Academics

Abstract 

Gm-ha1 is a protein of unknown function with the PDB ID: 4RNL. This protein is found in Streptomyces platensis, which is a bacteria species that produces two highly effective antibiotics. Despite the protein being of unknown function, it is hypothesized that this protein functions as a galactose mutarotase. Galactose mutarotase, also known as an aldose-1-epimerase, is an enzyme that commits the first step in the metabolism of galactose. In order to investigate the function of gm-ha1, various bioinformatic methods were used. ProMOL revealed that gm-ha1 had a homologous active site to PDB: 1snz, which is a human galactose mutarotase enzyme. Moreover, Pfam showed that gm-ha1 had conserved residues with the aldose-1-epimerase family and likely had a similar sequence to other proteins in that family. Further, Dali showed gm-ha1 had a high level of global alignment with other aldose-1-epimerase enzymes. Besides, Autodock and PyMOL showed that NAD was most likely an important ligand in relation to gm-ha1 enzyme catalysis. Lastly, purification was performed on a different protein (c8orf32) and confirmed the presence of purified protein in the gel. However, the kinetics experiment of the same protein proved that the data was invalid and could not be used in this study. The bioinformatic data obtained in this study do not reject the hypothesis that gm-ha1’s function as a galactose mutarotase enzyme. This was expected according to the data of the PDB profile of gm-ha1 (Tan et al. 2014). If the function of gm-ha1 is confirmed, it can give more knowledge of the Streptomyces platensis bacteria strain which can ultimately result in increased production of important antibiotics. 

Introduction 

The galactose mutarotase enzyme is a common protein found in both prokaryotes and eukaryotes. It is essential for the metabolism of galactose as it converts beta-D-galactose to alpha-D-galactose, which is the first step in normal galactose metabolism. This interchange can occur spontaneously in pure water in vitro, but organisms require the enzyme to perform this conversion in vivo because this reaction requires catalysis (Bouffard et al. 1994). In Escherichia Coli, galactose mutarotase is coded in the gal operon. This operon is induced by D-galactose, which indicates that the galactose mutarotase has some importance when D-galactose is present (Lee et al. 2008). Galactose mutarotase is essential for many prokaryotic cells as galactose is an abundant sugar and an excellent source of energy for bacteria. 

The PDB ID: 4RNL protein is found in Streptomyces platensis and is proposed to be a galactose mutarotase. However, this has not been confirmed as its function is unknown (Tan et al. 2014). It may prove beneficial to understand the function of the proteins in Streptomyces platensis as this species has a very important use in society. Streptomyces platensis is responsible for producing two key antibiotics, platensimycin and platencin. These two antibiotics have been shown to be effective against bacteria strains, such as MRSA and Streptococcus pneumoniae, that have developed resistance to many antibiotics (Falzone et al. 2017). Some researchers experimented and found a way for Streptomyces platensis to overproduce these two antibiotics tenfold the normal amount (Smanski et al. 2009). Learning how to overproduce antibiotics is a huge benefit to the medical field as antibiotics are in high demand, especially potent ones such as platensimycin and platencin (Smanski et al. 2009). A study that endeavored to determine the nutrients that increase Streptomyces platensis growth used a medium with high levels of glucose and lactose, which had growth of the bacteria on it. This indicates that Streptomyces platensis most likely utilizes galactose in its metabolism and growth. Therefore, determining the function of this protein may prove beneficial for providing media for culturing Streptomyces platensis. The protein was not named, so in this manuscript, the protein will be called gm-ha1. However, this study focused on the 4RNL-A chain for all sections of this report, so this name will be used when necessary. 

The original gm-ha1 study used E. coli which is one of the most common bacteria used for biochemical studies. One experiment had cloned the mutarotase gene of Acinetobacter calcoaceticus, which is another type of bacteria. This was performed by a complicated process of purifying the proteins, creating probes for the gene, cloning the gene, inserting the gene into a plasmid, and inserting the plasmids into E. coli (Gatz et al. 1986). In theory, this same process should be able to be performed on Streptomyces platensis to produce the same resulting E. coli with the mutarotase gene present. Another study that examined the mechanisms of galactose mutarotase had identified the amino acids and their positions that are crucial in performing the mutarotase functions. For example, it identified that Glu 304 and His 170 are the key peptides for catalysis (Thoden et al. 2003). The sequence of the 4RNL structure is listed in the PDB, so perhaps the sequence can be examined to see if the key peptides listed in this study are present in the same location in gm-ha1. We hypothesize that the gm-ha1 protein functions as a galactose mutarotase enzyme.

Read the Full Article Here