Background: Mercaptopyruvate sulfurtransferase (MST) generates H 2 S, a signaling molecule. Results: The detailed kinetics and crystal structure of human MST with bound substrate are reported. Conclusion: Thioredoxin is the preferred persulfide acceptor from MST. Significance: The structure provides molecular insights into activation and stabilization of MST reaction intermediates.

The catalytic potential for H 2 S biogenesis and homocysteine clearance converge at the active site of cystathionine β-synthase (CBS), a pyridoxal phosphate-dependent enzyme. CBS catalyzes β-replacement reactions of either serine or cysteine by homocysteine to give cystathionine and water or H 2 S, respectively. In this study, high-resolution structures of the full-length enzyme from Drosophila in which a carbanion (1.70 Å) and an aminoacrylate intermediate (1.55 Å) have been captured are reported. Electrostatic stabilization of the zwitterionic carbanion intermediate is afforded by the close positioning of an active site lysine residue that is initially used for Schiff base formation in the internal aldimine and later as a general base. Additional stabilizing interactions between active site residues and the catalytic intermediates are observed. Furthermore, the structure of the regulatory “energy-sensing” CBS domains, named after this protein, suggests a mechanism for allosteric activation by S -adenosylmethionine.

Ribonuclease P (RNase P) catalyzes the maturation of the 5′ end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.catalytic mechanism | magnesium | molecular recognition A ccording to the RNA world hypothesis, RNA played dual roles as carrier of genetic information and catalyst in a prebiotic world. However, over eons of evolution, proteins with their expanded 20-aa alphabet and greater structural and functional complexity took over many RNA-based functions. Structural insights into this evolutionary transition are limited because of the lack of examples of RNA and protein macromolecules that perform the same biological function in nature. One notable exception is ribonuclease P (RNase P) that catalyzes maturation of the 5′ end of tRNA across all domains of life. Until recently, all known RNase P enzymes included a catalytic RNA component. The discovery of a protein-only RNase P [proteinacous RNase P (PRORP)] from human mitochondria and Arabidopsis thaliana has dramatically shifted this paradigm (1-3). These enzymes represent a unique class of metallonucleases and are conserved among many eukaroytes (1, 2). A. thaliana encodes three PRORP enzymes (PRORP1, -2, and -3) that catalyze pretRNA processing (2). PRORP1 localizes to the mitochondria and chloroplast whereas PRORP2 and PRORP3 localize to the nucleus (2), suggesting that protein-based enzymes catalyze pretRNA maturation in these cellular locations (2, 3). To gain mechanistic and evolutionary insights into the PRORP...

An early step in the intracellular processing of vitamin B 12 involves CblC, which exhibits dual reactivity, catalyzing the reductive decyanation of cyanocobalamin (vitamin B 12 ), and the dealkylation of alkylcobalamins (e.g. methylcobalamin; MeCbl). Insights into how the CblC scaffold supports this chemical dichotomy have been unavailable despite it being the most common locus of patient mutations associated with inherited cobalamin disorders that manifest in both severe homocystinuria and methylmalonic aciduria. Herein, we report structures of human CblC, with and without bound MeCbl, which provide novel biochemical insights into its mechanism of action. Our results reveal that CblC is the most divergent member of the NADPHdependent flavin reductase family and can use FMN or FAD as a prosthetic group to catalyze reductive decyanation. Furthermore, CblC is the first example of an enzyme with glutathione transferase activity that has a sequence and structure unrelated to the GST superfamily. CblC thus represents an example of evolutionary adaptation of a common structural platform to perform diverse chemistries. The CblC structure allows us to rationalize the biochemical basis of a number of pathological mutations associated with severe clinical phenotypes.

Methyltransferases that employ cobalamin cofactors, or their analogues the cobamides, as intermediates in catalysis of methyl transfer play vital roles in energy generation in anaerobic unicellular organisms. In a broader range of organisms they are involved in the conversion of homocysteine to methionine. Although the individual methyl transfer reactions catalyzed are simple S N 2 displacements, the required change in coordination at the cobalt of the cobalamin or cobamide cofactors and the lability of the reduced Co +1 intermediates introduces the necessity for complex conformational changes during the catalytic cycle. Recent spectroscopic and structural studies on several of these methyltransferases have helped to reveal the strategies by which these conformational changes are facilitated and controlled.