Ketol‐acid reductoisomerase (EC 1.1.1.86) is involved in the biosynthesis of the branched‐chain amino acids. It is a bifunctional enzyme that catalyzes two quite different reactions at a common active site; an isomerization consisting of an alkyl migration, followed by an NADPH‐dependent reduction of a 2‐ketoacid. The 2‐ketoacid formed by the alkyl migration is not released. Using the pure recombinant Escherichia coli enzyme, we show that the isomerization reaction has a highly unfavourable equilibrium constant. The reductase activity is shown to be relatively nonspecific and is capable of utilizing a variety of 2‐ketoacids. The active site of the enzyme contains eight conserved polar amino acids and we have mutated each of these in order to dissect their contributions to the isomerase and reductase activities. Several mutations result in loss of the isomerase activity with retention of reductase activity. However, none of the 17 mutants examined have the isomerase activity only. We suggest a reason for this, involving direct reduction of a transition state formed during the isomerization, which is necessitated by the unfavourable equilibrium position of the isomerization. Our mechanism explains why the two activities must occur in a single active site without release of a 2‐ketoacid and provides a rationale for the requirement for NADPH by the isomerase.

Ketol-acid reductoisomerase (KARI; EC 1.1.1.86) catalyzes two steps in the biosynthesis of branchedchain amino acids. Amino acid sequence comparisons across species reveal that there are two types of this enzyme: a short form (Class I) found in fungi and most bacteria, and a long form (Class II) typical of plants. Crystal structures of each have been reported previously. However, some bacteria such as Escherichia coli possess a long form, where the amino acid sequence differs appreciably from that found in plants. Here, we report the crystal structure of the E. coli enzyme at 2.6 Å resolution, the first three-dimensional structure of any bacterial Class II KARI. The enzyme consists of two domains, one with mixed a/b structure, which is similar to that found in other pyridine nucleotide-dependent dehydrogenases. The second domain is mainly a-helical and shows strong evidence of internal duplication. Comparison of the active sites between KARI of E. coli, Pseudomonas aeruginosa, and spinach shows that most residues occupy conserved positions in the active site. E. coli KARI was crystallized as a tetramer, the likely biologically active unit. This contrasts with P. aeruginosa KARI, which forms a dodecamer, and spinach KARI, a dimer. In the E. coli KARI tetramer, a novel subunitto-subunit interacting surface is formed by a symmetrical pair of bulbous protrusions.Keywords: active site; domain duplication; enzyme structure; NADPH; X-ray crystallography It is over 10 years since Holm and Sander (1994) wrote "More and more frequently, a newly determined [protein] structure is similar in fold to a known one, even when no sequence similarity is detectable." It is implicit in this statement that when proteins show sequence similarity, the fold will be the same, and a host of examples have verified this belief. Nevertheless, some interesting variations have emerged. One example is the structures of the first pair of thiamine diphosphatedependent enzymes to be solved: transketolase (Lindqvist et al. 1992) and pyruvate oxidase (Muller and Schulz 1993). While the overall structure is very similar, consisting of three domains each of ,180 residues, the order of these domains along the primary structure differs; that is, one protein is a circular permutation of the other. This is but one of many examples where individual structural domains are conserved but may be shuffled into new combinations.Reprint requests to: Ronald G. Duggleby, School of Molecular and Microbial Sciences, The University of Queensland, Brisbane, QLD 4072, Australia; e-mail: ronald.duggleby@uq.edu.au; fax: +617-3365-4699.Abbreviations: KARI, ketol-acid reductoisomerase; rmsd, root mean square deviation.Article and publication are at

XIAP, a member of the inhibitor of apoptosis family of proteins, is a critical regulator of apoptosis. Inhibition of the BIR domain-caspase interaction is a promising approach towards treating cancer. Previous work has been directed towards inhibiting the BIR3-caspase-9 interaction, which blocks the intrinsic apoptotic pathway; selectively inhibiting the BIR2-caspase-3 interaction would also block the extrinsic pathway. The BIR2 domain of XIAP has successfully been crystallized; peptides and small-molecule inhibitors can be soaked into these crystals, which diffract to high resolution. Here, the BIR2 apo crystal structure and the structures of five BIR2-tetrapeptide complexes are described. The structural flexibility observed on comparing these structures, along with a comparison with XIAP BIR3, affords an understanding of the structural elements that drive selectivity between BIR2 and BIR3 and which can be used to design BIR2-selective inhibitors.

BackgroundSite-directed mutagenesis is an efficient method to alter the structure and function of genes. Here we report a rapid and efficient megaprimer-based polymerase chain reaction (PCR) mutagenesis strategy that by-passes any intermediate purification of DNA between two rounds of PCR.ResultsThe strategy relies on the use of a limiting concentration of one of the flanking primers (reverse or forward) along with the normal concentration of mutagenic primer, plus a prolonged final extension cycle in the first PCR amplification step. This first round of PCR generates a megaprimer that is used subsequently in the second round of PCR, along with the second flanking primer, but without the intermediate purification of the megaprimer. The strategy has been used successfully with four different plasmids to generate various mutants.ConclusionThis strategy provides a very rapid, inexpensive and efficient approach to perform site-directed mutagenesis. The strategy provides an alternative to conventional megaprimer based site-directed mutagenesis, which is based on an intermediate gel purification step. The strategy gives a high frequency of mutagenesis.

Imidazolonepropionase (HutI) (imidazolone-5-propanote hydrolase, EC 3.5.2.7) is a member of the amidohydrolase superfamily and catalyzes the conversion of imidazolone-5-propanoate to N-formimino-L-glutamate in the histidine degradation pathway. We have determined the three-dimensional crystal structures of HutI from Agrobacterium tumefaciens (At-HutI) and an environmental sample from the Sargasso Sea Ocean Going Survey (Es-HutI) bound to the product [ N-formimino-L-glutamate (NIG)] and an inhibitor [3-(2,5-dioxoimidazolidin-4-yl)propionic acid (DIP)], respectively. In both structures, the active site is contained within each monomer, and its organization displays the landmark feature of the amidohydrolase superfamily, showing a metal ligand (iron), four histidines, and one aspartic acid. A catalytic mechanism involving His265 is proposed on the basis of the inhibitor-bound structure. This mechanism is applicable to all HutI forms.