Analysis of Bacterial Genomes Begins to Unravel Complex Story of Metabolic Evolution

As any biochemist knows, genetic change is really chemical change, and so it follows that if you want to really see how evolution happens, you need to see how it affects biochemistry.

A genetic analysis searching for the evolutionary history of nitrogenase, the critical enzyme system that helps life use atmospheric nitrogen, has shown some interesting evolutionary relationships between the key metabolic processes of bacteria, and revealed some mysterious new chemical pathways that are not yet understood.

In a paper published in the current issue of the journal Molecular Biology and Evolution , Arizona State University biochemists Jason Raymond, Christopher Staples and Robert Blankenship and Rice University's Janet Siefert do an analysis of the genomes of a large group of bacteria and archaea, comparing in particular similar genes that produce the protein nitrogenase.The researchers find that similar or “homologous” nitrogenase genes exist across a broad range of organisms, and appear to be related to other similar genes coding for proteins involved in photosynthesis, as well as to other genes in archaea and bacteria that do neither photosynthesis nor “nitrogen fixation” (as the process of capturing atmospheric nitrogen is called).

Though biochemists have previously concluded that nitrogenase and enzymes involved in photosynthesis have structural similarities and thus appear to be related, these latter “uncharacterized” genes appear to reveal enzymes whose metabolic properties are as yet unknown.

“We found a group of homologous genes that doesn't correspond to any genes that go with photosynthesis or any that we know in nitrogen fixation - we found these in a wide range of organisms,” said Raymond.

The analysis suggests that the genes that code for neither nitrogenase nor enzymes in photosynthesis may be “relics,” coding for  metabolic pathways that are ancestral to both photosynthesis and nitrogen fixation. Horizontal gene transfer - the exchange of genes between different bacterial species -- appears to be responsible for the broad distribution of the original gene and for its subsequent divergence and specialization in the metabolic pathways of nitrogen fixation and photosynthesis.

“These enzymes are important evolutionary inventions,” said Blankenship. “Once they develop, they get passed between species quite a bit because they give the organisms that have them important advantages.”

Of all of evolution's great biochemical developments, the ability of life to break up and “fix” atmospheric nitrogen  was one of the most important accomplishments, and perhaps one of the most challenging.

“Without nitrogen, you can't have life as we know it,” said Blankenship. “In the very early earth, there was probably some available nitrogen in the form of ammonia or something similar, so early life forms didn't have to extract nitrogen from the atmosphere.

“At some point though, things reached a food crisis - you either find some way to get the atmosphere's molecular nitrogen into the cycle or you die. A minimum input of nitrogen can't sustain a big biosphere,” he noted.

“But it is hard to do. Nitrogen fixation is one of the most interesting biological processes because it's so difficult to do chemically. Nitrogenase is a very complex enzyme system that actually breaks molecular nitrogen's triple bond -- one of the strongest bonds in nature,” he said.

The nitrogenase system is so sophisticated and complex that it is difficult to reconstruct its evolutionary development. In some of its most sophisticated forms, such as versions incorporating the rare metal molybdenum, the system uses a network of complex enzymes to control and regulate the process and make it energy efficient. Such a system could have evolved gradually through a series of small changes, but the analysis suggests instead that it might have developed through duplication of the gene for a more primitive enzyme that has just now been discovered.

However, even the simplest enzyme capable of breaking nitrogen's triple bond requires great structural complexity that could not have evolved without earlier stages.

“Breaking molecular nitrogen required a lot of energy and was an evolutionarily complex transition,” Blankenship notes. “Even the most basic nitrogenase complex that we have today is amazingly sophisticated and energetically a very expensive system. It's not something that would have just popped up out of nowhere.”

Blankenship and colleagues propose that the ancestral nitrogenase enzyme must have evolved from a simpler enzyme that performed a simpler metabolic pathway and the researchers suggest that some of the “uncharacterized” nitrogenase homologue genes that they have discovered - genes that code for enzymes that neither perform nitrogen fixation or photosynthesis - may hold clues to what that pathway

“We've discovered a new group of enzymes that were not previously known and because of their position in the phylogenetic tree, we think of these as more ancient,” he said. “We think that these as yet uncharacterized enzymes are probably also doing some kind of similar chemical reduction - reducing a multiple bond to a single bond. The structure of most nitrogenase homologues is such that they take the input of a large amount of energy and use it to break multiple bonds. We're pretty sure that these enzymes are doing a similar sort of activity, but we don't yet know what.”

“Our idea is that the very early activity was something that was not nitrogen fixation per se, but some other, probably simpler, chemical transformation, probably also involving nitrogen compounds. Then, at a later time, the ability to do nitrogen fixation evolved from that.”

Since a number of the organisms that have the unknown enzymes live in exotic environments with toxic and/or hydrothermic conditions, the researchers hypothesize that the uncharacterized enzymes may be relics of an ancestral enzyme that derived nutrients from toxic compounds in the ancient environment.

“We think it may be something that may have been useful in an ancient biosphere where certain compounds were more abundant - perhaps an enzyme for cyanide (HN 3 ) or azide (N 3 - ) reduction… but we're trying to keep an open mind,” Blankenship said.

According to Blankenship, the team is currently testing the metabolic activity of some of the uncharacterized genes by splicing them into E. coli and exposing the modified bacteria to various proposed toxic compounds.

Regardless of what the uncharacterized enzymes are found to metabolize, Raymond, Blankenship and colleagues feel that the process of nitrogenase evolution that they have uncovered also exposes some basic truths concerning how evolution happens in biochemical processes.

“Evolution is a great recycler, a junkman who takes some piece that was invented for some other purpose and reuses it in a new way,” Blankenship said. “Once you do that, you can end up with some amazing things, like the nitrogenase complex - a stunningly complicated molecular machine that does extremely difficult chemistry.”