Purification and Characterization of the Alanine Aminotransferase from the Hyperthermophilic Archaeon Pyrococcus furiosus and Its Role in Alanine Production

Abstract

Alanine aminotransferase (AlaAT) was purified from cell extracts of the hyperthermophilic archaeon Pyrococcus furiosus by multistep chromatography. The enzyme has an apparent molecular mass of 93.5 kDa, as estimated by gel filtration, and consists of two identical subunits of 46 kDa, as deduced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the gene sequence. The AlaAT displayed a broader substrate specificity than AlaATs from eukaryal sources and exhibited significant activity with alanine, glutamate, and aspartate with either 2-oxoglutarate or pyruvate as the amino acceptor. Optimal activity was found in the pH range of 6.5 to 7.8 and at a temperature of over 95°C. The N-terminal amino acid sequence of the purified AlaAT was determined and enabled the identification of the gene encoding AlaAT (aat) in the P. furiosus genome database. The gene was expressed in Escherichia coli, and the recombinant enzyme was purified. The pH and temperature dependence, molecular mass, and kinetic parameters of the recombinant were indistinguishable from those of the native enzyme from P. furiosus. The k cat /K m values for alanine and pyruvate formation were 41 and 33 s ؊1 mM ؊1 , respectively, suggesting that the enzyme is not biased toward either the formation of pyruvate, or alanine. Northern analysis identified a single 1.2-kb transcript for the aat gene. In addition, both the aat and gdh (encoding the glutamate dehydrogenase) transcripts appear to be coregulated at the transcriptional level, because the expression of both genes was induced when the cells were grown on pyruvate. The coordinated control found for the aat and gdh genes is in good agreement with these enzymes acting in a concerted manner to form an electron sink in P. furiosus.The hyperthermophilic archaea are a group of phylogenetically related microorganisms which by definition grow optimally at or above 80°C, with a maximal growth temperature of 90°C or higher (5). The majority of these are strictly anaerobic heterotrophs, most of which are obligately dependent upon the reduction of elemental sulfur (S 0 ) to H 2 S. A limited number of facultative S 0 -reducing species are able to grow in the absence of S 0 by means of an alternative fermentative-type metabolism.

Insights on the Evolution of Metabolic Networks of Unicellular Translationally Biased Organisms from Transcriptomic Data and Sequence Analysis

“…In addition, ferredoxin has been suggested to be reoxidized by ferredoxin-NADP oxidoreductase (Silva et al 2000;Schut et al 2001). Moreover, NADPH can also be oxidized via the conversion of pyruvate to alanine, via glutamate dehydrogenase (CAI(PAB0391)=0.74) and alanine aminotransferase (CAI(PAB1810)=0.55) (Ward et al 2000). The high bias of all genes involved in ferrodoxin metabolism hints at the importance of this pathway for the organism.…”

Archaeal Fructose-1,6-bisphosphate Aldolases Constitute a New Family of Archaeal Type Class I Aldolase

“…To map the transcription start site of the fba-pfp transcript the 5Ј-32 P-labeled antisense oligonucleotide (5Ј-CCGTGCTCAATGCCGTGG-3Ј, position 72-89 of the fba gene) was used as primer for cDNA synthesis. For P. furiosus total RNA was isolated from maltose and pyruvate grown cells as described previously (40), and the transcription start was determined with a fluorescence (IRD800)-labeled antisense oligonucleotide (5Ј-CAAAGTCCG-TAGGGCCGTGC-3Ј (MWG), position 99 -118 of the fba gene). The primer extension reaction was performed using the Reverse Transcription System (Promega) according to the instructions of the manufacturer with the following modifications.…”

Isolation of a Mutant Auxotrophic forL-Alanine and Identification of Three Major Aminotransferases That SynthesizeL-Alanine inEscherichia coli

“…40) In addition, the seven other invariant residues in family I aminotransferase (Y70, P195, N194, P195, Y225, R266, and G268, the numbers of which are according to cytosolic pig AspAT) were also conserved in YfbQ and YfdZ. 41,42) The functional roles of these conserved residues are known to be binding with substrates and cofactor PLP. 36,41,42) Together with a phylogenetic analysis of these aminotransferases (data not shown), YfbQ and YfdZ are likely to belong to aminotransferase family I.…”

“…41,42) The functional roles of these conserved residues are known to be binding with substrates and cofactor PLP. 36,41,42) Together with a phylogenetic analysis of these aminotransferases (data not shown), YfbQ and YfdZ are likely to belong to aminotransferase family I.…”