Convergent Evolution of a Promiscuous 3-Hydroxypropionyl-CoA Dehydratase/Crotonyl-CoA Hydratase in Crenarchaeota and Thaumarchaeota

Abstract

The autotrophic 3-hydroxypropionate/4-hydroxybutyrate (HP/HB) cycle functions in thermoacidophilic, (micro)aerobic, hydrogen-oxidizing Crenarchaeota of the order Sulfolobales as well as in mesophilic, aerobic, ammonia-oxidizing Thaumarchaeota. Notably, the HP/HB cycle evolved independently in these two archaeal lineages, and crenarchaeal and thaumarchaeal versions differ regarding their enzyme properties and phylogeny. These differences result in altered energetic efficiencies between the variants. Compared to the crenarchaeal HP/HB cycle, the thaumarchaeal variant saves two ATP equivalents per turn, rendering it the most energy-efficient aerobic pathway for carbon fixation. Characteristically, the HP/HB cycle includes two enoyl coenzyme A (CoA) hydratase reactions: the 3-hydroxypropionyl-CoA dehydratase reaction and the crotonyl-CoA hydratase reaction. In this study, we show that both reactions are catalyzed in the aforementioned archaeal groups by a promiscuous 3-hydroxypropionyl-CoA dehydratase/crotonyl-CoA hydratase (Msed_2001 in crenarchaeon Metallosphaera sedula and Nmar_1308 in thaumarchaeon Nitrosopumilus maritimus). Although these two enzymes are homologous, they are closely related to bacterial enoyl-CoA hydratases and were retrieved independently from the same enzyme pool by the ancestors of Crenarchaeota and Thaumarchaeota, despite the existence of multiple alternatives. This striking similarity in the emergence of enzymes involved in inorganic carbon fixation from two independently evolved pathways highlights that convergent evolution of autotrophy could be much more widespread than anticipated. IMPORTANCE Inorganic carbon fixation is the most important biosynthetic process on Earth and the oldest type of metabolism. The autotrophic HP/HB cycle functions in Crenarchaeota of the order Sulfolobales and in ammonia-oxidizing Archaea of the phylum Thaumarchaeota that are highly abundant in marine, terrestrial, and geothermal environments. Bioinformatic prediction of the autotrophic potential of microorganisms or microbial communities requires identification of enzymes involved in autotrophy. However, many microorganisms possess several isoenzymes that may potentially catalyze the reactions of the cycle. Here, we studied the enzymes catalyzing 3-hydroxypropionyl-CoA dehydration and crotonyl-CoA hydration in Nitrosopumilus maritimus (Thaumarchaeota) as well as in Metallosphaera sedula (Crenarchaeota). We showed that both reactions were catalyzed by homologous promiscuous enzymes, which evolved independently from each other from their bacterial homologs. Furthermore, the HP/HB cycle is of applied value, and knowledge of its enzymes is necessary to transfer them to a heterologous host for synthesis of various value-added products.

(S)-3-Hydroxybutyryl-CoA Dehydrogenase From the Autotrophic 3-Hydroxypropionate/4-Hydroxybutyrate Cycle in Nitrosopumilus maritimus

“…The HP/HB cycle exists in two variants, and the apparent similarity in the intermediates of the cycle in AOA and in Sulfolobales is in sharp contrast with the results of the biochemical and/or phylogenetic analysis of the corresponding enzymes (Könneke et al, 2014;Otte et al, 2015;Liu et al, 2021). The difference in the autotrophic pathways in these two archaeal groups goes along with the difference in their ecology and environmental requirements.…”

“…Sulfolobales also possess a homologous crotonyl-CoA hydratase/( S )-3-hydroxybutyryl-CoA dehydrogenase. Although this protein is present in autotrophically grown Metallosphaera sedula (Sulfolobales) cells ( Ramos-Vera et al, 2011 ; Liu et al, 2021 ), it is responsible only for a minute flux in the crotonyl-CoA conversion to acetoacetyl-CoA in this archaeon ( Liu et al, 2020 , 2021 ). In contrast to the organisms using the DC/HB cycle, M. sedula and other Sulfolobales possess stand-alone crotonyl-CoA hydratase and ( S )-3-hydroxybutyryl-CoA dehydrogenase enzymes in the HP/HB cycle ( Liu et al, 2020 ).…”

“…In contrast to the organisms using the DC/HB cycle, M. sedula and other Sulfolobales possess stand-alone crotonyl-CoA hydratase and ( S )-3-hydroxybutyryl-CoA dehydrogenase enzymes in the HP/HB cycle ( Liu et al, 2020 ). Their crotonyl-CoA hydratase is a promiscuous protein that has also a high 3-hydroxypropionyl-CoA dehydratase activity and thus catalyzes two (de)hydratase reactions in the cycle ( Liu et al, 2021 ). Thaumarchaeota also use a similar promiscuous enzyme to catalyze these two reactions ( Liu et al, 2021 ).…”

“…Their crotonyl-CoA hydratase is a promiscuous protein that has also a high 3-hydroxypropionyl-CoA dehydratase activity and thus catalyzes two (de)hydratase reactions in the cycle ( Liu et al, 2021 ). Thaumarchaeota also use a similar promiscuous enzyme to catalyze these two reactions ( Liu et al, 2021 ). Interestingly, crenarchaeal and thaumarchaeal 3-hydroxypropionyl-CoA dehydratases/crotonyl-CoA hydratases are closely related to bacterial enoyl-CoA hydratases but were retrieved independently by the ancestors of Sulfolobales and AOA from different bacteria ( Liu et al, 2021 ).…”

“…Thaumarchaeota also use a similar promiscuous enzyme to catalyze these two reactions ( Liu et al, 2021 ). Interestingly, crenarchaeal and thaumarchaeal 3-hydroxypropionyl-CoA dehydratases/crotonyl-CoA hydratases are closely related to bacterial enoyl-CoA hydratases but were retrieved independently by the ancestors of Sulfolobales and AOA from different bacteria ( Liu et al, 2021 ). Here, we characterize ( S )-3-hydroxybutyryl-CoA dehydrogenase from the ammonia-oxidizing thaumarchaeon Nitrosopumilus maritimus , compare it with the enzyme from M. sedula and with the crotonyl-CoA hydratase/( S )-3-hydroxybutyryl-CoA dehydrogenases, and discuss the enzymological differences in these conversions in course of the DC/HB and HP/HB cycles.…”