Insights into Substrate and Metal Binding from the Crystal Structure of Cyanobacterial Aldehyde Deformylating Oxygenase with Substrate Bound

Buer, Benjamin C.; Paul, Bishwajit; Das, Debanu; Stuckey, Jeanne A.; Marsh, E. Neil G.

doi:10.1021/cb500343j

Cited by 32 publications

(41 citation statements)

References 49 publications

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“…However, in these structures, ADO exhibits an occluded conformation, therefore, we were not able to identify the entry point for the aldehyde to access the active site of ADO based on our AAR-ADO complex structures. Previously reported crystal structures of PmADO showed that a T-shaped channel is present inside ADO, with an opening between helices 7 and 8 at the ADO surface, which was suggested to represent the entry point for aldehyde 20 . The helices 7 and 8 of ADO are at the AAR-ADO interface and helix 7 is essential for the complex formation.…”

Section: Resultsmentioning

confidence: 95%

“…Thus, structure-based engineering of the two enzymes is a practical way to optimize the efficiency of alkane synthesis. Several crystal structures of ADO from different species of cyanobacteria were previously solved, revealing that ADO adopts a ferritin-like eighthelix (henceforth named helix 1-8) architecture, with a di-iron center buried inside the molecule [19][20][21][22] . An occluded substrate binding cavity accessing the di-iron center was identified in these structures 19,21,22 .…”

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confidence: 99%

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Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrier protein reductase

et al. 2020

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Long-chain alk(a/e)nes represent the major constituents of conventional transportation fuels. Biosynthesis of alkanes is ubiquitous in many kinds of organisms. Cyanobacteria possess two enzymes, acyl-acyl carrier protein (acyl-ACP) reductase (AAR) and aldehyde-deformylating oxygenase (ADO), which function in a two-step alkane biosynthesis pathway. These two enzymes act in series and possibly form a complex that efficiently converts long chain fatty acyl-ACP/fatty acyl-CoA into hydrocarbon. While the structure of ADO has been previously described, structures of both AAR and AAR-ADO complex have not been solved, preventing deeper understanding of this pathway. Here, we report a ligand-free AAR structure, and three AAR-ADO complex structures in which AARs bind various ligands. Our results reveal the binding pattern of AAR with its substrate/cofactor, and suggest a potential aldehydetransferring channel from AAR to ADO. Based on our structural and biochemical data, we proposed a model for the complete catalytic cycle of AAR.

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Section: Resultsmentioning

confidence: 95%

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confidence: 99%

Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrier protein reductase

et al. 2020

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“…The di-iron center is contained within an antiparallel four-α-helix bundle, where two histidines and four carboxylates (aspartate or glutamate) supply the protein ligands to the metal ions [1, 8–11]. The C1-derived co-product of the cADO-catalyzed reaction is formate (Fig.…”

Section: Introductionmentioning

confidence: 99%

Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship

et al. 2017

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BackgroundAldehyde-deformylating oxygenase (ADO) is a key enzyme involved in the biosynthetic pathway of fatty alk(a/e)nes in cyanobacteria. However, cADO (cyanobacterial ADO) showed extreme low activity with the k cat value below 1 min−1, which would limit its application in biofuel production. To identify the activity related key residues of cADO is urgently required.ResultsThe amino acid residues which might affect cADO activity were identified based on the crystal structures and sequence alignment of cADOs, including the residues close to the di-iron center (Tyr39, Arg62, Gln110, Tyr122, Asp143 of cADO-1593), the protein surface (Trp 178 of cADO-1593), and those involved in two important hydrogen bonds (Gln49, Asn123 of cADO-1593, and Asp49, Asn123 of cADO-sll0208) and in the oligopeptide whose conformation changed in the absence of the di-iron center (Leu146, Asn149, Phe150 of cADO-1593, and Thr146, Leu148, Tyr150 of cADO-sll0208). The variants of cADO-1593 from Synechococcus elongatus PCC7942 and cADO-sll0208 from Synechocystis sp. PCC6803 were constructed, overexpressed, purified and kinetically characterized. The k cat values of L146T, Q49H/N123H/F150Y and W178R of cADO-1593 and L148R of cADO-sll0208 were increased by more than two-fold, whereas that of R62A dropped by 91.1%. N123H, Y39F and D143A of cADO-1593, and Y150F of cADO-sll0208 reduced activities by ≤ 20%.ConclusionsSome important amino acids, which exerted some effects on cADO activity, were identified. Several enzyme variants exhibited greatly reduced activity, while the k cat values of several mutants are more than two-fold higher than the wild type. This study presents the report on the relationship between amino acid residues and enzyme activity of cADOs, and the information will provide a guide for enhancement of cADO activity through protein engineering.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-017-0351-8) contains supplementary material, which is available to authorized users.

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“…Bacterial multicomponent monooxygenases (BMMs), found in methanotrophs and Gammaproteobacteria that thrive on hydrocarbons, exemplify here the O 2 utilizers. Comprised with BMM are soluble methane monooxygenase (sMMO), soluble toluene monooxygenase (sTMO) and related varieties, as well as phenol hydroxylase (PH) and cyanobacterial enzymes that carry out deformylation of aldehydes . All these metalloproteins are virtually colorless when accurately purified.…”

Section: Introductionmentioning

confidence: 99%

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all‐Atom Molecular Dynamics Investigation in Water Solution

Pietra

2016

Chemistry & Biodiversity

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In a preliminary exploration of the dummy model for diiron proteins, random-acceleration molecular dynamics (RAMD) revealed that a pure four-helix bundle structure, like hemerythrin, constitutes an efficient cage for dioxygen (O ), which can only leave from defined, albeit very broad, gates. However, this well ordered structure does not constitute an archetype on which to compare O permeation of other diiron proteins, like the complex of soluble methane monooxygenase hydroxylase with the regulatory protein (sMMOH-MMOB). The reason is that with this complex, unlike hemerythrin, the four helices of the four-helix bundle are heavily bent, and RAMD showed that most traps for O lie outside them. It was also observed that, in spite of a nearly identical van der Waals radius for O and the natural substrate CH , the latter behaves under RAMD as a bulkier molecule than O , requiring a higher external force to be brought out of sMMOH-MMOB along trajectories of viable length. All that determined with sMMOH-MMOB multiple gates and multiple pathways to each of them through several binding pockets, for both O and CH . Of the two equally preferred pathways for O , at right angle with one another, one proved to be in accordance with the Xe-atom mapping for sMMOH. In contrast, none of the pathways identified for CH proved to be in accordance with such mapping, CH looking for more open avenues instead.

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Insights into Substrate and Metal Binding from the Crystal Structure of Cyanobacterial Aldehyde Deformylating Oxygenase with Substrate Bound

Cited by 32 publications

References 49 publications

Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrier protein reductase

Structural insights into catalytic mechanism and product delivery of cyanobacterial acyl-acyl carrier protein reductase

Identification of residues important for the activity of aldehyde-deformylating oxygenase through investigation into the structure-activity relationship

On Dioxygen and Substrate Access to Soluble Methane Monooxygenases: An all‐Atom Molecular Dynamics Investigation in Water Solution

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