Picture a factory assembly line where two machines receive the exact same raw material but somehow produce completely different finished products. Now shrink that factory down to a size invisible to the naked eye, place it inside a soil dwelling bacterium, and you have one of the most fascinating puzzles in modern biochemistry. How does nature build chemical diversity from a single starting point?
A new study published in Nature Chemistry has not only answered that question for a pair of antibiotic producing enzymes but has gone a step further and used that understanding to engineer molecules that have never existed before in nature. The implications reach well beyond academic curiosity. In an era when antibiotic resistance is one of the most pressing threats to global public health, the ability to rationally design new antibiotic structures could matter enormously.
The Antibiotics You May Never Have Heard Of
The story begins with a family of antibiotics called lincosamides. The most famous member of this family is lincomycin, a natural compound produced by a soil bacterium called Streptomyces lincolnensis. Its close relative clindamycin, a widely used clinical antibiotic prescribed today for serious infections, is a semisynthetic version of lincomycin.
Lincosamide antibiotics work by slipping inside bacterial ribosomes, the molecular machines that bacteria use to build their own proteins. Once inside, the antibiotic jams the ribosome and brings protein synthesis to a halt. No proteins, no growth, no infection.
A related lincosamide called celesticetin is produced by a different Streptomyces bacterium and carries a slightly different chemical structure. Specifically, the part of the molecule known as the S-alkyl group, a chemical attachment on the sulfur containing core of the antibiotic, differs between lincomycin and celesticetin. Previous research has shown that this structural difference matters considerably because it strongly influences how potent the antibiotic is. A more elaborate attachment at this position can dramatically boost antibacterial activity.
So the central question was this: how do bacteria build these structurally distinct lincosamides, and could scientists exploit that process to build new ones?
Two Enzymes, One Substrate, Two Different Outcomes
At the heart of lincosamide biosynthesis are two enzymes called LmbF and CcbF. Both belong to a broad category of biological catalysts that use a molecule called pyridoxal 5 phosphate as a helper. This compound is a form of vitamin B6, and enzymes that depend on it are among the most versatile chemical toolkits found in nature, capable of catalysing dozens of different types of reactions.
What makes LmbF and CcbF so intriguing is that they receive the same molecular input, a compound called S-glycosyl L-cysteine, and yet they process it in completely different ways. LmbF snips a chemical bond between carbon and sulfur through a reaction called beta elimination, generating a thiol group that gets further modified to produce lincomycin. CcbF, by contrast, strips off a carbon dioxide molecule and simultaneously performs an oxidative reaction involving molecular oxygen, a more complex transformation that eventually gives rise to the aldehyde group characteristic of the celesticetin pathway.
Same input, different chemistry, different antibiotics. A research team set out to understand precisely why.
"The reaction selectivity and oxygen utilization of LmbF and CcbF were rationally engineered through structure and calculation based mutagenesis. The catalytic function of CcbF was switched to that of LmbF, and remarkably, both LmbF and CcbF variants gained oxidative amidation activity to produce an unnatural S-acetamide derivative of lincosamide."
A Molecular Photograph at Atomic Resolution
The research team used X-ray crystallography to determine the three dimensional structures of both LmbF and CcbF at atomic resolution, achieving a precision of 1.7 and 1.8 angstroms respectively. An angstrom is one ten billionth of a metre. At this level of detail, individual atoms become visible and the positions of every chemical group inside the enzyme active site can be mapped precisely.
The two enzymes turned out to be highly similar in overall shape. Their molecular backbones can be superimposed on each other with a difference of only 1.6 angstroms across nearly 400 amino acid positions. Yet despite this overall similarity, four specific positions in the active site differ between LmbF and CcbF, and those four positions turn out to be everything.
To go beyond the static crystal structures and watch how each enzyme actually handles its substrate in motion, the team performed computational docking followed by 50 nanosecond molecular dynamics simulations. Think of this as a molecular movie, showing how the substrate settles into the enzyme and how each atom moves relative to the others over time. These simulations revealed that the substrate binds in a subtly different orientation inside each enzyme, and that difference in orientation is what steers the reaction down one chemical pathway or another.
The Molecular Switch: A Few Amino Acids Change Everything
The four residues that distinguish LmbF from CcbF are all aromatic amino acids, meaning they contain ring shaped chemical structures. In CcbF, positions occupied by smaller amino acids in LmbF are instead filled by tyrosine residues, which carry a bulky ring structure.
These tyrosine residues act as gatekeepers. In CcbF, their presence steers the substrate into a conformation where the bond to be broken during decarboxylation is positioned at exactly the right geometry for that reaction to proceed. At the same time, the tyrosines keep the reactive intermediate isolated from amino acids that would otherwise quench the reaction prematurely, leaving it free to react with oxygen and complete the oxidative transformation.
