Tuesday, February 26, 2019

Microbial Carcinogens in the Human Large Intestine

The microbial micro-environment in the human large intestine is intricate and complex. In fact, there are many hundreds of small molecules and metabolites produced by this diverse population of microflora that may profoundly influence human health. Many of these substances are produced by enzyme-directed pathways that have been shown to be programmed by so-called, “bacterial biosynthetic gene clusters.” 

One class of these molecules, colibactins, has been shown to be produced from a gene cluster called the, polyketide synthase island (PKS). PKS occurs in certain strains of Escherichia coli (E-coli) that seem to be prevalent in the microbiota of colorectal cancer (CRC) patients. Up until this time, despite many years of painstaking research, little has been discovered regarding the structure and the mode of action of colibactins.

Dr. Matthew Wilson and his colleagues at Vertex Pharmaceuticals have recently published a paper in the journal Science in which they describe the mode of action of colibactins. According to the author, “colibactin alkylates DNA in cultured cells and in vivo, forming covalent modifications known as DNA adducts. These colibactin-DNA adducts are chemical evidence of DNA damage and represent a detectable signature of exposure to colibactin. Misrepaired DNA adducts may generate mutations that contribute to colorectal tumorigenesis.”

In their research Wilson’s group identified the colibactin-DNA adducts as involving the cyclopropane ring and that the site of alkylation involves the nucleotide adenine within the DNA backbone (see diagrams below). Furthermore, it is believed that these adducts could lead to mutations in the oncogenes or tumor suppressor genes that drive CRC-related tumorigenesis.



Although, this model of colibactin involvement in DNA modification is significant, many questions remain unanswered in regard to how tumorigenesis is subsequently initiated in CRC. However, it is line of research that offers some promise in elucidating cancer-causing mechanisms.

Thursday, January 24, 2019

Evolution of an Enzyme from Short Peptide Pieces

Enzymes are complex protein molecules that are responsible for accelerating chemical reactions in the living cell that essentially make life possible. Each unique enzyme is responsible for a particular chemical transformation. The sum total of all of these reactions is what is referred to as cellular metabolism. The primary structure of enzymes – the sequence of amino acids embedded with the protein structure – is encoded within the particular gene responsible for the production of each unique enzyme.

It is of great interest to understand how such complex protein structures evolved from simpler structures that are known to have been available early in the evolution of life on planet earth – amino acids and peptide. In regard to proteins, the class of molecules that represent short pieces of protein is referred to as peptides. Peptides are short strands of amino acids tied together through peptide bonds. 



Enzymes generally require complex folding in their structures to foster their catalytic activity. Peptides are generally too short for this folding to occur. Recent research into the structure of metalloenzymes – enzymes that employ metal ions in their structures – suggest that metal ions may have helped induce folding in precursor peptides. Metalloenzymes are ubiquitous in nature and play fundamental roles in cellular biology and chemistry.

Sabine Studer from the Dana-Farber Cancer Institute, Boston and her colleagues have attempted to more fully understand the processes through which enzymes have evolved. They have done so by devising techniques for facilitating the transformation of a peptide capable of binding zinc into a functional enzyme with a complex globular structure.

According to the authors, “Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ∼ 10 6 M -1 s -1). The simultaneous optimization of structure and function in a na├»ve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.”

The techniques they successfully employed to accomplish this include (see diagram below)
Computational redesign
Cassette Mutagenesis – An in-vitro technique for altering genetic structure i.e. mutations.
DNA shuffling
Random mutagenesis
Alanine scan – Alanine is one of the amino acids that plays a critical role in protein structure.


Note: that the majority of the techniques employed involve manipulating the genetic information.

The work cited above is of seminal importance in the overall search for elucidating the molecular mechanisms that may account for the evolutionary development of life on planet earth from its elemental beginnings