Structural and biochemical characterization of the type-II LOG protein from Streptomyces coelicolor A3

Highlights

• Crystal structure of type-II LOG from Streptomyces coelicolor A3 (ScLOGII) was determined.

• The substrate binding mode of type-II LOG is suggested by docking calculations of AMP into the ScLOGII.

• ScLOGII has a long tail-like structure at the N-terminus that is involved in hexamerization.

Abstract

Streptomyces coelicolor A3 contains Sc5140, a gene coding for poorly understood bacterial LOG-like protein. In this study, we determined the crystal structure of Sc5140 and found it resembles the overall structure of other type-II LOGs. In addition, Sc5140 exhibited phosphoribohydrolase activity against adenosine monophosphate (AMP), indicating that it had the same function as known type-II LOGs. Based on these results, we designated Sc5140 as ScLOGII. We performed docking calculations of AMP into the ScLOGII structure, which suggested the mode of binding for type-II LOG with their AMP substrate. The ScLOGII structure uniquely exhibited a long tail-like structure at the N-terminus that was involved in hexamerization of the protein; the disordered N-terminal region (DNR). Truncation of DNR in ScLOGII negatively affected both the phosphoribohydrolase activity and the oligomerization of the protein, suggesting that this region functioned in enzyme stabilization. However, results from truncation experiments using ScLOGII and CgLOGII, a type-II LOG homologue from Corynebacterium glutamicum, were quite different, leaving uncertainty regarding the general functions of DNR in type-II LOGs. Overall, the current structural work may help in understand the significance of type-II LOG protein at the molecular level.

Introduction

Purine-derived cytokinins are N⁶-substituted adenines that are phytohormones with broad and important roles in the physiological processes of plants, such as root growth and branching [1], delay of senescence [2], chloroplast development [3], and the activation of pathogen-defense-related pathways [4]. Cytokinins are often conjugated with sugar moieties, such as nucleotides, nucleosides, and glucosides, but these forms are biologically less active or are inactive for plant cytokinin receptors [5]. Being released from their conjugates is the cytokinin-activating step, and is a key target for controlling active cytokinin levels. “Lonely guy” (LOG) was identified as an enzyme involved in this activation through direct hydrolysis of bonds between bases and phosphoribose moieties of cytokinin nucleotides in plants [6]. In addition to being plant enzymes, LOG-like proteins are also highly conserved in a variety of prokaryotes. The study of bacterial LOGs so far has been limited because they are also considered possible lysine decarboxylases [7,8].

Streptomycetes species compose the largest genus of Actinobacteria, and are well known for their multicellular mycelial life cycle, and antibiotic production from their complex secondary metabolism [9]. Antibiotics from Streptomycetes account for half of all the worlds' clinical antibiotics from natural origins [10]. In nature, Streptomycetes sp. inhabit soil, and are commonly saprophytic with many of them existing as endophytes and pathogens, especially of plants [11,12]. These are some of the most important groups for disclosing the ecosystems of microorganisms that exist in the ground. Streptomyces coelicolor A3 is a prominent model strain of Streptomycetes. As a soil dweller exposed to a wide variety of environmental conditions, S. coelicolor has many elaborate regulatory mechanism for environmental adaptation in relation to its unique existence [13]. Likewise, its genome has an abundancy of regulatory genes that respond to external stimuli and stresses [14].

Interestingly, S. coelicolor contains three genes that encode LOG-like proteins (Sc5140, Sc5491, and Sc5651), implying the microbial production of cytokinin and the interactions with plants. Among the LOG isoforms, the gene products of Sc5491 and Sc5140 have the highest homology to type-I and type-II LOGs, respectively. Type-I LOGs were the first LOGs characterized in plants to be cytokinin-activating enzymes, and to be physiologically active as a dimer [6,8,15]. In comparison, type-II LOGs adopt a unique hexamer formation through an additional α-helix [7,16]. The type-II LOGs have significantly different substrate-binding sites compared to those of type-I LOGs [16]. However, heterogeneous overexpression of type-II LOG in Escherichia coli produced detectable amounts of isopentenyladenine, a typical cytokinin, suggesting that type-II LOG may also function as cytokinin-activating protein [16]. This is highlighted by the differences in the prenyl-group binding sites, which implies that their optimum activities for substrates are likely to vary depending on the specific type of cytokinins. In this report, we describe the crystal structure of ScLOGII from S. coelicolor, which reveals a unique N-terminal structure. The functional implication of the N-terminal region is discussed.