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| Rossmann fold is a super-secondary structure that is characterized by an alternating motif of beta-strand-alpha helix-beta strand secondary structures. Hence this fold is also called a βαβ fold. The β-strands participate in the formation of a β-sheet. The βαβ fold structure is commonly observed in enzymes that have dinucleotide coenzymes, such as FAD, NAD and NADP.
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| ==History==
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| [[Image:FAD-NADH-Structures.png|500px|right|thumb| Fig. 1. Structures of FAD and NADH in vertical orientation.]]
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| In 1973 Rao and Rossmann reported that a βαβ fold super-secondary structure commonly appears in a variety of nucleotide binding proteins, such as lactate dehydrogenase and flavodoxin <ref name="R-R">PMID:4737475</ref>. In later studies this common structure was named after the author Michael G. Rossmann as Rossmann fold.
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| The βαβ fold structure was initially characterized in dinucleotide FAD and NADH binding proteins. Schulz et al. examining FAD binding domains of four enzymes noted that the βαβ fold structure was associated with a specific consensus sequence of Gly-x-Gly-x-x-Gly at the region of the tight loop between the first β-strand the α-helix <ref>PMID:7175934</ref>. Wierenga et al. systematically examined more structures and derived rules for a fingerprint sequence named "ADP-binding βαβ fold" <ref>PMID:3959077</ref>.
| | == '''Overview''' == |
| | '''Phosphotriesterase-Like Lactonase (PLL)''' family includes a group of enzymes that have main lactonase activity on lactones and acyl-homoserin lactones (AHLs) and, in addition, low promiscuous phosphotriesterase activity towards organophosphates compound (OPs). At the beginning most of them has been identified as putative phosphotriesterases and were called "Paraoxonases" (Pox) because able to degrade pesticides such as paraoxon <ref name='Merone'>PMID: 15909078</ref> <ref name='Porzio'>PMID:17337320</ref>. However, further structural, phylogenetic, and biochemical studies have revealed that these enzymes have a proficient lactonase activity, beside the weak phosphotriesterase activity <ref name='Afriat'>PMID:17105187</ref>. |
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| In 1989, Israel Hanukoglu found that NADPH binding βαβ fold in NADP dependent enzymes is characterized by a specific consensus sequence (briefly: Gly-x-Gly-x-x-Ala) that differs from the NADH binding site by one residue, with an alanine instead of the last glycine, and hypothesized that this single residue difference may determine the coenzyme specificity of the enzymes <ref name="HI-1989">PMID:2924777</ref>. Richard Perham and his colleagues confirmed this hypothesis by site-directed mutagenesis of glutathione reductase and showed that coenzyme specificity could be re-engineered from NAD to NADP <ref>PMID:2296288</ref>. The structural significance of the Ala for Gly substitution in NADP binding site was revealed by analysis of the crystal structure of NADP-dependent adrenodoxin-reductase <ref name="Ziegler-1999">PMID:10369776</ref>.
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| ==Dinucleotides that bind to Rossmann fold==
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| The βαβ fold is observed in numerous dinucleotide binding enzymes. The term "dinucleotide" may have two meanings: It may refer to an oligomer of two nucleotides such as A-G. In the present context, the term dinucleotide refers to a coenzyme that contains two distinct nucleotides. To emphasize the common structural aspects, the structures of two dinucleotides, FAD, NADH are shown in Figure 1.
| | == '''SsoPox''' == |
| | Sso Pox is a protein of 314 aa deriving from the hyperthermophilic archaeon ''Sulfolobus solfataricus'' and it is the first protein with phosphotriesterase activities to be identified in Archaea. It has an exceptional thermal stability with denaturation half-life of 4h and 90 min at 95 °C and 100 °C <ref name="Merone"/><ref name='Porzio'/>. |
| | Its activity depends on the presence of metal ions, with cobalt significantly enhancing catalysis. SsoPox have been reported to catalyse the hydrolysis of different N-acyl homoserine lactones AHLs; suggesting a physiological role as a quorum quencher lactonase. Infact the AHLs are natural molecules involved in the cell–cell communication process known as quorum sensing (QS) and any bacterial species may produce different AHLs, which vary in the length and substitution of the acyl chain. The anti-QS mechanisms of the enzyme works by the hydrolysis of the lactone bond of these AHLs. <ref name='Afriat'/> |
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| Note that both dinucleotides share at their base the common structure of adenosine diphosphate (ADP). The structure of FAD can be viewed either as a hybrid of AMP+FMN or as ADP+Riboflavin (see figure).
| | == References == |
| | | <references/> |
| NAD(P) is a two electron acceptor and donates the two electrons to FAD. The transfer of electrons takes place from C4 of NAD(P) to N5 of FAD. Both of these atoms are marked by their respective number in Figure 1.
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| ==Contact region between Rossmann fold and FAD==
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| [[Image:FAD-binding-sites.png|500px|right|thumb| Fig. 2. FAD binding sites of D-amino acid oxidase (2E48) and glutathione reductase (3GRS). FAD is shown in CPK mode.]]
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| In dinucleotide binding flavoproteins, FAD binding Rossmann fold is commonly located close to the amino terminus of the protein. Figure 2 shows the first Rossmann fold of two flavoproteins, D-amino acid oxidase (2e48)<ref>PMID:17303072</ref> and glutathione reductase (3GRS)<ref>PMID:3656429</ref>. In both enzymes, the first β-strand is followed by a tight loop that is connected to the N-terminal of the helix. The two highly conserved Gly residues in the consensus sequence are located in this turn to allow the sharp bending of the chain. At the end of the helix there is a wider turn that is followed by the second beta strand that runs parallel to the first strand.
