Paola E. Mera

Structural Characterization of the Active Site of the PduO-Type ATP:Co(I)rrinoid Adenosyltransferase from Lactobacillus reuteri

The three-dimensional crystal structure of the PduO-type corrinoid adenosyltransferase from Lactobacillus reuteri (LrPduO) has been solved to 1.68-Å resolution. The functional assignment of LrPduO as a corrinoid adenosyltransferase was confirmed by in vivo and in vitro evidence. The enzyme has an apparent \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{m}^{\mathrm{ATP}}\) \end{document} of 2.2 μm and \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(K_{m}^{\mathrm{Cobalamin}}\) \end{document} of 0.13 μm and a kcat of 0.025 s-1. Co-crystallization of the enzyme with Mg-ATP resulted in well-defined electron density for an N-terminal loop that had been disordered in other PduO-type enzyme structures. This newly defined N-terminal loop makes up the lower portion of the enzyme active site with the other half being contributed from an adjacent subunit. These results provide the first detailed description of the enzyme active site for a PduO-type adenosyltransferase and identify a unique ATP binding motif at the protein N terminus. The molecular architecture at the active site offers valuable new insight into the role of various residues responsible for the human disease methylmalonic aciduria.

Residue Phe112 of the human-type corrinoid adenosyltransferase (PduO) enzyme of Lactobacillus reuteri is critical to the formation of the four-coordinate Co(II) corrinoid substrate and to the activity of the enzyme.

ATP:Corrinoid adenosyltransferases (ACAs) catalyze the transfer of the adenosyl moiety from ATP to cob(I)alamin via a four-coordinate cob(II)alamin intermediate. At present, it is unknown how ACAs promote the formation of the four-coordinate corrinoid species needed for activity. The published high-resolution crystal structure of the ACA from Lactobacillus reuteri (LrPduO) in complex with ATP and cob(II)alamin shows that the environment around the alpha face of the corrin ring consists of bulky hydrophobic residues. To understand how these residues promote the generation of the four-coordinate cob(II)alamin, variants of the human-type ACA enzyme from L. reuteri (LrPduO) were kinetically and structurally characterized. These studies revealed that residue Phe112 is critical in the displacement of 5,6-dimethylbenzimidazole (DMB) from its coordination bond with the Co ion of the ring, resulting in the formation of the four-coordinate species. An F112A substitution resulted in a 80% drop in the catalytic efficiency of the enzyme. The explanation for this loss of activity was obtained from the crystal structure of the mutant protein, which showed cob(II)alamin bound in the active site with DMB coordinated to the cobalt ion. The crystal structure of an LrPduO(F112H) variant showed a DMB-off/His-on interaction between the corrinoid and the enzyme, whose catalytic efficiency was 4 orders of magnitude lower than that of the wild-type protein. The analysis of the kinetic parameters of LrPduO(F112H) suggests that the F112H substitution negatively impacts product release. Substitutions of other hydrophobic residues in the Cbl binding pocket did not result in significant defects in catalytic efficiency in vitro; however, none of the variant enzymes analyzed in this work supported AdoCbl biosynthesis in vivo.

Dihydroflavin-driven Adenosylation of 4-Coordinate Co(II) Corrinoids ARE COBALAMIN REDUCTASES ENZYMES OR ELECTRON TRANSFER PROTEINS?

The identity of the source of the biological reductant needed to convert cobalamin to its biologically active form adenosylcobalamin has remained elusive. Here we show that free or protein-bound dihydroflavins can serve as the reductant of Co2+Cbl bound in the active site of PduO-type ATP-dependent corrinoid adenosyltransferase enzymes. Free dihydroflavins (dihydroriboflavin, FMNH2, and FADH2) effectively drove the adenosylation of Co2+Cbl by the human and bacterial PduO-type enzymes at very low concentrations (1 μm). These data show that adenosyltransferase enzymes lower the thermodynamic barrier of the Co2+ → Co+ reduction needed for the formation of the unique organometalic Co–C bond of adenosylcobalamin. Collectively, our in vivo and in vitro data suggest that cobalamin reductases identified thus far are most likely electron transfer proteins, not enzymes.

Multiple Roles of ATP:Cob(I)alamin Adenosyltransferases in the Conversion of B12 to Coenzyme B12

Our mechanistic understanding of the conversion of vitamin B12 into coenzyme B12 (a.k.a. adenosylcobalamin, AdoCbl) has been substantially advanced in recent years. Insights into the multiple roles played by ATP:cob(I)alamin adenosyltransferase (ACA) enzymes have emerged through the crystallographic, spectroscopic, biochemical, and mutational analyses of wild-type and variant proteins. ACA enzymes circumvent the thermodynamic barrier posed by the very low redox potential associated with the reduction of cob(II)alamin to cob(I)alamin by generating a unique four-coordinate cob(II)alamin intermediate that is readily converted to cob(I)alamin by physiological reductants. ACA enzymes not only synthesize AdoCbl but also they deliver it to the enzymes that use it, and in some cases, enzymes in which its function is needed to maintain the fidelity of the AdoCbl delivery process have been identified. Advances in our understanding of ACA enzyme function have provided valuable insights into the role of specific residues, and into why substitutions of these residues have profound negative effects on human health. From an applied science standpoint, a better understanding of the adenosylation reaction may lead to more efficient ways of synthesizing AdoCbl.

Replication initiator DnaA binds at the Caulobacter centromere and enables chromosome segregation

Significance DnaA is an essential and conserved bacterial protein that enables the initiation of DNA replication. Although it is commonly held that the onset of bacterial chromosome segregation depends on the initiation of DNA replication, we have found that in Caulobacter crescentus, chromosome segregation can be induced in a DnaA-dependent, yet replication-independent manner. The chromosome replication origin, containing essential DnaA binding motifs, resides 8 kb from the centromere parS region that also contains DnaA binding motifs. The centromere parS region bound to the ParB partition protein initiates movement across the cell followed by the origin region. Mutations in a centromere DnaA motif that alter DnaA–centromere interaction exhibit aberrant patterns of ParB/parS translocation, implicating DnaA in the process of chromosome segregation. During cell division, multiple processes are highly coordinated to faithfully generate genetically equivalent daughter cells. In bacteria, the mechanisms that underlie the coordination of chromosome replication and segregation are poorly understood. Here, we report that the conserved replication initiator, DnaA, can mediate chromosome segregation independent of replication initiation. It does so by binding directly to the parS centromere region of the chromosome, and mutations that alter this interaction result in cells that display aberrant centromere translocation and cell division. We propose that DnaA serves to coordinate bacterial DNA replication with the onset of chromosome segregation.