Glucosamine N-Acetyltransferase (PDB ID: 5KF1), or GlmA, from the bacterium Clostridium acetobutylicum is an enzyme that exists physiologically as a dimer (1). GlmA has a total molecular weight of 76616.79 Daltons and an isoelectric point of 9.30 (2). GlmA does not complex with any prosthetic groups, does not have any associated metal ions, and does not engage in any drug interactions or alternate conformations (3). Each chain of GlmA complexes with acetyl coenzyme A (PDB ID: ACO), D-glucosamine, coenzyme A (PDB ID: COA), and N-acetyl-D-glucosamine, where the former two are substrates/ligands and the latter two are products of the transferase reaction (1). 

Clostridium acetobutylicum is a gram-positive bacteria, meaning that the peptidoglycan layer on the outside of the cell wall is not flanked on both sides by a lipid bilayer membrane (4). Although peptidoglycan is an integral part of the cell wall, its degradation accompanies cell growth; peptidoglycan degradation involves de-acetylation of its component N-acetylglucosamine (5). If these degradation products were not recycled into peptidoglycan, then a sizable amount of chemical capital would be wasted; thus, peptidoglycan recovery enzymes and their orthologs have been documented in gram-negative and gram-positive bacteria, respectively (6). GlmA is suspected to have an important role in cell wall recycling, as it catalyzes the transfer of the acetyl group on acetyl-CoA to the primary amino group on glucosamine (3). The resultant N-acetylglucosamine can then be used to rebuild peptidoglycan (7). A greater understanding of GlmA’s structure may lead to better comprehension of cell wall turnover, recovery, and structure. Increased knowledge in these areas will help chemical engineers more effectively use C. acetobutylicum in its role as a prominent producer of acetone, ethanol, and butanol (7).

GlmA is a dimer, and is composed of one A chain and one B chain, each containing 328 residues (1). Both subunits also bind glucosamine and acetyl-CoA. Each subunit has the same sequence and the same two domains: one from Met-1 to Ser-155 and the other from Gly-167 to Ala-316. However, only the C-terminal domain in each chain is catalytically competent. Each subunit of GlmA has 12 β-strands and 8 α-helices (with each domain containing 6 and 4, respectively), along with random coil regions spread over the structure. Additionally, the fourth β-strand in each domain contains a β-bulge, which may have an effect on CoA binding. The two chains’ primary structure equivalency suggests that modern day GlmA arose through gene duplication (3). This internal flanking of β-strands by α-helices, both of varying polarities, is a defining characteristic of GlmA’s protein family, named the general control non-depressible 5 (GCN5)-related N-acetyltransferases, or GNATs (8). Therefore, this specific secondary structure is directly related to the protein’s function, as that structure is highly conserved across GNATs.

Each chain also engages in numerous hydrogen bonds in order to facilitate substrate binding. For example, the C-3 and C-4 hydroxyl groups of glucosamine may bind to the carboxylate moiety of Asp-287. The pyrophosphoryl moiety of CoA hydrogen bonds in two locations; once to the amide backbone in residues Gly-265 and Arg-266, and another time to the carboxyl backbones of Ile-253 and Ile-255. Acetylated glucosamine also binds to the protein via hydrogen bonds with its own C-6 hydroxyl group and the aromatic side chain of Trp-193 and Glu-196. Trp-288 hydrogen bonds with the amino moiety of glucosamine (3). 

GlmA is abound with functionally important residues. Most of these residues are mainly involved in substrate and ligand binding via hydrogen bonding. However, a few more important functional residues exist. Asn-79 and Gly-251 function as β-bulge creators in the N-terminal and C-terminal domains, respectively. Finally, Tyr-297 and Asp-287 function as a catalytic acid and catalytic base respectively during the acetyl transfer in the active site pocket (3).

