Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a beta-agarase from the flavobacterium Zobellia galactanivorans

Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a beta-agarase from the flavobacterium Zobellia galactanivorans

Marine bacteria secrete specific glycoside hydrolases such as agarases to access polysaccharides from algal cell walls as a carbon and energy source. In an attempt to identify agarases with variable degradation patterns, a novel family GH16 beta-agarase from the marine bacterium Zobellia galactanivorans was expressed, purified and crystallized. The purified enzyme crystallized in two distinct forms that were grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant.

Hexagonal crystals belonging to space group P3(1)21 diffracted to 2.2 A resolution, whereas orthorhombic crystals belonging to space group P2(1)2(1)2(1) diffracted to 1.5 A resolution.

Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a beta-agarase from the flavobacterium Zobellia galactanivorans
Expression, purification and preliminary X-ray diffraction analysis of the catalytic module of a beta-agarase from the flavobacterium Zobellia galactanivorans

The regioselectively controlled introduction of chlorine into organic molecules is an important biological and chemical process. This importance derives from the observation that many pharmaceutically active natural products contain a chlorine atom.

Flavin-dependent halogenases are one of the principal enzyme families responsible for regioselective halogenation of natural products. Structural studies of two flavin-dependent tryptophan 7-halogenases (PrnA and RebH) have generated important insights into the chemical mechanism of halogenation by this enzyme family.

These proteins comprise two modules: a flavin adenine dinucleotide (FAD)-binding module and a tryptophan-binding module. Although the 7-halogenase studies advance a hypothesis for regioselectivity, this has never been experimentally demonstrated. PyrH is a tryptophan 5-halogenase that catalyzes halogenation on tryptophan C5 position.

We report the crystal structure of a tryptophan 5-halogenase (PyrH) bound to tryptophan and FAD. The FAD-binding module is essentially unchanged relative to PrnA (and RebH), and PyrH would appear to generate the same reactive species from Cl(-), O(2), and 1,5-dihydroflavin adenine dinucleotide.

We report additional mutagenesis data that extend our mechanistic understanding of this process, in particular highlighting a strap region that regulates FAD binding, and may allow communication between the two modules. PyrH has a significantly different tryptophan-binding module.

The data show that PyrH binds tryptophan and presents the C5 atom to the reactive chlorinating species, shielding other potential reactive sites. We have mutated residues identified by structural analysis as recognizing the tryptophan in order to confirm their role.

This work establishes the method by which flavin-dependent tryptophan halogenases regioselectively control chlorine addition to tryptophan. This method would seem to be general across the superfamily.

Tuberculosis (TB) is a major global health threat caused by Mycobacterium tuberculosis (Mtb). It is further fueled by the HIV pandemic and by increasing incidences of multidrug resistant Mtb-strains.

Rv2827c, a hypothetical protein from Mtb, has been implicated in the survival of Mtb in the macrophages of the host. The three-dimensional structure of Rv2827c has been determined by the three-wavelength anomalous diffraction technique using bromide-derivatized crystals and refined to a resolution of 1.93 A. The asymmetric unit of the orthorhombic crystals contains two independent protein molecules related by a non-crystallographic translation.

The tertiary structure of Rv2827c comprises two domains: an N-terminal domain displaying a winged helix topology and a C-terminal domain, which appears to constitute a new and unique fold. Based on structural homology considerations and additional biochemical evidence, it could be established that Rv2827c is a DNA-binding protein. Once the understanding of the structure-function relationship of Rv2827c extends to the function of Rv2827c in vivo, new clues for the rational design of novel intervention strategies may be obtained.

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