Affiliation:
1. Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal
Abstract
ABSTRACT
Glycerophosphoinositol (GPI) is a compatible solute present in a few hyperthermophiles. Interestingly, different GPI stereoisomers accumulate in
Bacteria
and
Archaea
, and the basis for this domain-dependent specificity was investigated herein. The archaeon
Archaeoglobus fulgidus
and the bacterium
Aquifex aeolicus
were used as model organisms. The synthesis of GPI involves glycerol phosphate cytidylyltransferase (GCT), which catalyzes the production of CDP-glycerol from CTP and glycerol phosphate, and di-
myo
-inositol phosphate-phosphate synthase (DIPPS), catalyzing the formation of phosphorylated GPI from CDP-glycerol and
l
-
myo
-inositol 1-phosphate. DIPPS of
A. fulgidus
recognized the two CDP-glycerol stereoisomers similarly. This feature and the ability of
31
P nuclear magnetic resonance (NMR) to distinguish the GPI diastereomers provided a means to study the stereospecificity of GCTs. The AF1418 gene and genes aq_185 and aq_1368 are annotated as putative GCT genes in the genomes of
A. fulgidus
and
Aq. aeolicus
, respectively. The functions of these genes were determined by assaying the activity of the respective recombinant proteins: AQ1368 and AQ185 are GCTs, while AF1418 has flavin adenine dinucleotide (FAD) synthetase activity. AQ185 is absolutely specific for
sn
-glycerol 3-phosphate, while AQ1368 recognizes the two enantiomers but has a 2:1 preference for
sn
-glycerol 3-phosphate. In contrast, the partially purified
A. fulgidus
GCT uses
sn
-glycerol 1-phosphate preferentially (4:1). Significantly, the predominant GPI stereoforms found in the bacterium and the archaeon reflect the distinct stereospecificities of the respective GCTs: i.e.,
A. fulgidu
s accumulates predominantly
sn
-glycero-1-phospho-3-
l
-
myo
-inositol, while
Aq. aeolicus
accumulates
sn
-glycero-3-phospho-3-l-
myo
-inositol.
IMPORTANCE
Compatible solutes of hyperthermophiles show high efficacy in thermal protection of proteins in comparison with solutes typical of mesophiles; therefore, they are potentially useful in several biotechnological applications. Glycerophosphoinositol (GPI) is synthesized from CDP-glycerol and
l
-
myo
-inositol 1-phosphate in a few hyperthermophiles. In this study, the molecular configuration of the GPI stereoisomers accumulated by members of the
Bacteria
and
Archaea
was established. The stereospecificity of glycerol phosphate cytidylyltransferase (GCT), the enzyme catalyzing the synthesis of CDP-glycerol, is crucial to the stereochemistry of GPI. However, the stereospecific properties of GCTs have not been investigated thus far. We devised a method to characterize GCT stereospecificity which does not require
sn
-glycerol 1-phosphate, a commercially unavailable substrate. This led us to understand the biochemical basis for the distinct GPI stereoisomer composition observed in archaea and bacteria.
Funder
Fundação para a Ciência e a Tecnologia
Publisher
American Society for Microbiology
Subject
Ecology,Applied Microbiology and Biotechnology,Food Science,Biotechnology
Cited by
8 articles.
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