The 2.1 Å structure of<i>Aerococcus viridans</i><scp>L</scp>-lactate oxidase (LOX)

Leiros, Ingar; Wang, Ellen; Rasmussen, Tonni; Oksanen, Esko; Repo, Heidi; Petersen, Steffen B.; Heikinheimo, Pirkko; Hough, Edward

doi:10.1107/s1744309106044678

Cited by 48 publications

(64 citation statements)

References 39 publications

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“…The given amino acid positions refer to the enzyme from Nostoc (No-LOX, All0170; the full alignment is shown Supplemental Figure 1 online). The residues involved in the binding of FMN are highly conserved between both enzyme types (Lindqvist and Brä ndé n, 1989;Maeda-Yorita et al, 1995;Stenberg et al, 1995;Stenberg and Lindqvist, 1997;Leiros et al, 2006). Amino acids presumably involved in substrate binding are marked in gray.…”

Section: Biochemical Analyses Of Gox-like Proteins In Vitromentioning

confidence: 99%

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N₂ Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

et al. 2011

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Glycolate oxidase (GOX) is an essential enzyme involved in photorespiratory metabolism in plants. In cyanobacteria and green algae, the corresponding reaction is catalyzed by glycolate dehydrogenases (GlcD). The genomes of N 2 -fixing cyanobacteria, such as Nostoc PCC 7120 and green algae, appear to harbor genes for both GlcD and GOX proteins. The GOX-like proteins from Nostoc (No-LOX) and from Chlamydomonas reinhardtii showed high L-lactate oxidase (LOX) and low GOX activities, whereas glycolate was the preferred substrate of the phylogenetically related At-GOX2 from Arabidopsis thaliana. Changing the active site of No-LOX to that of At-GOX2 by site-specific mutagenesis reversed the LOX/GOX activity ratio of No-LOX. Despite its low GOX activity, No-LOX overexpression decreased the accumulation of toxic glycolate in a cyanobacterial photorespiratory mutant and restored its ability to grow in air. A LOX-deficient Nostoc mutant grew normally in nitrate-containing medium but died under N 2 -fixing conditions. Cultivation under low oxygen rescued this lethal phenotype, indicating that N 2 fixation was more sensitive to O 2 in the Dlox Nostoc mutant than in the wild type. We propose that LOX primarily serves as an O 2 -scavenging enzyme to protect nitrogenase in extant N 2 -fixing cyanobacteria, whereas in plants it has evolved into GOX, responsible for glycolate oxidation during photorespiration.

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Section: Biochemical Analyses Of Gox-like Proteins In Vitromentioning

confidence: 99%

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N₂ Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

et al. 2011

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“…The region of residues near the range 199-204 is a good candidate for participation in such a conformational rearrangement as a result of its high apparent mobility. Various residues in this segment are often missing in crystal structures and those that are present usually exhibit high temperature factors [3][4][5][6][7][27][28][29]. In the A95G mutant subunits, certain residues in this region cannot be observed in the electron density: A, 203-204; B, 199-204; C, 203-204; D, 203-204.…”

Section: Reactivity With O 2 and Alternative Electron Acceptors In Thmentioning

confidence: 99%

“…In the present study, we have focused on the LOX from Aerococcus viridans (avLOX), a prototypical oxidase-type member of the a-hydroxy acid-oxidizing flavoenzyme family that has been well characterized biochemically [9,21,25,26] and structurally [6,[27][28][29]. The enzyme is biotechnologically important for its commercialized use in analytical devices for blood L-lactate determination [30,31].…”

mentioning

confidence: 99%

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O₂ and other electron acceptors

