that these few electron transfer components are able to interact with numerous oxygenase components and the conserved LigXc and LigXd orthologs are important in sphingomonads.

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