A comparable protein to GlmA is the multi-domain glycoside hydrolase family 3 β-N-acetylglucosaminidase from Rhizomucor miehei (PDB ID: 4ZM6), or RmNag (1). RmNag was selected as a comparison protein by querying PSI-BLAST and the Dali Server. BLAST, or the Basic Local Alignment Search Tool, searches through a selected database of biological sequences in order to find biological entries that are similar to an input sequence. PSI-BLAST is an extension of BLAST which deals with protein sequences. PSI-BLAST entries found to have similar regions with the query sequence will be returned with an Expect value (E-value), which is a parameter that relates the similarity between sequences by assigning gaps in their sequences and comparing their alignment. The E-value is a measure of significance, and the lower the E-value, the more significant the similarity between the query and result sequences; an E-value lower than 0.05 is considered a strong match. For example, RmNag’s E-value is 10^-20 (9). The Dali server, in contrast, is a tool that returns proteins which have similar tertiary structures (“folds”) as a query protein using a “sum-of-pairs” method. These proteins are returned with Z-scores, which are also measures of significance; a Z-score above 2.0 is deemed a strong match. For example, RmNag’s Z-score is 34.8 (10). 

Each subunit of RmNag has 4 distinct domains, and exhibits an N-terminal β-N-acetylglucosaminidase region (NTR) over domains A and B, and a C-terminal N-acetyltransferase region (CTR) over domains C and D. Since the NTR and CTR are monomeric and dimeric, respectively, in solution, RmNag’s overall dimeric nature is suspected to be due to interactions in the CTR (8). This implies that native RmNag is composed of two equivalent chains, like GlmA (1). Thus, in terms of tertiary structure, RmNag and GlmA are extremely similar. An analysis of the RmNag CTR and GlmA shows a high degree of similarity in terms of secondary structure. Each chain’s CTR has 4 α-helices and 6 β-strands — just like each domain of each chain of GlmA — but there are no β-bulges, unlike in GlmA (8). Both RmNag’s CTR and GlmA exhibit many parallel and antiparallel β-strands, surrounded by α-helices — both of mixed polarities. In fact, this flanking of β-strands on all sides by α-helices is a defining characteristic of N-acetyltransferases (8). In terms of primary structure, the two proteins are extremely different; RmNag’s chains each consist of 858 residues, which is a far greater number than GlmA’s 328 residues per chain (1). Also, whereas RmNag binds acetyl-CoA only in the CTR, GlmA binds acetyl-CoA in both its NTR and CTR. However, whereas only the CTR in GlmA is catalytically competent, both the NTR and CTR are catalytically competent in RmNag (3, 8). Additionally, RmNag has another function: the catalysis of the hydrolysis of β-N-acetylglucosamine residues from polysaccharides (8). Overall, the functions of each protein are categorically different; GlmA acts as a peptidoglycan recovery mechanism in C. acetobutylicum whereas RmNag metabolizes the structural polysaccharide chitin — which consists of N-acetylglucosamine residues in β(1, 4) linkages (4). However, due to RmNag’s CTR region being similar to GlmA, RmNag may also act as a chitin recovery agent simultaneously through its glucosamine re-acetylation motif (8).

Compared to various other enzymes and proteins, Glucosamine N-acetyltransferase from Clostridium acetobutylicum, or GlmA, is a relatively large protein with its 2 subunits of 328 residues each and total molecular weight 76616.79 Daltons (1, 2). These numbers are comparable to proteins on the larger end of the scale, such as human serine albumin or yeast hexokinase (4). However, each part of GlmA — from its catalytic acids and bases in its primary structure and distinctive GNAT secondary structure to its tertiary structure binding pockets — is vital to its function as an acetylating mechanism in bacteria (3). In fact, GlmA’s catalytically competent CTR region, along with its substrates of D-glucosamine and Acetyl-CoA, may provide gram-positive bacteria the avenue through which they may continuously regenerate arguably their most important defense and structure-defining organelle— the cell wall, which is composed of peptidoglycan which in turn is composed of N-acetylglucosamine (5). Therefore, the structure and function of GlmA are intimately related.