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| FAD structure is shown in CPK format. The atoms can be identified by their colors: C: grey; O: red, P: orange and N: purple. The turn at β-α border is in contact with the negatively charged oxygens (red colored) of the two phosphate (orange colored) groups.
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| To see the locations of the conserved glycines in the consensus sequence, click the following residues
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| Gly-x-Gly-x-x-Gly
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| ==Contact region between Rossmann fold and NAD(P)==
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| [[Image:NAD-binding-sites.png|500px|right|thumb| Fig. 3. NAD binding sites of 3-phosphoglycerate dehydrogenase (2P9E) and lactate dehydrogenase (1I0Z). NAD is shown in CPK mode..]]
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| Figure 3 shows the Rossmann fold of two NAD binding proteins, 3-phosphoglycerate dehydrogenase (2P9E)<ref>PMID:17459882</ref> and lactate dehydrogenase (1I0Z)<ref>PMID:11276087</ref>. In enzymes that have just an NAD binding site, the site may be close to the N terminus of the protein as in lactate dehydrogenase. In flavoproteins that bind two dinucleotides, such as glutathione reductase and adrenodoxin reductase <ref name="HI-1989" />, the NAD(P) binding site appears in the middle of the protein.
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| The βαβ fold has a structure similar to the fold shown above for FAD. For both enzymes, the first β-strand is followed by a tight turn that is connected to the N-terminal of the helix. The same Gly-x-Gly-x-x-Gly consensus sequence appears at the turn between the first strand and the helix. Again, similar to FAD site, the turn region is in contact with the negatively charged oxygens (red colored) of the two phosphate (orange colored) groups.
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| ==A βαβ fold example in ferredoxin reductase ==
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| To illustrate a βαβ fold in a complete protein, a 3D example (PDB ID: 1F3P) is shown below. The example protein is a ferredoxin reductase from Pseudomonas that binds both an FAD and NADH <ref>PMID:11090282</ref>. This enzyme binds NADH which transfers its two electrons to the FAD coenzyme of ferredoxin reductase. These electrons are then transferred to a ferredoxin that is an iron sulfur electron transfer protein. This ferredoxin then donates the electrons to an oxygenase that uses the electrons in a dioxygenase reaction.
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| Below the two core β-strands of the FAD binding site of the enzyme (PDB ID: 1F3P) are shown in cyan colored "rocket" format, with a red colored helix in between the two strands.
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| <Structure load='1f3p' size='500' frame='true' align='left' caption='Fig. 4. FAD binding site of ferredoxin reductase. PDB ID: 1F3P.' scene='59/595757/Ferredoxin-reductase-fad/2' />
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| The following scenes illustrate some aspects of the structure.
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| To rotate the molecule click and hold left mouse button.
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| To zoom in or out use the mouse wheel.
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| Click the following green links for the action indicated:
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| : * <scene name='59/595757/Ferredoxin-reductase-fad-gly14/2'>Display the first conserved glycine in space filling CPK format at the end of the first beta-strand of the FAD binding site.</scene>
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| : * <scene name='59/595757/Ferredoxin-reductase-fad-gly16/4'>Display the second conserved glycine in space filling CPK format at the beginning of the helix of the FAD binding site.</scene>
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| : * <scene name='59/595757/Ferredoxin-reductase-fad-5-78/1'>Display the structure of the residues 5-78.</scene> Note that in between the second β-strand and the third one there are four α-helical segments.
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| : * <scene name='59/595757/Ferredoxin-reductase-full/3'>Display the full structure of ferredoxin reductase.</scene> Note that FAD has been colored a yellowish green, and NADP is shown also in CPK format that neighbors FAD.
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| {{clear}}
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| ==Extension of the beta sheet by additional strands==
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| [[Image:3-phosphoglycerate_dehydrogenase-2P9E-sheet.png|400px|right|thumb| Fig. 5. 3-phosphoglycerate dehydrogenase (2P9E) beta sheet in the NAD binding domain. The two beta-strands that form the core of the Rossmann fold are marked in dark-blue color.]]
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| As seen in the above example of ferredoxin reductase the β-sheet that is in the nucleotide domain may have more than two strands. In many (but not all) proteins with βαβ fold, the β-strands may be part of a larger β-sheet with up to seven β-strands. Figure 4 shows five strands forming a β-sheet in phosphoglycerate dehydrogenase (2P9E). Note that the segment connecting the second strand to the third is in coiled confirmation and not helical. Whereas the subsequent connections between strands include α-helix segments.
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| ==Evolutionary origin of the βαβ fold ==
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| A myriad of proteins include the βαβ Rossmann fold. Many of these proteins can be grouped in a hierarchy of families based on their sequence similarities <ref>PMID:11514662</ref>,<ref>PMID:8749365</ref>,<ref>PMID:17658942</ref>. Yet, many of these families do not show any significant sequence homology across families.
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| The observation that the βαβ structure and its consensus sequence is observed in many seemingly unrelated proteins raises the question whether the origin of the βαβ fold of all these proteins is a common ancestral sequence. Alternatively, there is also a possibility that this structure emerged in different proteins independently.
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| The basic nucleus of the βαβ fold is about 30 residues. In many proteins that do not share any significant sequence homology, there exists an extensive tertiary structural homology beyond this 30 residue segment, particularly in specific domains that bind dinucleotides. Therefore, the probability that this type of extensive structural homology evolved independently is very low. Thus, most likely βαβ fold represents an ancient structure that left its vestige in numerous proteins. Certainly, this conclusion does not exclude the possibility of independent convergent evolution.
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| ==References== | |
| <references /> | |