Pectate lyases harness anti beta-elimination chemistry to cleave the alpha-1,4 linkage in the homogalacturonan region of plant cell wall pectin. We have studied the binding of five pectic oligosaccharides to Bacillus subtilis pectate lyase in crystals of the inactive enzyme in which the catalytic base is substituted with alanine (R279A). We discover that the three central subsites (-1, +1, and +2) have a profound preference for galacturonate but that the distal subsites can accommodate methylated galacturonate. It is reasonable to assume therefore that pectate lyase can cleave pectin with three consecutive galacturonate residues. The enzyme in the absence of substrate binds a single calcium ion, and we show that two additional calcium ions bind between enzyme and substrate carboxylates occupying the +1 subsite in the Michaelis complex. The substrate binds less intimately to the enzyme in a complex made with a catalytic base in place but in the absence of the calcium ions and an adjacent lysine. In this complex, the catalytic base is correctly positioned to abstract the C5 proton, but there are no calcium ions binding the carboxylate at the +1 subsite. It is clear, therefore, that the catalytic calcium ions and adjacent lysine promote catalysis by acidifying the alpha-proton, facilitating its abstraction by the base. There is also clear evidence that binding distorts the relaxed 2(1) or 3(1) helical conformation of the oligosaccharides in the region of the scissile bond.

Of the 30 biosynthetic steps necessary for the production of cobalamin (vitamin B12), eight involve the addition of S-adenosylmethionine-derived methyl groups to the tetrapyrrole framework. These eight methyl additions are catalysed by six canonical methyltransferase domains and one noncanonical methyltransferase domain. Recombinant forms of four methyltransferases from Rhodobacter capsulatus, CobJ, CobM, CobF and CobL, and of the C-terminal noncanonical domain of CobL (CobL-C) have been crystallized, some in more than one crystal form. Most of the crystals diffracted to beyond 2.5 Å resolution and all are suitable for structure determination. Crystals of CobM and CobJ, which are involved in ring contraction, and of CobL, which is involved in two methylations and decarboxylation, are reported for the first time.

In preliminary work, a millimeter-wave vector network analyzer is used to analyze pectate lyase and a sequenced concentration of glucose solutions. Data reveal concentration-dependant spectra for glucose and Ca 2+ -sensitive spectra for pectate lyase.

No abstract

No abstract