Molecular evolution of gas cavity in [NiFeSe] hydrogenases resurrected in silico

Abstract

Oxygen tolerance of selenium-containing [NiFeSe] hydrogenases (Hases) is attributable to the high reducing power of the selenocysteine residue, which sustains the bimetallic Ni-Fe catalytic center in the large subunit. Genes encoding [NiFeSe] Hases are inherited by few sulphate-reducing δ-proteobacteria globally distributed under various anoxic conditions. Ancestral sequences of [NiFeSe] Hases were elucidated and their three-dimensional structures were recreated in silico using homology modelling and molecular dynamic simulation, which suggested that deep gas channels gradually developed in [NiFeSe] Hases under absolute anaerobic conditions, whereas the enzyme remained as a sealed edifice under environmental conditions of a higher oxygen exposure risk. The development of a gas cavity appears to be driven by non-synonymous mutations, which cause subtle conformational changes locally and distantly, even including highly conserved sequence regions.

Figure 1. Decoding an in-frame opal codon (UGA) by selenosome.

Sec is directed by the in-frame opal codon (UGA), and is synthesized on a specific tRNA^UGA (selC product) that is initially charged with L-serine [1]. A reactive selenium donor compound, monoselenophosphate, is synthesized from ATP and selenide by the catalysis of selenophosphate synthetase, the selD product [2]. L-Sec-tRNA^UGA is synthesized by L-selenocysteine synthase, selA product, which converts L-Ser to L-Sec on the tRNA^UGA [3]. SelB is an elongation factor, which binds to L-Sec-tRNA and the stem-loop structure, SECIS element, designating the position to incorporate L-Sec in a growing polypeptide chain [4].

Figure 2. The C-terminal sequences of extant [NiFeSe] Hases, in which the in-frame opal codon was read through as Sec (U).

Upper: Amino acid sequence annotated in GenBank/NCBI. Lower: Alternative translation of the in-frame opal codon as Sec (U).

Figure 3

Phylogenetic relationships of (a) [NiFeSe] Hase and (b) [NiFe] Hase assemblages in a ML phylogenetic tree. Genes from sulphate-reducing bacteria of terrestrial origin (1–3), from clinical isolates (4,5), of marine vent origin (7–9), and of sediment origin (10–12) are numbered in red, brown, blue, and green, respectively. Scores designated at each branch represent the percent of the 300-times Bootstrap test.

Figure 4. Physical genome maps.

Location and coding directions are indicated by colored triangles representing selA (purple), selB (blue), selC (orange), selD (yellow), [NiFeSe] Hase (red) and [NiFe] Hase (white). Genomes of B. wadsworthia 3_1_6, D. piger and D. phosphitoxidans are draft sequences in which contigs are interrupted by gaps that remain to be elucidated for the completed genome sequencing.

Figure 5. Molecular evolution of gas cavities in ancestral and extant [NiFeSe] Hases.

(a) Diagram representation using cavity identity based on location and amino acid residues involved in the gas cavity and the designation of cavity size, dent (d) < crack (k) < crevice (v). The internal cavity (i) is an isolated cage near the Ni-Fe metal center. (b) Structures of modelled extant [NiFeSe] Hases including the MD models for D. vulgaris Hildenborough (2wpn.pdb) and D. baculatum (1cc1.pdb), which were also annealed on MD calculation. Gas cavities are designated as cavity identity and cavity size representation.

Figure 6. Geographic positions where the SRB strains were isolated are located on the current world map

(a) and on the PANGEA continent 200 Mya (b). Maps were created with GPlates visualisation software as described previously.

Figure 7. Synonymous and non-synonymous mutations along the aligned codons for the small subunit (1–361) and large subunit (362–777).

Codons with more non-synonymous substitutions (δN − δS > 0) are positive in the blue bars. Negative bars implicate highly conserved sites that accumulated synonymous mutations. The red graph of cavity scores represents the number of amino acid residues involved in cavity formation.

Figure 8. Regional conformation changes during cavity development from ancestral 136 to D. salexigens [NiFeSe] Hase.

The development of cavity #2 is shown in a close-up view. The amino acid residues that have changed through non-synonymous mutation (δN > δS) are depicted as sticks, whereas highly conserved residues (δS > δN) are shown as cartoon representation. Left panels represent cavities around the Ni-Fe active site.