The building blocks of enzymatic and functional structures in living organisms are proteins created by linking amino acids into peptides (sub units of proteins). The mechanisms for creating peptides in proteins and some coenzymes are the clues that White and colleagues are following. "Enzymes that mechanistically do the same thing are included into a family, and we believe that there is an ancestral enzyme for this family," says David Graham, who was an NSF postdoctoral fellow in microbial biology at Virginia Tech.
In their attempt to reconstruct biochemical history, White's group has discovered two enzymes in Methanococcus jannaschii that may predate the cell's use of ribosome to build proteins. Their research will be reported in the Proceedings of the National Academy of Science (PNAS) by Hong Li, a post doc at Virginia Tech; Huimin Xu, a Virginia Tech technician, Graham, now at the University of Texas at Austin, and White, professor of biochemistry. The article (#3391), "Glutathione synthetase homologs encode a-L-glutamate ligases for methanogenic coenzyme F420 and tetrahydrosarcinapterin biosyntheses," will be published in the PNAS online Early Edition during the week of Monday, Aug. 4 - Friday, Aug. 8, 2003.
"We found two enzymes, MptN and CofF, which are descendants of the ATP-grasp superfamily," says White.
The ATP superfamily is a group of enzymes that use ATP -- the nucleotide energy source for the cell. "ATP-grasp" refers to a shared nucleotide-binding method. Every self-sustaining, living organism has ATP superfamily enzymes. "We are interested in determining the functions of genes and how coenzymes are made," says Graham.
The two newly discovered genes share a common ancestor with the ribosomal protein S6:glutamate ligase and a putative a-aminoadipate ligase, defining the first group of ATP-grasp enzymes with a shared amino acid substrate specificity.
"Most people learn in high school biology about ribosomes' role in making protein, but there is a whole other world without ribosomes - interesting predecessors to how peptides were formed before ribosomes," says Graham.
White's group studies archaea, one of the earliest forms of life -- from when the earth was hot and soupy. Archaea are now found in such places as ocean vents and camel guts. "We are looking at present metabolism to extrapolate to ancient life," says White.
"MptN and CofF both produce alpha glutamate bonds (the same as in proteins), so we infer that an ancestor protein was also making alpha glutamate bonds," says Graham. "The mechanism is the same, but the substrate that the glutamate is attaching to is really different."
The compounds range from a protein to a small molecule, says White.
"We have defined a family that shares the same ability to add alpha glutamate," says Graham. "But we don't know why, yet."
Li also discovered another enzyme, CofE, which may predate ribosome. It makes gamma-linked glutamate bonds. Her article, "CofE catalyzes the addition of two glutamates to F420-0 in F420 coenzyme biosynthesis in Methanococcus jannaschii" is forthcoming in the journal Biochemistry.
"Our initial interest in how F420 is made led to discovery of one new enzyme in sarcinpterin and two enzymes in F420 that are mechanistically related. They all have glutamate in their chemical structure and share a common reaction method for adding this amino acid," says White. "This work has shown how changes in members of a superfamily of enzymes can lead to a wider diversity in their function - in this case the biosynthesis of coenzymes."
After millions of years, the genealogy of life is more like a spider web, White says. "You never know where you will end up, which makes it exciting. We are working on one spoke of the spider web and want to go back to the center.
"In the meantime, we have expanded our knowledge of gene function, which is a central goal of our work."
The reviewers of the PNAS article commented that the research increased the understanding of the ATP superfamily and appreciated the elucidation of the relationships between two members in terms of coenzyme biosynthesis.
Li received her Ph.D. in biochemistry from Virginia Tech in May 2002 and plans to continue her research.
Hong Li, firstname.lastname@example.org, can also be reached at Dr. White's number.
David Graham, email@example.com
PR CONTACT at Virginia Tech: Susan Trulove, 540-231-5646, STrulove@vt.edu
Elucidation of methanogenic coenzyme biosyntheses: from spectroscopy to genomics (1971 to 2001) David E. Graham, Robert H. White, published in the Royal Society of Chemistry journal, Natural Product Reports, 2002 issue 2.
Glutathione synthetase homologs encode a-L-glutamate ligases for methanogenic coenzyme F420 and tetrahydrosarcinapterin biosyntheses
Hong Li, Huimin Xu, David E. Graham, and Robert H. White
Department of Biochemistry (0308), Virginia Polytechnic Institute and State University, Blacksburg, VA 24061
Edited by Stephen J. Benkovic, Pennsylvania State University, University Park Campus, University Park, PA, and approved June 30, 2003 (received for review June 4, 2003)
Proteins in the ATP-grasp superfamily of amide bond-forming ligases have evolved to function in a number of unrelated biosynthetic pathways. Previously identified homologs encoding glutathione synthetase, D-alanine:D-alanine ligase and the bacterial ribosomal protein S6:glutamate ligase have been vertically inherited within certain organismal lineages. Although members of this specificity-diverse superfamily share a common reaction mechanism, the non-overlapping set of amino acid and peptide substrates recognized by each family provided few clues as to their evolutionary history. Two members of this family have been identified in the hyperthermophilic marine archaeon Methanococcus jannaschii and shown to catalyze the final reactions in two coenzyme biosynthetic pathways. The MJ0620 (mptN) locus encodes a tetrahydromethanopterin:a-L-glutamate ligase that forms tetrahydrosarcinapterin, a single carbon-carrying coenzyme. The MJ1001 (cofF) locus encodes a g-F420-2:a-L-glutamate ligase, which caps the g-glutamyl tail of the hydride carrier coenzyme F420. These two genes share a common ancestor with the ribosomal protein S6:glutamate ligase and a putative a-aminoadipate ligase, defining the first group of ATP-grasp enzymes with a shared amino acid substrate specificity. As in glutathione biosynthesis, two unrelated amino acid ligases catalyze sequential reactions in coenzyme F420 polyglutamate formation: a g-glutamyl ligase adds 1-3 L-glutamate residues and the ATP-grasp type ligase described here caps the chain with a single a-linked L-glutamate residue. The analogous pathways for glutathione, F420, folate and murein peptide biosyntheses illustrate convergent evolution of non-ribosomal peptide biosynthesis through the recruitment of single-step amino acid ligases.