Structure of a pullulanase from Bacillus acidopullulyticus

Abstract

The industrial hydrolysis of polysaccharides continues to be of considerable interest and, with the drive towards green chemical processes and renewable sources of fuel, the enzymatic hydrolysis of plant carbohydrates is only set to increase. One of the most widely used plant derived polysaccharides are the starches; predominantly a-1,4 linked D-glucopyranoside polymers with, to varying degrees, a-1,6 branches. Complete starch hydrolysis requires a consortium of enzymes including endo-amylases, glucoamylases and a-glucosidases as well as diverse a-1,6 cleaving enzymes including ‘‘pullulanases’’. Many of these a-glucan active enzymes are found in the largest of the CArbohydrate-active enZYmes (CAZY) sequence families, family GH13 (see www.cazy.org1,2 recently reviewed in a historical context in Ref. 3). Considering the diversity of GH13 enzymes4 and their burgeoning applications in environmental-friendly processes, there remains a need to study these enzymes further particularly to probe their 3-D structures and often complex modular architectures. Here we report the 3-D structure, refined in two crystal forms at resolutions of 1.7 and 2.1 A, respectively, of a pullulanase from the bacterium Bacillus acidopullulyticus. The substrate, pullulan, is a polysaccharide in which repeating maltotriosyl units (a-1,4 linked) are joined through a-1,6 links, Figure 1. Pullulanases cleave the a1,6 links to liberate maltotriose. In industrial applications, these enzymes find use not merely for the breakdown of pullulan but also for the hydrolysis of the a-1,6 linkages in amylopectin starch. The B. acidopullulyticus enzyme (hereafter BaPul13A) thus finds commercial application in the starch industry where its ‘‘debranching’’ activity is utilized in the production of high fructose corn syrup5 and in the production of high maltose content syrups but also in the brewing industry, especially in the production of low calorie and ‘‘light’’ beers where it allows more complete attenuation (i.e., more complete fermentation of the mash and less residual sugar). The 3-D structure of BaPul13A was solved by single isomorphous replacement using an ‘‘in-house’’ uranyl derivative and subsequently refined in two different crystal forms. Pullulanases are frequently characterized by complex multi-domain architectures in which the catalytic module is appended to several carbohydrate-binding domains (CBMs) as well as many domains of unknown function (termed ‘‘X’’ modules). The structure of BaPul13a indeed forms an unusual domain organization, in which the N-terminal CBM41 domain is disordered/partially absent in-crystal but in which the X45a-X25-X45b-CBM48-GH13 multi-domain architecture is both clear and not, to our knowledge, previously observed. Comparison with the known Klebsiella pneumoniae pullulanase6 and the pullulanase/debranching enzyme from Bacillus subtilis reveals a conserved active centre and similar polysaccharide binding surfaces consistent with a1,6 hydrolase activity within the a-amylase fold.