Solution Behavior and Activity of a Halophilic Esterase under High Salt Concentration

Background: Halophiles are extremophiles that thrive in environments with very high concentrations of salt. Although the salt reliance and physiology of these extremophiles have been widely investigated, the molecular working mechanisms of their enzymes under salty conditions have been little explored. Methodology/Principal Findings: A halophilic esterolytic enzyme LipC derived from archeaon Haloarcula marismortui was overexpressed from Escherichia coli BL21. The purified enzyme showed a range of hydrolytic activity towards the substrates of p-nitrophenyl esters with different alkyl chains (n = 2−16), with the highest activity being observed for p-nitrophenyl acetate, consistent with the basic character of an esterase. The optimal esterase activities were found to be at pH 9.5 and [NaCl] = 3.4 M or [KCl] = 3.0 M and at around 45°C. Interestingly, the hydrolysis activity showed a clear reversibility against changes in salt concentration. At the ambient temperature of 22°C, enzyme systems working under the optimal salt concentrations were very stable against time. Increase in temperature increased the activity but reduced its stability. Circular dichroism (CD), dynamic light scattering (DLS) and small angle neutron scattering (SANS) were deployed to determine the physical states of LipC in solution. As the salt concentration increased, DLS revealed substantial increase in aggregate sizes, but CD measurements revealed the maximal retention of the α-helical structure at the salt concentration matching the optimal activity. These observations were supported by SANS analysis that revealed the highest proportion of unimers and dimers around the optimal salt concentration, although the coexistent larger aggregates showed a trend of increasing size with salt concentration, consistent with the DLS data. Conclusions/Significance: The solution α-helical structure and activity relation also matched the highest proportion of enzyme unimers and dimers. Given that all the solutions studied were structurally inhomogeneous, it is important for future work to understand how the LipC's solution aggregation affected its activity

Phylogenetic and Evolutionary Patterns in Microbial Carotenoid Biosynthesis Are Revealed by Comparative Genomics

BACKGROUND: Carotenoids are multifunctional, taxonomically widespread and biotechnologically important pigments. Their biosynthesis serves as a model system for understanding the evolution of secondary metabolism. Microbial carotenoid diversity and evolution has hitherto been analyzed primarily from structural and biosynthetic perspectives, with the few phylogenetic analyses of microbial carotenoid biosynthetic proteins using either used limited datasets or lacking methodological rigor. Given the recent accumulation of microbial genome sequences, a reappraisal of microbial carotenoid biosynthetic diversity and evolution from the perspective of comparative genomics is warranted to validate and complement models of microbial carotenoid diversity and evolution based upon structural and biosynthetic data. METHODOLOGY/PRINCIPAL FINDINGS: Comparative genomics were used to identify and analyze in silico microbial carotenoid biosynthetic pathways. Four major phylogenetic lineages of carotenoid biosynthesis are suggested composed of: (i) Proteobacteria; (ii) Firmicutes; (iii) Chlorobi, Cyanobacteria and photosynthetic eukaryotes; and (iv) Archaea, Bacteroidetes and two separate sub-lineages of Actinobacteria. Using this phylogenetic framework, specific evolutionary mechanisms are proposed for carotenoid desaturase CrtI-family enzymes and carotenoid cyclases. Several phylogenetic lineage-specific evolutionary mechanisms are also suggested, including: (i) horizontal gene transfer; (ii) gene acquisition followed by differential gene loss; (iii) co-evolution with other biochemical structures such as proteorhodopsins; and (iv) positive selection. CONCLUSIONS/SIGNIFICANCE: Comparative genomics analyses of microbial carotenoid biosynthetic proteins indicate a much greater taxonomic diversity then that identified based on structural and biosynthetic data, and divides microbial carotenoid biosynthesis into several, well-supported phylogenetic lineages not evident previously. This phylogenetic framework is applicable to understanding the evolution of specific carotenoid biosynthetic proteins or the unique characteristics of carotenoid biosynthetic evolution in a specific phylogenetic lineage. Together, these analyses suggest a "bramble" model for microbial carotenoid biosynthesis whereby later biosynthetic steps exhibit greater evolutionary plasticity and reticulation compared to those closer to the biosynthetic "root". Structural diversification may be constrained ("trimmed") where selection is strong, but less so where selection is weaker. These analyses also highlight likely productive avenues for future research and bioprospecting by identifying both gaps in current knowledge and taxa which may particularly facilitate carotenoid diversification

Tradeoff between robustness and elaboration in carotenoid networks produces cycles of avian color diversification

BACKGROUND: Resolution of the link between micro- and macroevolution calls for comparing both processes on the same deterministic landscape, such as genomic, metabolic or fitness networks. We apply this perspective to the evolution of carotenoid pigmentation that produces spectacular diversity in avian colors and show that basic structural properties of the underlying carotenoid metabolic network are reflected in global patterns of elaboration and diversification in color displays. Birds color themselves by consuming and metabolizing several dietary carotenoids from the environment. Such fundamental dependency on the most upstream external compounds should intrinsically constrain sustained evolutionary elongation of multi-step metabolic pathways needed for color elaboration unless the metabolic network gains robustness - the ability to synthesize the same carotenoid from an additional dietary starting point. RESULTS: We found that gains and losses of metabolic robustness were associated with evolutionary cycles of elaboration and stasis in expressed carotenoids in birds. Lack of metabolic robustness constrained lineage's metabolic explorations to the immediate biochemical vicinity of their ecologically distinct dietary carotenoids, whereas gains of robustness repeatedly resulted in sustained elongation of metabolic pathways on evolutionary time scales and corresponding color elaboration. CONCLUSIONS: The structural link between length and robustness in metabolic pathways may explain periodic convergence of phylogenetically distant and ecologically distinct species in expressed carotenoid pigmentation; account for stasis in carotenoid colors in some ecological lineages; and show how the connectivity of the underlying metabolic network provides a mechanistic link between microevolutionary elaboration and macroevolutionary diversification. REVIEWERS: This article was reviewed by Junhyong Kim, Eugene Koonin, and Fyodor Kondrashov. For complete reports, see the Reviewers' reports section.This item is part of the UA Faculty Publications collection. For more information this item or other items in the UA Campus Repository, contact the University of Arizona Libraries at [email protected]

Biocatalytic production of enantiopure cyclohexane-trans-1,2-diol using extracellular lipases from Bacillus subtilis

Two extracellular lipases from Bacillus subtilis, B. subtilis lipase A and lipase B, have been expressed in the heterologous host Escherichia coli, biochemically characterized and used for the kinetic resolution of (rac)-trans-1,2-diacetoxycyclohexane. Both enzymes were selectively acting on the (R,R)-enantiomer of the racemic substrate, highly specifically hydrolyzing only one of the two ester groups present, thus allowing the preparation of enantiopure (R,R)- and (S,S)-cyclohexane-trans-1,2-diol. The reaction conditions for the use of purified enzyme and crude cell lyophilizate were optimized and reactions in batch and repetitive batch modes were carried out on a preparative scale to yield enantiopure product (>99% enantiomeric excess)