Fe-S cluster biogenesis by the bacterial Suf pathway.

Biogenesis of iron-sulfur (FeS) clusters in an essential process in living organisms due to the critical role of FeS cluster proteins in myriad cell functions. During biogenesis of FeS clusters, multi-protein complexes are used to drive the mobilization and protection of reactive sulfur and iron intermediates, regulate assembly of various FeS clusters on an ATPase-dependent, multi-protein scaffold, and target nascent clusters to their downstream protein targets. The evolutionarily ancient sulfur formation (Suf) pathway for FeS cluster assembly is found in bacteria and archaea. In Escherichia coli, the Suf pathway functions as an emergency pathway under conditions of iron limitation or oxidative stress. In other pathogenic bacteria, such as Mycobacterium tuberculosis and Enterococcus faecalis, the Suf pathway is the sole source for FeS clusters and therefore is a potential target for the development of novel antibacterial compounds. Here we summarize the considerable progress that has been made in characterizing the first step of mobilization and protection of reactive sulfur carried out by the SufS-SufE or SufS-SufU complex, FeS cluster assembly on SufBC2D scaffold complexes, and the downstream trafficking of nascent FeS clusters to A-type carrier (ATC) proteins. Cell Biology of Metals III edited by Roland Lill and Mick Petris.

Kemp Eliminase Activity of Ketosteroid Isomerase.

Kemp eliminases represent the most successful class of computationally designed enzymes, with rate accelerations of up to 109-fold relative to the rate of the same reaction in aqueous solution. Nevertheless, several other systems such as micelles, catalytic antibodies, and cavitands are known to accelerate the Kemp elimination by several orders of magnitude. We found that the naturally occurring enzyme ketosteroid isomerase (KSI) also catalyzes the Kemp elimination. Surprisingly, mutations of D38, the residue that acts as a general base for its natural substrate, produced variants that catalyze the Kemp elimination up to 7000-fold better than wild-type KSI does, and some of these variants accelerate the Kemp elimination more than the computationally designed Kemp eliminases. Analysis of the D38N general base KSI variant suggests that a different active site carboxylate residue, D99, performs the proton abstraction. Docking simulations and analysis of inhibition by active site binders suggest that the Kemp elimination takes place in the active site of KSI and that KSI uses the same catalytic strategies of the computationally designed enzymes. In agreement with prior observations, our results strengthen the conclusion that significant rate accelerations of the Kemp elimination can be achieved with very few, nonspecific interactions with the substrate if a suitable catalytic base is present in a hydrophobic environment. Computational design can fulfill these requirements, and the design of more complex and precise environments represents the next level of challenges for protein design.

Kemp Elimination in Cationic Micelles: Designed Enzyme-Like Rates Achieved through the Addition of Long-Chain Bases.

The Kemp elimination is prototypical reaction used to study proton abstraction from carbon. Several hydrophobic systems are known to accelerate this reaction, including two classes of computationally-designed enzymes. However, it is unclear whether these computationally-designed enzymes establish specific interactions with their substrates, as natural enzymes do, or if most of the rate acceleration is due to the hydrophobicity of the substrate. We used a simple system composed of cationic micelles and a long chain base (such as lauryl phosphate or lauric acid) to measure the rate acceleration for the Kemp elimination. Remarkably, we found that this simple system can accelerate the reaction by 4 orders of magnitude, approaching the rates of more complex designed systems. Use of different substrates suggests that the reaction takes place at the interface between the micellar head and water (the Stern layer) with the long-chain base embedded in the micelle and the substrate in the aqueous solution. Thus, we suggest that significant rate accelerations can be achieved regardless of the precise positioning of substrates. Because natural enzymes use specific interactions to position their substrates, we propose that acceleration of the Kemp elimination is not a suitable benchmark for the success of the design process, and we suggest that more complex reactions should be used.