In LmbF, the absence of those bulky tyrosine residues allows the substrate to sit in a different orientation, favouring the beta elimination reaction instead.
In essence, a handful of amino acid substitutions in the active site acts as a molecular traffic director, routing the same chemical raw material down two completely different reaction pathways.
Rewriting the Rules of Two Enzymes
Once the structural logic was clear, the team did something remarkable. They used this understanding to redesign the enzymes, swapping amino acids at key positions to alter which reaction each enzyme would carry out.
By substituting tyrosine residues in CcbF with smaller amino acids at three positions simultaneously, they converted CcbF into an enzyme that performs the beta elimination reaction characteristic of LmbF. The catalytic identity of one enzyme had been switched to that of another through rational, structure guided protein engineering.
But the most striking discovery came from a different set of mutations. When specific residues in either LmbF or CcbF were altered in a particular way, both enzymes gained an entirely new activity that neither wild type enzyme possesses. The engineered variants began producing a compound called an S-acetamide derivative of lincosamide, a molecule that has never been reported before in nature.
Oxygen isotope labelling experiments confirmed that molecular oxygen is the source of the oxygen atom in this new product. Essentially, the engineered enzymes learned to use oxygen in a novel way, producing a brand new chemical group on the antibiotic scaffold.
KEY FACTS
What is a lincosamide antibiotic? A class of antibiotics produced by Streptomyces bacteria that work by binding to bacterial ribosomes and blocking protein synthesis. Clindamycin, derived from lincomycin, is a widely used clinical antibiotic.
What is pyridoxal 5 phosphate? A form of vitamin B6 that serves as a cofactor for a large family of enzymes. These enzymes catalyse an enormous variety of chemical reactions including amino acid metabolism and natural product biosynthesis.
What did the researchers discover? That just four amino acid differences in the active sites of LmbF and CcbF determine whether the enzyme performs beta elimination or oxidative deamination, explaining how two structurally similar enzymes can produce chemically distinct antibiotics.
What is the new molecule? An S-acetamide derivative of lincosamide, a compound not previously found in nature, produced when engineered variants of both LmbF and CcbF gained a novel oxidative amidation activity.
Why This Matters for the Antibiotic Crisis
Antibiotic resistance is a growing global emergency. Bacteria evolve resistance to existing drugs, and the pipeline of new antibiotics has been alarmingly thin for decades. One of the major bottlenecks is chemical diversity. Most antibiotics in clinical use belong to a relatively small number of structural families, and bacteria have had decades to develop resistance mechanisms against them.
What this research demonstrates is a more elegant approach to that problem. By understanding the molecular logic of how enzymes build structural diversity into natural products, scientists can use that logic as a design blueprint. The ability to rationally switch an enzyme from one reaction to another, or to engineer it to perform a reaction that does not exist in nature, opens a path toward creating libraries of new antibiotic structures for testing.
The S-acetamide derivative produced by the engineered enzymes represents precisely that kind of novel structure. Whether it has useful antibacterial properties remains to be tested, but the principle it demonstrates, that enzyme engineering guided by structural understanding can generate genuinely new chemistry, is what the field has been working toward.
A Broader Window Into Enzyme Evolution
PLP dependent enzymes are extraordinarily widespread in biology, found in organisms from bacteria to humans, and they catalyse an enormous variety of reactions. The mechanistic insights gained from studying LmbF and CcbF, particularly the understanding of how active site geometry controls reaction selectivity and how oxygen utilization is governed by specific residues, add meaningfully to the general understanding of this entire enzyme class.
The study also provides a clear demonstration that structure and calculation based enzyme engineering is maturing as a discipline. Rather than relying purely on trial and error mutagenesis, the researchers used crystal structures and molecular dynamics simulations as predictive tools, making targeted changes that produced expected outcomes. This kind of rational, computation assisted engineering is becoming an increasingly powerful strategy in the creation of new biocatalysts for medicine, agriculture, and industrial chemistry.
For now, a pair of bacterial enzymes from soil microbes have shown us that the molecular machinery of life contains untapped chemical creativity, and that with the right tools we are beginning to unlock it.
Publication Details: Year of publication: 2025 Journal: Nature Chemistry Publisher: Springer Nature Volume / Pages: Volume 17, February 2025, pp. 256–264 DOI: https://doi.org/10.1038/s41557-024-01687-7
Credit & Disclaimer: This article is based on the peer reviewed research paper. All scientific facts, findings, and conclusions presented here are drawn directly from the original study and remain unchanged. This popular science article is intended purely for general educational purposes. Readers are strongly encouraged to consult the full research article for complete experimental data, methodology, and detailed scientific analysis.
