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'''Cytokinin deshydrogenase'''([http://en.wikipedia.org/wiki/Cytokinin_dehydrogenase Wikipedia]), also called CKX, is an enzyme which '''degrades cytokinin''' ([http://en.wikipedia.org/wiki/Cytokinin Wikipedia]) '''hormones''' in plants and which is encoded by the gene '''ZmCKX1'''. The one is located in '''Zea Maize''' on the chromosome 3 (NCBI Gene ID: 542585) and belongs to a multigene family called '''V'''anillyl-'''A'''lcohol '''O'''xidase (VAO) flavoprotein family. The gene is more particularly expressed in the kernel of maize, mainly in the embryo since it may protect the embryo from too much cytokinin and permit the correct development. <ref name="Frebortova">PMID: 19912568 </ref>

Cytokine deshydrogenase are '''extracellular''' and '''monomeric''' proteins with a molecular weight of 63kDa.<ref name="Kopečnýa">PMID: 20478354 </ref><ref name="Kopečnýa2">PMID: 18571199 </ref> For protein production purposes, ZmCKO1 precursor protein was truncated by deletion of 18 N-terminal amino acids to produce the expected mature enzyme. <ref name="Kopečnýa3">PMID: 15927342 </ref>

The Enzyme Classification number of CKX is EC 1.5.99.12 and this indicates that the enzyme is an '''oxydo reductase''' which acts on the CH-NH group of the donor. Consequently the reaction of CKX with its cytokinin substrate is a '''transfer of two electrons''' from the cytokinin to an '''electron acceptor''' which is in the case of CKX the '''F'''lavine '''A'''denine '''N'''ucleotide (FAD) cofactor. <ref name="Kopečnýa">PMID: 20478354 </ref>

In some papers the denomination '''CKO''' can be found for cytokinin deshydrogenase. Indeed in the vanillyl-alcohol oxidase flavoprotein family most of the enzyme use '''molecular oxygen''' as electron acceptor to '''reoxidize''' the FAD cofactor. That’s why the enzyme was first called '''Cytokinin Oxidase''' (CKO). CKX is an exception in the family since the enzyme uses '''other compounds''' ,such as quinone, for electron acceptor and poorly reacts with oxygen. Consequently the enzyme is now called '''CKX''' and enters the category of '''dehydrogenase'''. <ref name="Frebortova">PMID: 19912568 </ref> <ref name="Malitoa">PMID: 15321719 </ref>

== '''Structure & Function''' ==

=== '''Motifs & Domains''' ===

CKX has a '''two main domains''' structure, with an '''FAD-binding domain''' (residues 33–244 and 492–534) and a '''substrate-binding domain''' (residues 245–491). <ref name="Kopečnýa">PMID: 20478354 </ref>

The active site contains very '''highly conserved residues''' except for one residue which is located at the entrance of the active site (where a few amino acids such as Glu, Asp, Ser, Gly and other aliphatic amino acids can be found). <ref name="Kopečnýa">PMID: 20478354 </ref>

Three residues, Asp169, Glu288 and Glu381 may have an importance in '''cytokinin binding''' and '''enzyme action''' <ref name="Kopečnýa3">PMID: 15927342 </ref>

The presence of a conserved '''GHS''' at position 104-106 domain has been shown in the '''FAD binding domain''' which may have been highlighted in CKX structure like GlWeVPHPWLNL motif around position 390 and PGQxIF signature at the C-terminal ends. These sequences are specifically found in CKX family enzymes and their '''very high conservation inside the family''' shows that they have an important role for the enzyme functioning, such as in this case '''substrate recognition and electron transport'''. <ref name="Schmülling">PMID: 12721786 </ref>

==='''Three-dimensional structure '''===

===='''Catalytic centre'''====

Thanks to a X-ray analysis the architecture of the '''catalytic centre''' has been determined in details. The one is made of a '''funnel-region''' on the surface of the protein and an internal cavity where we can find the '''flavine ring'''. The cavity has a volume of about 400 Angtröm and in addition to the FAD it will also be able '''to bind the reactive part of the substrate'''. This cavity has a connection to the outside thanks to a '''narrow pore''' which has an entrance containing '''three conserved amino acids''': Asp169, Trp397 and Leu458.

The funnel region and the cavity are also connected thanks to a '''pore''' with a 4 Angström diameter. <ref name="Malitoa">PMID: 15321719 </ref>

===='''FAD cofactor binding domain'''====

The FAD cofactor has a '''covalent''' way of binding with the enzyme. The attachment takes place at the His105 in the '''GHS domain''' of the enzyme. The one binds the '''8-methyl group of the flavin ring''' (isoalloxazine ring).

