Elsevier

Biochimie

Volume 87, Issue 11, November 2005, Pages 1011-1022
Biochimie

High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

https://doi.org/10.1016/j.biochi.2005.04.006Get rights and content

Abstract

Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z. mays CKO1 cDNA was cloned in the yeast Yarrowia lipolytica to achieve heterologous protein expression. The recombinant ZmCKO1 was recovered from cultures of transformed yeasts and purified using several chromatographic steps. The enzyme was obtained as a homogeneous protein in a remarkably high-yield and its molecular and kinetic properties were characterized. The enzyme showed a molecular mass of 69 kDa, pI was 6.3. Neutral sugar content of the molecule was 22%. Absorption and fluorescence spectra were in accordance with the presence of FAD as a cofactor. Peptide mass fingerprinting using MALDI-MS correctly assigned the enzyme in MSDB protein database. The enzyme showed a relatively high degree of thermostability (T50 = 55 °C for 30 min incubation). The following pH optimum and Km values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (Km = 0.5 μM), pH 7.6 for isopentenyl adenosine (Km = 1.9 μM), pH 7.9 for zeatin (Km = 1.5 μM) and pH 7.3 for zeatin riboside (Km = 2.0 μM). ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the existence of dual enzyme functionality (oxygen serves as a cosubstrate in the absence of better electron acceptors).

Introduction

While cytokinin oxidase/dehydrogenase (CKO/CKX), which irreversibly degrades cytokinins, was partially purified from maize kernels as early as 1974 [1], the first gene in maize (ZmCKX1, EMBL/GenBank accession code AF044603) and its mRNA (ZmCKO1, EMBL/GenBank accession code Y18377) were isolated only in 1999 [2], [3]. The deduced protein sequence comprises 534 amino acids, including eight possible N-glycosylation sites and a putative signal peptide of 18 amino acids. The ZmCKX1/ZmCKO1-encoded protein has enzymatic activity, and is secreted and glycosylated [2], [3]. Another CKO from Zea mays (ZmCKO3) was also shown to be secreted, when expressed in its native form (with its native signal peptide) in the yeast Yarrowia lipolytica [4].

ZmCKO1 contains FAD as a cofactor covalently bound to the conserved His105 residue of a GHS motif [5]. Recently Malito et al. [6] published the crystal structure of ZmCKO1 expressed in the yeast Pichia pastoris showing that FAD is linked through the 8-methyl group of the flavin ring. The ZmCKO1 molecule exhibits a topology defined by cofactor and substrate domains. Two active-site residues, Asp169 and Glu288, seem to be crucial for both cytokinin binding and enzyme action.

The enzyme, which oxidatively degrades cytokinins in plants, has been named cytokinin oxidase since the dawn of its study [7]. It was suggested and later demonstrated that the reaction could not proceed without oxygen [1], [8]. In 1999, two independent groups reported that CKO is able to reduce 2,6-dichlorophenol indophenol (DCPIP) as an electron acceptor [2], [9]. Later, Galuszka et al. [10] reported that oxygen is not required and hydrogen peroxide is not produced during the catalytic reaction of purified CKOs from wheat and barley grains. They also proposed to reclassify the enzyme to cytokinin dehydrogenase and this has been approved (EC 1.5.99.12). Moreover, H2O2 could not be detected in activity assays performed with a recombinant maize enzyme [11].

When isopentenyl adenine (iP) undergoes oxidation by CKO, adenine and 3-methyl-2-butenal are formed as reaction products [12]. Substrate degradation proceeds via an unstable imine intermediate [13]. During the first half reaction, the oxidized FAD cofactor (having absorption maxima at around 360 and 450 nm) is reduced to FADH2 via two-electron transfer that leads to a temporary bleaching and the subsequent appearance of a new maximum at 315 nm. Reoxidation of the reduced FAD is achieved in two ways. In the oxidase mode, electrons are transferred to oxygen and hydrogen peroxide is expected to be released. Alternatively, in the dehydrogenase mode, electrons are transferred to other electron acceptors, e.g. quinones [14]. The reductive half reaction is fast (k = 950 s–1 for iP), therefore, the reoxidation of FAD seems to be the rate limiting step in the catalytic cycle. In the presence of different artificial electron acceptors, iP shows the best substrate properties (kcat/Km) [5], [14]. Some diphenylureas, which are known as potent cytokinin agonists, inhibit CKO activity [8], [13] in a competitive manner towards substrates [5].

We recently reported the crystallization of recombinant ZmCKO1 expressed in the yeast Y. lipolytica [15]. Since its expression and its purification were reported only briefly, we describe here in detail all steps concerning cloning, high-level expression and purification. Additionally, other enzyme characteristics such as pI, thermostability (T50), pH optima and Km values for natural substrates were determined. The recombinant ZmCKO1 was also digested by trypsin or chymotrypsin and the respective peptide fragments were analyzed by mass spectrometry. For the first time, the reaction stoichiometry of CKO functioning in the oxidase mode (i.e. in the absence of any artificial electron acceptor) was determined. It emerged that the obtained stoichiometric ratio was significant for the confirmation of dual enzyme functionality.

Section snippets

Chemicals and proteins

Isopentenyl adenine (iP), isopentenyl adenosine (iPR), zeatin riboside (ZR), 4-aminophenol, bicinchoninic acid–protein assay kit, 2,6-dichlorophenol indophenol (DCPIP) and FAD sodium salt were purchased from Sigma-Aldrich Chemie (Steinheim, Germany). Zeatin (Z) was from ICN Biochemicals (Cleveland, OH, USA). All other chemicals were of analytical purity grade.

Bovine serum albumin (BSA), catalase from bovine liver, glucose oxidase (GOD) from Aspergillus niger, horseradish peroxidase (HRP),

Production of recombinant ZmCKO1

The putative 18 a.a.-long signal peptide of ZmCKO1 precursor protein (as predicted by PSORT and TargetP programs) was replaced by the prepro region (XPR2 prepro) of Y. lipolytica alkaline extracellular protease gene in order to direct the secretion of mature protein into the yeast culture medium. ZmCKO1 expression was under control of the recombinant promoter hp4d in the expression/secretion vector pINA1267 [16]. A scheme of the corresponding ZmCKO1-producing plasmid (pINA6703) is given in Fig.

Discussion

The kernel is a major source of CKO in maize despite the low content of active enzyme. This has to be correlated with a low concentration of gene transcripts even if, among the CKO multigene family, CKO1 is predominantly expressed in the kernel [4]. Therefore, heterologous expression was highly desirable on the way to obtaining reasonable amounts of pure ZmCKO1.

A fusion protein comprising 37 N-terminal amino acids, which originated from the pBluescript sequence upstream of ZmCKO1 protein

Conclusions

ZmCKO1 expressed in Y. lipolytica may be obtained as a homogeneous protein in a high yield. Upon purification, the recombinant enzyme shows a high value of specific activity, when compared to lengthy and difficult isolation of authentic CKOs from plant material, which yields only negligible amounts of purified protein. ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the

Acknowledgments

We thank Dr. Henrik Thomas from Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany as well as Dr. Christian Malosse from Unité de Phytopharmacie et Médiateurs Chimiques, INRA, Versailles, France for their assistance in MALDI-MS measurements. This work was supported in part by the grants MSM 6198959216 and MSM ME 664 from the Ministry of Education, Youth and Sports, Czech Republic. Research stay of D. Kopečný at INRA, Versailles, France, was supported by a French

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    Permanent address: Department of Biochemistry, Faculty of Science, Palacký University, Šlechtitelů 11, CZ-783 71 Olomouc, Czech Republic.

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