The structure of Medicago truncatula δ(1)-pyrroline-5-carboxylate reductase provides new insights into regulation of proline biosynthesis in plants.

Milosz Ruszkowski,Boguslaw Nocek,Giuseppe Forlani,Zbigniew Dauter

doi:10.3389/fpls.2015.00869

Abstract

The two pathways for proline biosynthesis in higher plants share the last step, the conversion of δ1-pyrroline-5-carboxylate (P5C) to L-proline, which is catalyzed by P5C reductase (P5CR, EC 1.5.1.2) with the use of NAD(P)H as a coenzyme. There is increasing amount of evidence to suggest a complex regulation of P5CR activity at the post-translational level, yet the molecular basis of these mechanisms is unknown. Here we report the three-dimensional structure of the P5CR enzyme from the model legume Medicago truncatula (Mt). The crystal structures of unliganded MtP5CR decamer, and its complexes with the products NAD+, NADP+, and L-proline were refined using x-ray diffraction data (at 1.7, 1.85, 1.95, and 2.1 Å resolution, respectively). Based on the presented structural data, the coenzyme preference for NADPH over NADH was explained, and NADPH is suggested to be the only coenzyme used by MtP5CR in vivo. Furthermore, the insensitivity of MtP5CR to feed-back inhibition by proline, revealed by enzymatic analysis, was correlated with structural features. Additionally, a mechanism for the modulation of enzyme activity by chloride anions is discussed, as well as the rationale for the possible development of effective enzyme inhibitors.

Highlights

In plant cells the function of proline goes much further than being a building block for proteins (Szabados and Savouré, 2010)
This work was focused on description of the main features of a plant P5CR from a functional and structural perspective
Even though P5CR sequences differ between species from different domains of life, we showed that the overall fold of MtP5CR is similar to those of the bacterial and human orthologs, which have had their threedimensional structures determined (Nocek et al, 2005; Pike et al, unpublished)

Summary

Introduction

In plant cells the function of proline goes much further than being a building block for proteins (Szabados and Savouré, 2010). Many studies have shown that free proline can trigger signal transduction pathways associated with different stress responses. The signaling function of proline was first observed in bacteria and was related to osmotic stress (Csonka, 1988; Csonka et al, 1988; Csonka and Hanson, 1991). Proline was reported to be a key player in plant adaptation to adverse environmental conditions (Verslues and Sharma, 2010), such as high salinity (Yoshiba et al, 1995), drought (Choudhary et al, 2005), abnormal doses of UV radiation (Saradhi et al, 1995), exposure to heavy metals (Schat et al, 1997), reactive oxygen species (Yang et al, 2009), and pathogens (Fabro et al, 2004; Haudecoeur et al, 2009). It was recently reported that free proline can regulate development, flowering and reproduction (Mattioli et al, 2008, 2009; Funck et al, 2012).

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