Crystal structure of the polyextremophilic alpha-amylase AmyB from Halothermothrix orenii: details of a productive enzyme-substrate complex and an N domain with a role in binding raw starch

J Mol Biol. 2008 May 9;378(4):852-70. doi: 10.1016/j.jmb.2008.02.041. Epub 2008 Feb 29.

Abstract

The gene for a membrane-bound, halophilic, and thermostable alpha-amylase, AmyB, from Halothermothrix orenii was cloned and sequenced. The crystal structure shows that, in addition to the typical domain organization of family 13 glycoside hydrolases, AmyB carries an additional N-terminal domain (N domain) that forms a large groove--the N-C groove--some 30 A away from the active site. The structure of AmyB with the inhibitor acarbose at 1.35 A resolution shows that a nonasaccharide has been synthesized through successive transglycosylation reactions of acarbose. Unexpectedly, in a complex of wild-type AmyB with alpha-cyclodextrin and maltoheptaose at 2.2 A resolution, a maltotetraose molecule is bound in subsites -1 to +3, spanning the cleavage point at -1/+1, with the -1 glucosyl residue present as a (2)S(o) skew boat. This wild-type AmyB complex was obtained in the presence of a large excess of substrate, a condition under which it is possible to capture Michaelis complexes, which may explain the observed binding across -1/+1 and ring distortion. We observe three methionine side chains that serve as "binding platforms" for glucosyl rings in AmyB, a seemingly rare occurrence in carbohydrate-binding proteins. The structures and results from the biochemical characterization of AmyB and AmyB lacking the N domain show that the N domain increases binding of the enzyme to raw starch. Furthermore, theoretical modeling suggests that the N-C groove can accommodate, spatially and chemically, large substrates such as A-starch.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acarbose / chemistry
  • Acarbose / metabolism
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism*
  • Binding Sites
  • Cloning, Molecular
  • Crystallography, X-Ray
  • Glucans / chemistry
  • Glucans / metabolism
  • Hydrogen-Ion Concentration
  • Models, Molecular
  • Protein Binding
  • Protein Structure, Tertiary
  • Sodium Chloride
  • Starch / chemistry*
  • Starch / metabolism*
  • Structural Homology, Protein
  • Substrate Specificity
  • Temperature
  • alpha-Amylases / chemistry*
  • alpha-Amylases / genetics
  • alpha-Amylases / metabolism*

Substances

  • Bacterial Proteins
  • Glucans
  • maltoheptaose
  • Sodium Chloride
  • Starch
  • alpha-Amylases
  • Acarbose

Associated data

  • PDB/3BC9
  • PDB/3BCD
  • PDB/3BCF