Substrate-induced Assembly of Methanococcoides burtonii D-Ribulose-1,5-bisphosphate Carboxylase/Oxygenase Dimers into Decamers

Abstract

Like many enzymes, the biogenesis of the multi-subunit CO2-fixing enzyme ribulose-1,5-bisphosphate (RuBP) carboxylase/oxygenase (Rubisco) in different organisms requires molecular chaperones. When expressed in Escherichia coli, the large (L) subunits of the Rubisco from the archaeabacterium Methanococcoides burtonii assemble into functional dimers (L2). However, further assembly into pentamers of L2 (L10) occurs when expressed in tobacco chloroplasts or E. coli producing RuBP. In vitro analyses indicate that the sequential assembly of L2 into L10 (via detectable L4 and L6 intermediates) occurs without chaperone involvement and is stimulated by protein rearrangements associated with either the binding of substrate RuBP, the tight binding transition state analog carboxyarabinitol-1,5-bisphosphate, or inhibitory divalent metal ions within the active site. The catalytic properties of L2 and L10 M. burtonii Rubisco (MbR) were indistinguishable. At 25 °C they both shared a low specificity for CO2 over O2 (1.1 mol·mol-1) and RuBP carboxylation rates that were distinctively enhanced at lowpH(∼4 s-1 at pH 6, relative to 0.8 s-1 at pH 8) with a temperature optimum of 55 °C. Like other archaeal Rubiscos, MbR also has a high O2 affinity (Km(O2)=∼2.5 μM). The catalytic and structural similarities of MbR to other archaeal Rubiscos contrast with its closer sequence homology to bacterial L2 Rubisco, complicating its classification within the Rubisco superfamily.