Structural basis of the substrate specificity of the FPOD/FAOD family revealed by fructosyl peptide oxidase from<i>Eupenicillium terrenum</i>

Gan, Weiqiong; Gao, Feng; Xing, Kun; Jia, Minghao; Liu, Haiping; Gong, Weimin

doi:10.1107/s2053230x15003921

Cited by 15 publications

(20 citation statements)

References 37 publications

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“…Their two‐domain architecture comprises a classic FAD‐binding motif formed by four α‐helices packed against two β‐sheets, and a catalytic domain formed by an 8‐stranded mixed β‐sheet and two long α‐helices, one of which is located on top of the active site. To date, the only known structures available of this family are the structure of Amadoriase II, both in its free form (PDB code: 3DJD) and in complex with the inhibitor fructosyl‐thyoacetate (FSA) (PDB code: 3DJE), and the structure of FPOX‐E in free form (PDB code: 4RSL) . However, the Amadoriase II in free form could only be partially traced in the electron density map, thus affording an incomplete structural model that did not allow some of the most crucial mechanistic details of the enzyme to be clarified.…”

Section: Resultsmentioning

confidence: 99%

“…The lack of such crucial piece of information has so far been one of the major limiting factors for the development of novel Amadoriase‐like enzymes for diagnostic or therapeutic applications. This structural limit has been overcome in part by the successful determination of the free and the inhibitor‐bound crystal structures of Amadoriase II from Aspergillus fumigatus, and by the more recent structure of the free fructosyl peptide oxidase from Eupenicillium terrenum (known as FPOX‐E or EtFPOD) which, to date, are the only known FAOX structures available in the literature. However, Amadoriase II has been reported to be mostly active on backbone fructosyl amines and on hydrophobic substrates (for example, glycated glycines), whereas FPOX‐E is active mostly on α‐fructosyl amino acids …”

Section: Introductionmentioning

confidence: 99%

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Crystal structure of the deglycating enzyme Amadoriase I in its free form and substrate‐bound complex

et al. 2016

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Amadoriases, also known as fructosyl amine oxidases (FAOX), are enzymes that catalyze the de-glycosylation of fructosyl amino acids. As such, they are excellent candidates for the development of enzyme-based diagnostic and therapeutic tools against age- and diabetes-induced protein glycation. However, mostly because of the lack of a complete structural characterization of the different members of the family, the molecular bases of their substrate specificity have yet to be fully understood. The high resolution crystal structures of the free and the substrate-bound form of Amadoriase I shown herein allow for the identification of key structural features that account for the diverse substrate specificity shown by this class of enzymes. This is of particular importance in the context of the rather limited and partially incomplete structural information that has so far been available in the literature on the members of the FAOX family. Moreover, using molecular dynamics simulations, we describe the tunnel conformation and the free energy profile experienced by the ligand in going from bulk water to the catalytic cavity, showing the presence of four gating helices/loops, followed by an "L-shaped" narrow cavity. In summary, the tridimensional architecture of Amadoriase I presented herein provides a reference structural framework for the design of novel enzymes for diabetes monitoring and protein deglycation. Proteins 2016; 84:744-758. © 2016 Wiley Periodicals, Inc.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Crystal structure of the deglycating enzyme Amadoriase I in its free form and substrate‐bound complex

et al. 2016

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“…A search for structurally similar proteins using the PDBeFold server yielded the fructosamine oxidase from A. fumigatus (PDB entry 4WCT ) ( 8 ) and the fructosyl peptide oxidase from Eupenicillium terrenum (PDB entry 4RSL ) ( 9 ) as the closest structural homologs with Q scores greater than 0.35 and root-mean-square deviations (RMSDs) of ∼2 Å. A superposition of FsqB with these two structures is shown in Fig.…”

