Time-resolved and static-ensemble structural chemistry of hydroxymethylbilane synthase

Area of Science:

• Biochemistry
• Structural Biology
• Enzymology

Background:

• Hydroxymethylbilane synthase (HMBS), also known as porphobilinogen deaminase (PBGD), catalyzes tetrapyrrole synthesis.
• Previous crystallographic studies of HMBS, including time-resolved analyses, have provided insights into its structure.
• A specific polypeptide loop (residues 47-58) near the active site has been consistently absent in electron density maps of determined HMBS structures.

Purpose of the Study:

• To review existing HMBS structures and present new molecular graphics analyses.
• To present a new HMBS cryo-structure (1.66 A resolution) obtained at 100 K, aiming to trap the elusive polypeptide loop.
• To compare the structures of *E. coli* HMBS with *Mycobacterium tuberculosis* (MTb) HMBS to guide future studies on the pathogenic form.

Main Methods:

• Review of existing crystallographic data and molecular graphics.
• X-ray diffraction data collection at cryo-temperature (100 K) for HMBS.
• Refinement of the cryo-structure model, including protein, cofactor, and water molecules.
• Sequence and structural comparison between *E. coli* and *M. tuberculosis* HMBS.

Main Results:

• A new HMBS cryo-structure was refined to 1.66 A resolution (PDB code: 1GTK).
• The elusive polypeptide loop (residues 47-58) remained unobserved in the electron density maps, even at cryo-temperature.
• The *M. tuberculosis* HMBS shows a 41% sequence identity to *E. coli* HMBS, with a notable two-residue deletion in the flexible loop, potentially aiding its visualization.

Conclusions:

• The 1.66 A cryo-structure expands the ensemble of known HMBS structures.
• Despite cryo-temperature, the flexible loop remains challenging to crystallographically resolve.
• Structural differences in the loop of MTb HMBS offer a promising avenue for future structural investigations of this medically relevant enzyme.