et al. 2014

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Aerococcus viridans L-lactate oxidase (avLOX) is a biotechnologically important flavoenzyme that catalyzes the conversion of L-lactate and O 2 into pyruvate and H 2 O 2. The enzymatic reaction underlies different biosensor applications of avLOX for blood L-lactate determination. The ability of avLOX to replace O 2 with other electron acceptors such as 2,6-dichlorophenol-indophenol (DCIP) allows the possiblity of analytical and practical applications. The A95G variant of avLOX was previously shown to exhibit lowered reactivity with O 2 compared to wild-type enzyme and therefore was employed in a detailed investigation with respect to the specificity for different electron acceptor substrates. From stopped-flow experiments performed at 20°C (pH 6.5), we determined that the A95G variant (fully reduced by L-lactate) was approximately three-fold more reactive towards DCIP (1.0 AE 0.1 9 10 6 M À1 Ás À1 ) than O 2 , whereas avLOX wildtype under the same conditions was 14-fold more reactive towards O 2 (1.8 AE 0.1 9 10 6 M À1 Ás À1 ) than DCIP. Substituted 1,4-benzoquinones were up to five-fold better electron acceptors for reaction with L-lactate-reduced A95G variant than wild-type. A 1.65-A crystal structure of oxidized A95G variant bound with pyruvate was determined and revealed that the steric volume created by removal of the methyl side chain of Ala95 and a slight additional shift in the main chain at position Gly95 together enable the accomodation of a new active-site water molecule within hydrogen-bond distance to the N5 of the FMN cofactor. The increased steric volume available in the active site allows the A95G variant to exhibit a similar trend with the related glycolate oxidase in electron acceptor substrate specificities, despite the latter containing an alanine at the analogous position.Database Atomic coordinates and related experimental data concerning the structure of the A95G variant of A. viridans lacate oxidase have been deposited in the Protein Data Bank under the identifier 4RJE.Abbreviations avLOX, Aerococcus viridans L-lactate oxidase; DCIP, 2,6-dichlorophenol-indophenol; GOX, glycolate oxidase; LOX, L-lacate oxidase; PDB, Protein Data Bank.

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“…The protein sequence of lactate oxidase in A. viridans (BAA09172), the predicted protein sequence of the lactate oxidase previously described in S. iniae (accession number Y07622), and the predicted protein sequences of the corrected type 1 lactate oxidase and the novel type 2 variant from S. iniae described in this paper were aligned in Clustal X (28). Enzymatically active sites for A. viridans as described in previous publications (10,16,17,35,36) that are conserved in S. iniae were marked in the alignment. The Protein Data Bank (PDB) file of 2DU2 (30) on the resolved crystal structure of lactate oxidase in A. viridans was downloaded from the Research Collaboratory for Structural Bioinformatics PDB (http://www.pdb.org/pdb/home/home.do) and subunits 2 and 3 were visualized using PyMOL, version 1.0 (8).…”

Section: Methodsmentioning

confidence: 99%

“…The protein models were viewed in PyMOL, and two identical subunits were placed adjacent to each other using Adobe Photoshop. Subunit interactions and predicted active sites were as previously described for A. viridans (10,16,17,35,36).…”

Section: Methodsmentioning

confidence: 99%

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi ( Lates calcarifer )

Nawawi

Baiano

Kvennefors

et al. 2009

Appl Environ Microbiol

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In Streptococcus iniae, lactate metabolism is dependent upon two proteins, lactate permease that mediates uptake and lactate oxidase, a flavin mononucleotide-dependent enzyme that catalyzes oxidation of ␣-hydroxyacids. A novel variant of the lactate oxidase gene, lctO, in Australian isolates of S. iniae from diseased barramundi was found during a diagnostic screen using LOX-1 and LOX-2 primers, yielding amplicons of 920 bp instead of the expected 869 bp. Sequencing of the novel gene variant (type 2) revealed a 51-nucleotide insertion in lctO, resulting in a 17-amino-acid repeat in the gene product, and three-dimensional modeling indicated formation of an extra loop in the monomeric protein structure. The activities of the lactate oxidase enzyme variants expressed in Escherichia coli were examined, indicating that the higher-molecular-weight type 2 enzyme exhibited higher activity. Growth rates of S. iniae expressing the novel type 2 enzyme were not reduced at lactate concentrations of 0.3% and 0.5%, whereas a strain expressing the type 1 enzyme exhibited reduced growth rates at these lactate concentrations. During a retrospective screen of 105 isolates of S. iniae from Australia, the United States, Canada, Israel, Réunion Island, and Thailand, the type 2 variant arose only in isolates from a single marine farm with unusually high tidal flow in the Northern Territory, Australia. Elevated plasma lactate levels in the fish, resulting from the effort of swimming in tidal flows of up to 3 knots, may exert sufficient selective pressure to maintain the novel, high-molecular-weight enzyme variant.

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The 2.1 Å structure ofAerococcus viridansL-lactate oxidase (LOX)

Cited by 48 publications

References 39 publications

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N₂ Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N₂ Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O₂ and other electron acceptors

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi ( Lates calcarifer )

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The 2.1 Å structure ofAerococcus viridansL-lactate oxidase (LOX)

Cited by 48 publications

References 39 publications

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N2 Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N2 Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

Host-Directed Evolution of a Novel Lactate Oxidase in Streptococcus iniae Isolates from Barramundi ( Lates calcarifer )

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Cyanobacterial Lactate Oxidases Serve as Essential Partners in N₂ Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

Cyanobacterial Lactate Oxidases Serve as Essential Partners in N₂ Fixation and Evolved into Photorespiratory Glycolate Oxidases in Plants

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O₂ and other electron acceptors