Oxaloacetate synthesis in the methanarchaeon Methanosarcina barkeri: pyruvate carboxylase genes and a putative Escherichia coli-type bifunctional biotin protein ligase gene (bpl/birA) exhibit a unique organization.
Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO(2) fixation reaction is catalyzed by an apparent alpha(4)beta(4)-type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits. The purified enzyme was most active at 70 degrees C, insensitive to aspartate and glutamate, mildly inhibited by alpha-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg(2+). It showed negative cooperativity towards bicarbonate at 70 degrees C but not at 37 degrees C. The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin. pycA, pycB, and a putative bpl gene formed a novel operon-like arrangement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M. barkeri and the closely related euryarchaeon Archaeoglobus fulgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL). We found the element Tyr(Phe)ProX(5)Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their "swinging arms."
Mukhopadhyay
B
B
Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801, USA. biswarup@life.uiuc.edu
Purwantini
E
E
Kreder
C L
CL
Wolfe
R S
RS
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D017124:Conserved Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D006361:Heating; D017021:Methanosarcina barkeri / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D009876:Operon; D062907:Oxaloacetic Acid / Q000378:metabolism*; D011766:Pyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D062907:Oxaloacetic Acid; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli; D011766:Pyruvate Carboxylase