Section: Resultsmentioning

confidence: 99%

Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family

Lahham

Pavkov‐Keller

Fuchs

et al. 2018

Journal of Biological Chemistry

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Flavin-dependent enzymes catalyze many oxidations, including formation of ring structures in natural products. The gene cluster for biosynthesis of fumisoquins, secondary metabolites structurally related to isoquinolines, in the filamentous fungus Aspergillus fumigatus harbors a gene that encodes a flavoprotein of the amine oxidase family, termed fsqB (fumisoquin biosynthesis gene B). This enzyme catalyzes an oxidative ring closure reaction that leads to the formation of isoquinoline products. This reaction is reminiscent of the oxidative cyclization reported for berberine bridge enzyme and tetrahydrocannabinol synthase. Despite these similarities, amine oxidases and berberine bridge enzyme–like enzymes possess distinct structural properties, prompting us to investigate the structure–function relationships of FsqB. Here, we report the recombinant production and purification of FsqB, elucidation of its crystal structure, and kinetic analysis employing five putative substrates. The crystal structure at 2.6 Å resolution revealed that FsqB is a member of the amine oxidase family with a covalently bound FAD cofactor. N-methyl-dopa was the best substrate for FsqB and was completely converted to the cyclic isoquinoline product. The absence of the meta-hydroxyl group, as e.g. in l-N-methyl-tyrosine, resulted in a 25-fold lower rate of reduction and the formation of the demethylated product l-tyrosine, instead of a cyclic product. Surprisingly, FsqB did not accept the d-stereoisomer of N-methyltyrosine, in contrast to N-methyl-dopa, for which both stereoisomers were oxidized with similar rates. On the basis of the crystal structure and docking calculations, we postulate a substrate-dependent population of distinct binding modes that rationalizes stereospecific oxidation in the FsqB active site.

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“…This limit has been overcome in part by the successful determination of the free and the inhibitor-bound crystal structures of Amadoriase II from Aspergillus fumigatus 13 , by the more recent crystal structure determination of the free fructosyl peptide oxidase from Eupenicillium terrenum (also known as FPOX-E or EtFPOD) 14 and, lately, by the crystal structure of the free and the substrate-bound form of Amadoriase I from the templates were selected based on a balance between: (i) the lowest expectation value (e-value) and (ii) the highest percentage of sequence identity. The e-value is an indicator that provides an estimation of the significance of the alignment (the lower the expectation value the higher the significance).…”

Section: Introductionmentioning

confidence: 99%

Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members

et al. 2016

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Enzymatic assays based on Fructosyl Amino Acid Oxidases (FAOX) represent a potential, rapid and economical strategy to measure glycated hemoglobin (HbA1c), which is in turn a reliable method to monitor the insurgence and the development of diabetes mellitus. However, the engineering of naturally occurring FAOX to specifically recognize fructosyl-valine (the glycated N-terminal residue of HbA1c) has been hindered by the paucity of information on the tridimensional structures and catalytic residues of the different FAOX that exist in nature, and in general on the molecular mechanisms that regulate specificity in this class of enzymes. In this study, we use molecular dynamics simulations and advanced modeling techniques to investigate five different relevant wild-type FAOX (Amadoriase I, Amadoriase II, PnFPOX, FPOX-E and N1-1-FAOD) in order to elucidate the molecular mechanisms that drive their specificity towards polar and nonpolar substrates. Specifically, we compare these five different FAOX in terms of overall folding, ligand entry tunnel, ligand binding residues and ligand binding energies. Our work will contribute to future enzyme structure modifications aimed at the rational design of novel biosensors for the monitoring of blood glucose levels.

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Structural basis of the substrate specificity of the FPOD/FAOD family revealed by fructosyl peptide oxidase fromEupenicillium terrenum

Cited by 15 publications

References 37 publications

Crystal structure of the deglycating enzyme Amadoriase I in its free form and substrate‐bound complex

Crystal structure of the deglycating enzyme Amadoriase I in its free form and substrate‐bound complex

Oxidative cyclization of N-methyl-dopa by a fungal flavoenzyme of the amine oxidase family

Molecular dynamics simulations provide insights into the substrate specificity of FAOX family members

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