Potential anti-infective targets in pathogenic yeasts: structure and properties of 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans.
A synthetic gene specifying a putative 3,4-dihydroxy-2-butanone 4-phosphate synthase of Candida albicans directed the synthesis of a 22.5 kDa peptide in a recombinant Escherichia coli strain. The recombinant protein was purified to apparent homogeneity by two chromatographic steps and was shown to catalyze the formation of L-3,4-dihydroxy-2-butanone 4-phosphate from ribulose 5-phosphate at a rate of 332 nmol mg(-1) min(-1). Hydrodynamic studies indicated a native molecular mass of 41 kDa in line with a homodimer structure. The protein was crystallized in its apoform. Soaking yielded crystals in complex with the substrate ribulose 5-phosphate. The structures were solved at resolutions of 1.6 and 1.7 angstroms, respectively, using 3,4-dihydroxy-2-butanone 4-phosphate synthase of E. coli for molecular replacement. Structural comparison with the orthologs of Magnaporthe grisea and Methanococcus jannaschii revealed a hitherto unknown conformation of the essential acidic active-site loop.
Echt
Stefanie
S
Lehrstuhl für Organische Chemie und Biochemie, Technische Universität München, Garching, Germany.
Bauer
Stefanie
S
Steinbacher
Stefan
S
Huber
Robert
R
Bacher
Adelbert
A
Fischer
Markus
M
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology*; D001483:Base Sequence; D002176:Candida albicans / Q000201:enzymology*; D003001:Cloning, Molecular; D004271:DNA, Fungal; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010453:Peptide Synthases / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D000935:Antifungal Agents; D004271:DNA, Fungal; C490100:3,4-dihydroxy-2-butanone 4-phosphate synthase, C albicans; D010453:Peptide Synthases