Redundant synthesis of cysteinyl-tRNACys in Methanosarcina mazei

Abstract

A subset of methanogenic archaea synthesize the cysteinyl-tRNA(Cys) (Cys-tRNA(Cys)) needed for protein synthesis using both a canonical cysteinyl-tRNA synthetase (CysRS) as well as a set of two enzymes that operate via a separate indirect pathway. In the indirect route, phosphoseryl-tRNA(Cys) (Sep-tRNA(Cys)) is first synthesized by phosphoseryl-tRNA synthetase (SepRS), and this misacylated intermediate is then converted to Cys-tRNA(Cys) by Sep-tRNA:Cys-tRNA synthase (SepCysS) via a pyridoxal phosphate-dependent mechanism. Here, we explore the function of all three enzymes in the mesophilic methanogen Methanosarcina mazei. The genome of M. mazei also features three distinct tRNA(Cys) isoacceptors, further indicating the unusual and complex nature of Cys-tRNA(Cys) synthesis in this organism. Comparative aminoacylation kinetics by M. mazei CysRS and SepRS reveals that each enzyme prefers a distinct tRNA(Cys) isoacceptor or pair of isoacceptors. Recognition determinants distinguishing the tRNAs are shown to reside in the globular core of the molecule. Both enzymes also require the S-adenosylmethione-dependent formation of (m1)G37 in the anticodon loop for efficient aminoacylation. We further report a new, highly sensitive assay to measure the activity of SepCysS under anaerobic conditions. With this approach, we demonstrate that SepCysS functions as a multiple-turnover catalyst with kinetic behavior similar to bacterial selenocysteine synthase and the archaeal/eukaryotic SepSecS enzyme. Together, these data suggest that both metabolic routes and all three tRNA(Cys) species in M. mazei play important roles in the cellular physiology of the organism.

FIGURE 1.

A, dependence of reaction rate on tRNA concentration for aminoacylation by M. mazei SepRS. Replot of concentration versus initial velocities determined from time courses. The inset shows the imaged TLC plate of a single time course for . B, the secondary structures of M. mazei tRNA^Cys isoacceptors , , and , are depicted from left to right. The 7-66 base pair at the bottom of the acceptor stem, which distinguishes from , is shown in boldface type on the cloverleaf at the center. Nucleotides distinguishing (left) from both and are shown in boldface type on the cloverleaf. Mutations of (right) made in this study are circled. The correspondence between the nucleotides and the mutations described under “Results and Discussion” and in Table 1 is as follows: tRNA^CysΔ2, introduction of A33 from into ; tRNA^CysΔ3, introduction of A57 from into ; tRNA^CysΔ5, introduction of both C49 and U51 from into ; tRNA^CysΔ4, introduction of the five D-loop and variable loop nucleotides U20, C21, U44, A46, and G47 from into .

FIGURE 2.

Time course for plateau cysteinylation by M. mazei CysRS in the absence or presence of ^m1G37 in . The effect of methylating and is nearly identical (not shown).

FIGURE 3.

A, UV-visible absorbance spectrum of SepCysS revealing the presence of bound PLP in the overexpressed recombinant enzyme. B, imaged TLC plate showing time-dependent formation of Cys-tRNA^Cys catalyzed by SepCysS. Conditions were as follows: 50 mm Tris (pH 7.5), 20 mm KCl, 10 mm MgCl₂, 5 mm ATP, 3 mm phosphoserine, 5 mm DTT, 10 μm tRNA^Cys, and 20 μm SepRS. After 5 min, Cys-tRNA^Cys formation was initiated by the addition of SepCysS to 20 μm and sodium sulfide to 5 mm. The percentage conversion to Cys-tRNA^Cys is determined by the ratio of the intensities for Sep-AMP and Cys-AMP and converted to molarity by multiplying by the concentration of Sep-tRNA^Cys determined from the plateau aminoacylation value. C, plot of the velocity of Cys-tRNA^Cys synthesis catalyzed by SepCysS versus enzyme concentration under steady-state conditions. No activity is observed at enzyme concentrations below 50 nm. A constant concentration of 5 μm was used.

FIGURE 4.

A, time courses of Cys-tRNA^Cys synthesis. Sigmoidal behavior is observed under conditions of enzyme excess. The conditions used were 0.10–30 μm SepRS as indicated by the different symbols, 5 mm sodium sulfide, and 5 μm Sep-tRNA^Cys. Error bars are omitted for clarity but are similar to those depicted in Fig. 3C. B, plot of the decrease in Sep-tRNA^Cys concentration together with Cys-tRNA^Cys formation. Quantitation of spots on the imaged TLC plate resulting in concentration of each species present was plotted against time. The conditions were as follows: 50 mm Tris (pH 7.5), 20 mm KCl, 10 mm MgCl₂, 5 mm ATP, 3 mm phosphoserine, 5 mm DTT, 6 μm Sep-tRNA^Cys, and 20 μm SepCysS. A small amount of an intermediate species is observed that forms quickly and then disappears as Cys-tRNA^Cys formation increases.

FIGURE 5.

Proposed mechanism for the synthesis of Cys-tRNA^Cys by SepCysS. The mechanism is based upon a proposed sulfur relay system and on similarity to Sec-tRNA^[Ser]Sec biosynthesis. The movement of electrons into PLP has been omitted. The inclusion of bisulfide as the sulfur donor indicates how the enzyme functions in this in vitro study (bottom right; the PLP generated would reform the internal aldimine with Lys²²²). The identification of Lys²²² as the likely side chain that ligates PLP is based on a structure-based sequence alignment with A. fulgidus SepCysS.