Serine 363 of a Hydrophobic Region of Archaeal Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase from Archaeoglobus fulgidus and Thermococcus kodakaraensis Affects CO2/O2 Substrate Specificity and Oxygen Sensitivity.

Archaeal ribulose 1, 5-bisphospate carboxylase/oxygenase (RubisCO) is differentiated from other RubisCO enzymes and is classified as a form III enzyme, as opposed to the form I and form II RubisCOs typical of chemoautotrophic bacteria and prokaryotic and eukaryotic phototrophs. The form III enzyme from archaea is particularly interesting as several of these proteins exhibit unusual and reversible sensitivity to molecular oxygen, including the enzyme from Archaeoglobus fulgidus. Previous studies with A. fulgidus RbcL2 had shown the importance of Met-295 in oxygen sensitivity and pointed towards the potential significance of another residue (Ser-363) found in a hydrophobic pocket that is conserved in all RubisCO proteins. In the current study, further structure/function studies have been performed focusing on Ser-363 of A. fulgidus RbcL2; various changes in this and other residues of the hydrophobic pocket point to and definitively establish the importance of Ser-363 with respect to interactions with oxygen. In addition, previous findings had indicated discrepant CO2/O2 specificity determinations of the Thermococcus kodakaraensis RubisCO, a close homolog of A. fulgidus RbcL2. It is shown here that the T. kodakaraensis enzyme exhibits a similar substrate specificity as the A. fulgidus enzyme and is also oxygen sensitive, with equivalent residues involved in oxygen interactions

Retention of carboxylase activity in the presence of oxygen.

<p>Wild-type (●), M295D (○), S363I (▼), S363V (∇), M295D/S363I (■) and M295D/S363V (□) homogeneous enzymes were exposed to varying amounts of oxygen and assayed for carboxylase activity. As described in Materials and Methods, enzymes were exposed to oxygen for a minimum of 10 min, with no discernible change in activity after this time period. The percent activity retained is the difference in activity between the anaerobic samples compared to the oxygen exposed samples. The M295D/S363I and M295D/S363V enzymes retained significantly more activity than all the other enzymes when all enzymes were incubated with concentrations of oxygen ranging from 10% (42.1 μM) to 100% (421 μM) in the gas phase. Points represent the average of three determinations.</p

Kinetic properties of homogeneous recombinant wild-type and mutant RubisCOs from <i>A</i>. <i>fulgidus</i> RbcL2 and wild-type RubisCO from <i>T</i>. <i>kodakaraensis</i> RbcL assayed at 83°C.

<p>^a Average of at least three independent assays.</p><p>^b Determined at 20 x K_RuBP except for the S363V and M295D/S363I and M295D/S363V mutant enzymes which were determined at 4–6 x K_RuBP.</p><p>^c Values for WT and M295D previously reported (17) and run in parallel with the other mutant <i>A</i>. <i>fulgidus</i> and <i>T</i>. <i>kodakaraensis</i> enzymes for comparison</p><p>Kinetic constants were determined as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138351#pone.0138351.ref017" target="_blank">17</a>].</p

Carboxylase activity at 83°C of highly purified recombinant <i>T</i>. <i>kodakaraensis</i> RbcL wild-type and mutant enzymes under anaerobic and oxygen exposed conditions.

<p>^a Average of duplicate assays</p><p>Carboxylase activity at 83°C of highly purified recombinant <i>T</i>. <i>kodakaraensis</i> RbcL wild-type and mutant enzymes under anaerobic and oxygen exposed conditions.</p

Anion exchange chromatographic separation of RubisCO reaction products [³H]3-PGA and [³H]2-PG generated from a completed reaction mixture containing [1-³H] RuBP after 2 h reaction at 83°C.

<p><i>T</i>. <i>kodakaraensis</i> RubisCO was incubated in the presence of both molecular oxygen and CO₂ to generate [³H] 3-PGA and [³H] 2-PG under (A) defined conditions as described in Materials and Methods and (B) under conditions previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138351#pone.0138351.ref020" target="_blank">20</a>]. In (C), wild-type <i>A</i>. <i>fulgidus</i> RbcL2 was assayed under the same conditions as previously described [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138351#pone.0138351.ref020" target="_blank">20</a>]. Peaks at the beginnings of the chromatographic profiles represent degraded RuBP produced in this reaction mixture at high temperatures.</p

Complementation and growth of <i>R</i>. <i>capsulatus cbbLS</i>/<i>cbbM</i> knockout strain SBI/II^- using <i>A</i>. <i>fulgidus</i> wild-type and mutant Rubisco (<i>rbcL2</i>) genes in plasmid pRPS-MCS3MA.

<p>Photoautotrophic growth was performed under an atmosphere of 20% CO₂/80% H₂ in glass tubes prepared under anaerobic conditions. Wild-type <i>R</i>. <i>capsulatus</i> SB1003 (●); <i>R</i>. <i>capsulatus</i> strain SBI-II- containing plasmid pRPS-MCS3 and <i>A</i>. <i>fulgidus rbcL2</i> wild type (○) or mutants M295D (▼); S363I (■); and S363V (▽). Points represent the average of two to three cultures.</p

Comparison of side chain interactions of Ser-366 with Gly-316 and Thr-317 in the solved crystal structure of <i>T</i>. <i>kodakaraensis</i> RbcL with corresponding residues in the model structure of <i>A</i>. <i>fulgidus</i> RbcL2.

<p>Ser-366 in <i>T</i>. <i>kodakaraensis</i> RbcL (A) is situated on <i>β</i>-strand 6 pointing away from the active site, situated in a highly conserved hydrophobic pocket, and interacts with a highly conserved residue, Gly-316, depicted by dashed purple lines. Ser-366 does not interact with the other highly conserved residue, Thr-317. Conversely, the model structure of <i>A</i>. <i>fulgidus</i> RbcL2 (B) suggests that the identical residues in this region, Ser-363, Gly-313 and Thr-314 all interact to form hydrogen bonds (dashed purple lines).</p

Partial amino acid sequence alignment of <i>A</i>. <i>fulgidus</i> and <i>T</i>. <i>kodakaraensis</i> archaeal form III RubisCOs.

<p>Multiple sequence alignments were performed by using ClustalW [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0138351#pone.0138351.ref039" target="_blank">39</a>]. Residue identities are marked with an asterisk, conserved substitutions are marked with a colon, and semiconserved substitutions are marked with a period. Known active-site and highly conserved residues are labeled C for catalytic and R for RuBP binding properties and colored red. Amino acids colored blue are identical to the position of either Met-295 or Ser-363 residues in the model structure of <i>A</i>. <i>fulgidus</i> RbcL2.</p

Amino acid residues involved in the formation of a hydrophobic pocket in a specific region of the RubisCO enzyme and sequence comparison of these residues between forms I, II and III RubisCOs.

<p>^a Catalytic residue</p><p>Amino acid residues involved in the formation of a hydrophobic pocket in a specific region of the RubisCO enzyme and sequence comparison of these residues between forms I, II and III RubisCOs.</p

The hydrophobic pocket of <i>Archaeoglobus fulgidus</i> RbcL2 RubisCO showing interactions of Se∇r-363 with conserved residues Gly-313 and Thr-314.

<p>Ile-312 is situated on <i>β</i>–strand 6 between a highly conserved residue, His-311, necessary for the binding of RuBP and the Gly-313 residue that interacts with Ser-363. Ser-363 is directed away from the active site towards <i>α</i>–helix 6. The loop 6 structure is important by virtue of folding over the active site during catalysis and is between <i>β</i>–strand 6 and <i>α</i>–helix 6, shown on the upper left of the figure.</p