Identifying enzyme’s regioselectivity from an indicator diagram

This paper was part of my journal club recently. I touched upon LPMOs, short for Lytic Polysaccharide Monooxygenases, in my previous post that are basically oxidative enzymes.

These interesting group of enzymes have three basic types: Type I, Type II, and Type III, classified based on the site of attack, namely LPMO1 (Type I) when oxidation occurs at C1 carbon, LPMO2 (Type II) when oxidation occurs at C4 carbon, and LPMO3 (Type III) if either C1 or C4 carbons are attacked. These subtypes are part of four CAZy families to which LPMOs are categorized into (AA9, AA10, AA11, and AA13).

Having said this, the identification of the types in LPMO is not a trivial task. This specificity to cleave a particular bond, or regiospecificity, is characterized by time-consuming chromatography experiments (HPAEC-PAD), as they are time course studies that involve incubating with the substrate for longer periods. If aldonic acids are discovered in the experiments, then it is C1 cleaving or Type I; and if 4-gemdiol-aldose is detected than it is C4 cleaving of Type II LPMOs.

Given this complex identification protocol, any shortcut to identify the regiospecificity is welcome and that’s what Danneels et al have attempted in their paper published in PLOS ONE. Specifically, using an indicator diagram based identification, they give a solution to identify regiospecificity.

To test they used Hypocrea jecorina‘s LPMO9A (having both C1/C4 cleavage) and did site-directed mutagenesis on key aromatic residues that are involved in substrate binding to create mutants that are either selective to C1 or C4 cleavage. Comparing the activity of the wildtype with the mutants by plotting the speed of release of aldonic acids with respect to 4-gemdiol-aldose the authors plot it as an indicator diagram. Basically, if one calculate the slope of the line, and it is closer to x-axis (release of aldonic acid) then the enzyme’s regiospecificity is for C1 oxidation or consisting of Type I LPMO activity. If closer to y-axis, then Type II LPMO activity.

It would be interesting to see this type of indicator diagram applied for enzyme activity identification for new LPMO enzymes, and also for enzyme engineering studies on LPMO.

Reference: B. Danneels, M. Tanghe, H. Joosten, T. Gundinger, O. Spadiut, I. Stals and T. Desmet, “A quantitative indicator diagram for lytic polysaccharide monooxygenases reveals the role of aromatic surface residues in HjLPMO9A regioselectivity“, 2017. .

What about Lignin?

Image Courtesy: i.bnet.com

Biofuel prodcution involves removing Lignin from the biomass, in fact efficient removal so that Lignin and its by-products do not inhibit the enzymatic process that follows. But, what happens to the Lignin? Well, it can used in laying roads and thus creating bioasphalt.

Usually, after the sugars, cellulose, and other more useful materials have been extracted from plant matter to make biofuels or paper, the leftover lignin is tossed aside and burned. In principle, the economics are therefore promising: Paper companies could profit from what had been a waste product, and biofuel makers could similarly use the proceeds of selling lignin to bring down fuel production costs.

FYI, Lignin looks something like this.

Lignin. Image Courtesy: Wikimedia Commons

Read more here

 R. Chris Williams, a materials engineer at the Iowa State University Institute for Transportation, has developed a way to turn lignin-rich stover, leftover from biofuel production, into bioasphalt. The Iowa team uses a process called fast pyrolysis that turns plant waste into a charcoal-like fertilizer, natural gas, and an oily mixture that can be made into bioasphalt.

If you live in Iowa, you could see the bioasphalt paved bike path in Des Moines as the article mentions.

Update: My friend Ethy, pointed out that the bike trail (~1 mile long) between University Avenue and Franklin Avenue, on the west side of Glendale Cemetery has this bioasphalt bike path. See map below. Link: http://www.news.iastate.edu/news/2010/oct/bioasphalt

Google Maps

Bourzac, K. (2015). Inner Workings: Paving with plants Proceedings of the National Academy of Sciences, 112 (38), 11743-11744 DOI: 10.1073/pnas.1509010112

Enzyme specificity – It may all be in the loop

This post is about an article that got published last week in Journal of Biological Chemistry (JBC). Let me tell you why I found this very interesting. Metagenomic sequences are filling and going to fill the databases with lot of new sequences. In the case of enzymes there is going to be a huge list of sequences from new genomic sequences that on a preliminary screening shows as having a potential enzymatic activity. However, most of them do not register any activity on substrates. This becomes problematic for two reasons:

1. There are hardly any distinguishing features in homolog sequences that can be used to identify active vs. non active
2. In most cases, homologs act on different substrates, either exclusively or have mixed specificity

Thus, any identification/feature that can shed light on substrate specificity (which can tell whether the enzyme will be active or not) would be of immense help to screen true-positives more effectively. In this paper, Sukharnikov et al have used the Glycosyl Hydrolase 48 (GH48) family of enzymes that have shown to have cellulolytic activity. Basically, they are endoglucanases that cleave an internal glycosidic bond.

Cellulose to Glucose
Image Courtesy: http://discovery.kcpc.usyd.edu.au/9.2.2-short/9.2.2_CellulosetoEthene.html

So, they took the sequences of GH48 with known activity from CAZy and other sequences that were picked from NCBI’s nr database using the Pfam GH48 domain information, did a multiple sequence alignment and built a tree. Using this one can easily find orthologs (one copy per genome and come from a phyla that shares the same ancestor with another species), paralogs (two or more copies per genome), and horzontal gene transferred (based on phyletic distribution and probabilistic approach) genes (HTG).

It was clearly seen that the prokaryotic GH48 sequences shared a common ancestor; paralogs retained the conserved residues in the catalytic domain and showed “innovation” with the auxillary domains (like Carbohydrate binding module or CBM). The insteresting outcome of this analysis was the horizontally transferred genes (HTG) from the prokaryotic genome to eukaryotic genome (Fungi and Insects). To test this, one of the HTG genes from Hahella chejuensis when tested on amorphous cellulose, it showed cellulase activity.

By this time, you might wonder where I am leading all this too. I left the best part for the last, since the authors solved the structure of the HGT GH48, and when compared with other structural homologs, a particular omega loop facing the substrate binding part of the protein has a change in conformation. In other GH48 sturctures, this loop has identical conformation, but not in the HTG GH48! Moreover, the insect GH48 sequences (obtained from metagenomic sequences) were seen to lack cellulolytic activity and had chitinase activity and this was seen due to absence of the omega loop.

In summary, the authors suggest two things:

• for GH48 sequences in the prokaryotic to be cellulolytic the conserved residues from the prokarytes can be used as a genomic signature
• The GH48 from metagenomic insect sequences have evolved to accommodate the bulkier chitin. For which, they probably had to lose the omega loop.

So, its all in the loop…

UPDATE: The structure details of H. chejuensis can be found here – http://www.rcsb.org/pdb/explore/explore.do?structureId=4fus

Sukharnikov, L., Alahuhta, M., Brunecky, R., Upadhyay, A., Himmel, M., Lunin, V., & Zhulin, I. (2012). Sequence, structure, and evolution of cellulases in the glycoside hydrolase family 48 Journal of Biological Chemistry DOI: 10.1074/jbc.M112.405720