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Journal of Bacteriology, June 2001, p. 3804-3810, Vol. 183, No. 12
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.12.3804-3810.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

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

Biswarup Mukhopadhyay,* Endang Purwantini,dagger Cynthia L. Kreder,Dagger and Ralph S. Wolfe

Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

Received 15 December 2000/Accepted 7 March 2001

Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO2 fixation reaction is catalyzed by an apparent alpha 4beta 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°C, insensitive to aspartate and glutamate, mildly inhibited by alpha -ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg2+. It showed negative cooperativity towards bicarbonate at 70°C but not at 37°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)ProX5Phe(Tyr) to be fully conserved in biotin-dependent enzymes; it might function as the hinge for their "swinging arms."

* Corresponding author. Mailing address: University of Illinois at Urbana-Champaign, Department of Microbiology, B103 Chemical and Life Sciences Laboratory, 601 S. Goodwin Ave., Urbana, IL 61801. Phone: (217) 333-1397. Fax: (217) 244-6697. E-mail: biswarup{at}life.uiuc.edu.

dagger Present address: Department of Pharmacy, Institut Teknologi Bandung, Bandung 40173, Indonesia.

Dagger Present address: Rigel, Inc., South San Francisco, CA 94080.

Journal of Bacteriology, June 2001, p. 3804-3810, Vol. 183, No. 12
0021-9193/01/$04.00+0   DOI: 10.1128/JB.183.12.3804-3810.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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