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Co-occurrence of both l-asparaginase subtypes in Arabidopsis: At3g16150 encodes a K+-dependent l-asparaginase

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Abstract

l-asparaginases (EC 3.5.1.1) are hypothesized to play an important role in nitrogen supply to sink tissues, especially in legume-developing seeds. Two plant l-asparaginase subtypes were previously identified according to their K+-dependence for catalytic activity. An l-asparaginase homologous to Lupinus K+-independent enzymes with activity towards β-aspartyl dipeptides, At5g08100, has been previously characterized as a member of the N-terminal nucleophile amidohydrolase superfamily in Arabidopsis. In this study, a K+-dependent l-asparaginase from Arabidopsis, At3g16150, is characterized. The recombinants At3g16150 and At5g08100 share a similar subunit structure and conserved autoproteolytic pentapeptide cleavage site, commencing with the catalytic Thr nucleophile, as determined by ESI-MS. The catalytic activity of At3g16150 was enhanced approximately tenfold in the presence of K+. At3g16150 was strictly specific for l-Asn, and had no activity towards β-aspartyl dipeptides. At3g16150 also had an approximately 80-fold higher catalytic efficiency with l-Asn relative to At5g08100. Among the β-aspartyl dipeptides tested, At5g08100 had a preference for β-aspartyl-His, with catalytic efficiency comparable to that with l-Asn. The phylogenetic analysis revealed that At3g16150 and At5g08100 belong to two distinct subfamilies. The transcript levels of At3g16150 and At5g08100 were highest in sink tissues, especially in flowers and siliques, early in development, as determined by quantitative RT-PCR. The overlapping spatial patterns of expression argue for a partially redundant function of the enzymes. However, the high catalytic efficiency suggests that the K+-dependent enzyme may metabolize l-Asn more efficiently under conditions of high metabolic demand for N.

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Abbreviations

Ntn:

N-terminal nucleophile

GUS:

β-Glucoronidase

MES:

2-(N-morpholino)ethanesulfonic acid

AGI:

Arabidopsis genome initiative

ABRC:

Arabidopsis Biological Resource Center

IPTG:

Isopropyl β-D-1-thiogalactopyranoside

DAA:

Day after anthesis

FW:

Fresh weight

DEPC:

Diethyl pyrocarbonate

TAIR:

The Arabidopsis Information Resource

NCBI:

National Center for Biotechnology Information

TIGR:

The Institute for Genome Research

CBR:

Canadian Bioinformatics Resource

LSD:

Least significant difference

EST:

Expressed sequence tag

HsGA:

Human glycosylasparaginase

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Acknowledgements

We thank the ABRC (Ohio State University, Colombus, OH) for the At5g08100 cDNA clone, and Dr. Soon Park (Agriculture and Agri-Food Canada, Greenhouse and Processing Crops Research Centre, Harrow, Ontario) for seeds of white navy bean (P. vulgaris) cv. AC Compass. We also thank Alex Molnar for his expert graphical assistance in the preparation of figures.

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  1. Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, 1391 Sandford St, N5V 4T3, London, Ontario, Canada

    Luanne Bruneau, Ralph Chapman & Frédéric Marsolais

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  1. Luanne Bruneau
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  2. Ralph Chapman
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Correspondence to Frédéric Marsolais.

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Bruneau, L., Chapman, R. & Marsolais, F. Co-occurrence of both l-asparaginase subtypes in Arabidopsis: At3g16150 encodes a K+-dependent l-asparaginase. Planta 224, 668–679 (2006). https://doi.org/10.1007/s00425-006-0245-9

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