Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

<p>Abstract</p> <p>Background</p> <p>β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured <it>in vitro </it>using His-tag purified proteins and <it>in vivo </it>in a β-carotene-accumulating <it>E. coli </it>system.</p> <p>Results</p> <p>Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our <it>ab initio </it>model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.</p> <p>Conclusions</p> <p>Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.</p

Microbial diversity and biogeochemical cycling in soda lakes

Soda lakes contain high concentrations of sodium carbonates resulting in a stable elevated pH, which provide a unique habitat to a rich diversity of haloalkaliphilic bacteria and archaea. Both cultivation-dependent and -independent methods have aided the identification of key processes and genes in the microbially mediated carbon, nitrogen, and sulfur biogeochemical cycles in soda lakes. In order to survive in this extreme environment, haloalkaliphiles have developed various bioenergetic and structural adaptations to maintain pH homeostasis and intracellular osmotic pressure. The cultivation of a handful of strains has led to the isolation of a number of extremozymes, which allow the cell to perform enzymatic reactions at these extreme conditions. These enzymes potentially contribute to biotechnological applications. In addition, microbial species active in the sulfur cycle can be used for sulfur remediation purposes. Future research should combine both innovative culture methods and state-of-the-art ‘meta-omic’ techniques to gain a comprehensive understanding of the microbes that flourish in these extreme environments and the processes they mediate. Coupling the biogeochemical C, N, and S cycles and identifying where each process takes place on a spatial and temporal scale could unravel the interspecies relationships and thereby reveal more about the ecosystem dynamics of these enigmatic extreme environments