Phylogenetic and X-ray crystallographic studies suggest that ribulose 1,5 bisphosphate carboxylase/oxygenase (RubisCO) and RubisCO-Like-Proteins (RLPs) are structurally related, though RLPs lack the catalytic properties of bonafide RubisCO. Previous studies implicate a role for RLP in metabolism of 5-methylthioadenosine (MTA) in Bacillus subtilis, where MTA is an intermediate of a sulfur (methionine) salvage pathway. Interestingly, Rhodospirillum rubrum RubisCO weakly catalyzes an enolase reaction with 2,3-diketo-5-methylthio-pentyl-1-phosphate (DKMTP), similar to the RLP of Bacillus subtilis. R. rubrum is an intriguing model as this organism contains both RubisCO and RLP and each protein seems to be utilized for MTA metabolism under distinct growth conditions.
The questions addressed in this dissertation were: (1) Is RubisCO involved in simultaneous carbon and sulfur metabolism; (2) Are all forms of RubisCO capable of performing an MTA-dependent enolase reaction; (3) Do the substrates RuBP and DKMTP share the same active site?
Results from molecular, genetic and in vivo experiments indicate several sources of form I, form II, and form III RubisCO complement RubisCO/RLP knockout strains in R. rubrum to MTA dependent growth under specified physiological conditions. All forms of RubisCO utilized in this dissertation appear to catalyze the enolization of DKMTP via a reaction that bears similarity to the enolization reaction catalyzed by RubisCO during CO2 fixation.
In vitro and in vivo studies suggest R. rubrum RubisCO plays an important role in the sulfur salvage pathway that is distinct from its previously well-characterized carboxylation/oxygenation reaction. Structure-function studies in combination with molecular modeling studies of various mutant forms of RubisCO revealed that two known ligands of RubisCO (i.e., DKMTP, RuBP) do not share conserved interactions with RubisCO, suggesting distinct residues are differentially involved in the carboxylation/oxygenation or enolase reactions.
In summary, using genetic and molecular tools, this dissertation advances our knowledge on the mechanism and regulation of novel, wide-spread sulfur salvage roles of the well characterized enzyme RubisCO. Important insight was obtained into the structure-function relationships of the enzyme in its two different metabolic roles. The discovery of RLP and the elucidation of its function, gives an entirely new dimension to the function and evolution of RubisCO, i.e., this one enzyme functions to catalyze two distinct metabolic pathways.