Biochemistry and Crystal Structure of Ectoine Synthase: A Metal-Containing Member of the Cupin Superfamily.

Nils Widderich,Andreas Seubert,Erhard Bremer,Ramona Riclea,Sander H J Smits,Jeroen S Dickschat,Stefanie Kobus,Johann Heider,Astrid Höppner,Andreas Hofmann

doi:10.1371/journal.pone.0151285

Abstract

Ectoine is a compatible solute and chemical chaperone widely used by members of the Bacteria and a few Archaea to fend-off the detrimental effects of high external osmolarity on cellular physiology and growth. Ectoine synthase (EctC) catalyzes the last step in ectoine production and mediates the ring closure of the substrate N-gamma-acetyl-L-2,4-diaminobutyric acid through a water elimination reaction. However, the crystal structure of ectoine synthase is not known and a clear understanding of how its fold contributes to enzyme activity is thus lacking. Using the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis (Sa), we report here both a detailed biochemical characterization of the EctC enzyme and the high-resolution crystal structure of its apo-form. Structural analysis classified the (Sa)EctC protein as a member of the cupin superfamily. EctC forms a dimer with a head-to-tail arrangement, both in solution and in the crystal structure. The interface of the dimer assembly is shaped through backbone-contacts and weak hydrophobic interactions mediated by two beta-sheets within each monomer. We show for the first time that ectoine synthase harbors a catalytically important metal co-factor; metal depletion and reconstitution experiments suggest that EctC is probably an iron-dependent enzyme. We found that EctC not only effectively converts its natural substrate N-gamma-acetyl-L-2,4-diaminobutyric acid into ectoine through a cyclocondensation reaction, but that it can also use the isomer N-alpha-acetyl-L-2,4-diaminobutyric acid as its substrate, albeit with substantially reduced catalytic efficiency. Structure-guided site-directed mutagenesis experiments targeting amino acid residues that are evolutionarily highly conserved among the extended EctC protein family, including those forming the presumptive iron-binding site, were conducted to functionally analyze the properties of the resulting EctC variants. An assessment of enzyme activity and iron content of these mutants give important clues for understanding the architecture of the active site positioned within the core of the EctC cupin barrel.

Highlights

Compatible solutes are exploited by members of all three domains of life as versatile cyto-protectants [1], in particular against cellular stress elicited by high osmolarity environments [2,3,4,5]
The two crystal structures that we report here for the (Sa)EctC protein, and data derived from extensive sitedirected mutagenesis experiments targeting evolutionarily highly conserved residues within the extended EctC protein family, provide a first view into the architecture of the catalytic core of the ectoine synthase
We focused our biochemical and structural studies on the ectoine synthase from S. alaskensis [(Sa)EctC], a cold-adapted marine ultra-microbacterium [40], from which we recently determined the crystal structure of the ectoine hydroxylase (EctD) in complex with either its substrate or its reaction product [27]

Summary

Introduction

Compatible solutes are exploited by members of all three domains of life as versatile cyto-protectants [1], in particular against cellular stress elicited by high osmolarity environments [2,3,4,5]. The ectoine derivative 5-hydroxyectoine, a highly effective stress protectant in its own right [22,23,24], is synthesized by a substantial subgroup of the ectoine producers [13, 14] This stereospecific chemical modification of ectoine (Fig 1) is catalyzed by the ectoine hydroxylase (EctD) (EC 1.14.11) [25, 26], a member of the non-heme containing iron(II) and 2-oxoglutarate-dependent dioxygenase superfamily [27]. The remarkable function preserving effects of ectoines for macromolecules and cells [28,29,30,31], frequently addressed as chemical chaperones, led to a substantial interest in exploiting these compounds for biotechnological purposes and medical applications [12, 32, 33]

Methods

Results

Conclusion