The '''two negative charges''' brought by the '''pyrophosphate of the FAD cofactor''' is compensated by the '''nitrogen atoms''' of a few amino acids such as Gly102, Arg103, Gly104, His105, Ser106 and Thr174. <ref name="Malitoa">PMID: 15321719 </ref>

===='''Substrate binding domain'''====

The substrate binds by following a '''“plug-into-socket”''' mode and this way of binding will cause no conformational change during binding at the active site in contrary to a lot of other dehydrogenases and oxidases. This binding mode will '''seal the active site'''. In order to have no need of movements the CKX enzyme shows a very good pre-organisation and complementary with the cytokinin substrate. There are three amino acids which play a key role in the enzyme activation: Asp169, Glu288 and Glu381. The Asp169 residue of the enzyme is '''Hydrogen-bonded to the N10 atom of the substrate''' (various studies have shown that it is involved in the '''recognition of the substrate''') and to the Glu288 residue. The Asp169-Glu288 pair implies that a '''proton is shared between the two side-chains'''. We can’t know the exact protonation state but the pair suppose Asp169 is an active-site base and possibly involved in proton abstraction from the substrate N10 atom. The substrate is also recognized by Glu381 which is '''Hydrogen-bonded to the N7 atom of the cytokinin'''. The others N-atoms are Hydrogen-bonded '''with the solvent except''' for N1 atom. The end of the substrate aromatic chain is located between the N10 atom of FAD, Leu492 and Asn399. These interactions allow the positioning the '''C11 atom of the cytokinin substrate''' very close to the '''flavin N5 atom''' (at about 3.0 A°). Indeed it is this carbon atom which will be the site of the '''oxydative attack of the cytokinin by the FAD cofactor''' <ref name="Malitoa">PMID: 15321719 </ref> <ref name="Kopečnýa">PMID: 20478354 </ref>

=='''Mechanism'''==

The positioning of the C11 close to the N5 of the flavine suggests that electrons and protons are exchanged through a '''direct transfer from the substrate to the flavine'''. This implies a '''short-lived radical intermediate''' or the direct tranfer of a '''hydride anion'''.<ref name="Malitoa">PMID: 15321719 </ref> The attack of the N5 to the C11 create a '''carbocation''', on the cytokinin, which is stabilized by the N10–Asp 169 Hydrogen-bond interaction and the resonance effect in the oxidised imine product.

The reduced enzyme-product complex (FADH2) is '''reoxidised''' by using an '''organic electron acceptor''', the hydroxamic acid 2,4-dihydroxy-7-methoxy-1,4-benzoxazin-3-on (DIMBOA) or more precisely, the free radicals generated by the reaction of laccase and peroxydase on the DIMBOA. The reoxidation of FADH2 allows the product (adenine and 3 methyl 2 butanal) release.<ref name="Malitoa">PMID: 15321719 </ref>

=='''Regulation'''==

The reductive part of reaction is '''fast''', as a consequence it is the '''reoxidation of the FAD cofactor''' which constitute the '''rate limiting step''' for the catalytic action of the CKX enzyme. <ref name="Kopečnýa3">PMID: 15927342 </ref> In absence of organic electron acceptor, oxygen can be used but it is a poor electron acceptor, so this shows that the regeneration of free radicals is an important step. The coupling of CKX with laccase or peroxidase involves that the regulation of the CKX activity is '''highly dependent of the regulation of these two enzymes.'''

Different in-vivo '''competitive inhibitors''' of the CKX have been identified such as diphenylureas and derivatives such as CPPU and thidiazuron <ref name="Frebortova">PMID: 19912568 </ref>, which are known as cytokinin agonists <ref name="Kopečnýa3">PMID: 15927342 </ref> or Urea-type cytokinins.<ref name="Kopečnýa">PMID: 20478354 </ref>

Another type of regulation is made by '''glycosylation''' : CKX has indeed '''five asparagine''' residues which can be glycosylated: Asn63, Asn134, Asn294, Asn323 and Asn338. Protein glycosylation is particularly important since it regulates the '''enzymatic activity''' and the '''protein stability''' <ref name="Kopečnýa">PMID: 20478354 </ref><ref name="Schmülling">PMID: 12721786 </ref> and it was suggested, it has a role for '''enzyme localization'''. <ref name="Frebortova">PMID: 19912568 </ref>

It has been shown that the cytokinin has a '''positiv feedback''' on the CKX activity. This indicates that the gene expression or the enzyme activity is '''enhanced by its substrate'''. It has also been shown that phyto-hormon auxin activates CKX activity and in contrary abscisic acid inhibits its activity but the relation has not been observed in the whole plant but only in certain tissues. <ref name="Frebortova">PMID: 19912568 </ref>

== '''References''' ==

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