Abstract
Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Here, we demonstrate that in vivo activity-based protein profiling (ABPP) may be used to sensitively detect either native or heterogeneously expressed active enzymes in living archaea even under these extreme conditions. In combination with the development of a genetically engineered archaeal screening strain, ABPP can furthermore be used in functional enzyme screenings from (meta)genome samples. We anticipate that our ABPP approach may therefore find application in basic archaeal research but also in the discovery of novel enzymes from (meta)genome libraries.
Highlights
Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies
We provide a proof-of-concept study with archaeal serine hydrolases because they are (i) ubiquitously expressed in all domains of life and represent a well-characterized enzyme family which features an active serine residue critically involved in substrate hydrolysis[28,29], (ii) known to be robust and reliable targets in activity-based protein profiling (ABPP) approaches[30,31] and (iii) biocatalysts of great biotechnological interest[32]
To establish a suitable protocol for in vivo ABPP under these harsh conditions, we focused our attention on phosphonate-based serine hydrolase probes because these probes are among the best characterized activity-based probes (ABPs) and are known to display robust labelling[30,31]
Summary
Archaea are characterized by a unique life style in often environmental extremes but their thorough investigation is currently hampered by a limited set of suitable in vivo research methodologies. Many of the so far cultured archaea thrive under environmental extremes of temperature, pH or salinity They combine biochemical and cellular properties of bacteria and eukarya and display exceptional features like the use of distinct membrane lipids or an exclusive cell wall composition[9,12]. Their overall metabolic complexity resembles bacteria and lower eukarya, they lack many of the classical central metabolic pathways but instead harbour modified variants that involve enzymes with only weak or even no similarity to bacterial or eukaryotic counterparts[13]. The use of such probes in ABPP experiments requires a two-step labelling methodology: in the first step, the ABP is added in vivo to the biological system to enable ABPP labelling; in the second step, a ‘standard’ reporter tag such as a fluorophore moiety is attached after cell lysis via bioorthogonal conjugation chemistries such as a Staudinger ligation or a Huisgen-type Cu(I)-catalysed (3 þ 2)
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