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.

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.

Discovery of a substrate selectivity switch in tyrosine ammonia-lyase, a member of the aromatic amino acid lyase family.

Tyrosine ammonia-lyase (TAL) is a recently described member of the aromatic amino acid lyase family, which also includes phenylalanine (PAL) and histidine ammonia-lyases (HAL). TAL is highly selective for L-tyrosine, and synthesizes 4-coumaric acid as a protein cofactor or antibiotic precursor in microorganisms. In this report, we identify a single active site residue important for substrate selection in this enzyme family.

Preparation, characterization, and optimization of an in vitro C30 carotenoid pathway.

The ispA gene encoding farnesyl pyrophosphate (FPP) synthase from Escherichia coli and the crtM gene encoding 4,4'-diapophytoene (DAP) synthase from Staphylococcus aureus were overexpressed and purified for use in vitro. Steady-state kinetics for FPP synthase and DAP synthase, individually and in sequence, were determined under optimized reaction conditions. For the two-step reaction, the DAP product was unstable in aqueous buffer; however, in situ extraction using an aqueous-organic two-phase system resulted in a 100% conversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate into DAP.

Identification of a carotenoid oxygenase synthesizing acyclic xanthophylls: combinatorial biosynthesis and directed evolution.

A carotenoid desaturase homolog from Staphylococcus aureus (CrtOx) was identified. When expressed in engineered E. coli cells synthesizing linear C(30) carotenoids, polar carotenoid products were generated, identified as aldehyde and carboxylic acid C(30) carotenoid derivatives.

Directed evolution of Escherichia coli farnesyl diphosphate synthase (IspA) reveals novel structural determinants of chain length specificity.

Directed evolution of farnesyl diphosphate (FPP, C15) synthase (IspA) of Escherichia coli was carried out by error-prone PCR with a color complementation screen utilizing C40 carotenoid pathway enzymes. This allowed IspA mutants with enhanced production of the C40 carotenoid precursor geranylgeranyl diphosphate (GGPP, C20) to be readily identified. Analysis of these mutants was carried out in order to better understand the mechanisms of product chain length specificity in this enzyme.

Alteration of product specificity of Aeropyrum pernix farnesylgeranyl diphosphate synthase (Fgs) by directed evolution.

Directed evolution of the C25 farnesylgeranyl diphosphate synthase of Aeropyrum pernix (Fgs) was carried out by error-prone PCR with an in vivo color complementation screen utilizing carotenoid biosynthetic pathway enzymes. Screening yielded 12 evolved clones with C20 geranylgeranyl diphosphate synthase activity which were isolated and characterized in order to understand better the chain elongation mechanism of this enzyme. Analysis of these mutants revealed three different mechanisms of product chain length specificity.

Investigation of factors influencing production of the monocyclic carotenoid torulene in metabolically engineered Escherichia coli.

Factors influencing production of the monocyclic carotenoid torulene in recombinant Escherichia coli were investigated by modulating enzyme expression level, culture conditions, and engineering of the isoprenoid precursor pathway. The gene dosage of in vitro evolved lycopene cyclase crtY2 significantly changed the carotenoid profile. A culture temperature of 28 degrees C showed better production of torulene than 37 degrees C while initial culture pH had no significant effect on torulene production.

Engineering of secondary metabolite pathways.

Nature produces an astonishing wealth of secondary metabolites with important biological functions. To access this diversity of structurally complex chemical compounds for industrial and biomedical applications, cells have been engineered to produce higher levels and/or novel compounds that were previously inaccessible. Recent examples of metabolic and combinatorial engineering illustrate different strategies for the production of secondary metabolites in microbial cells.

Crystallization of a novel alpha-amylase, AmyB, from the thermophilic halophile Halothermothrix orenii.

This is a report on the structure determination of AmyB, the second alpha-amylase from Halothermothrix orenii, by X-ray crystallography. This bacterium was isolated from saltpans where conditions consisted of both high temperatures and high NaCl content. AmyB is a 599-residue protein which is stable and significantly active at 358 K in starch solution containing up to 10%(w/v) NaCl.

Biosynthesis of structurally novel carotenoids in Escherichia coli.

Previously, we utilized in vitro evolution to alter the catalytic functions of several carotenoid enzymes and produce the novel carotenoids tetradehydrolycopene and torulene in Escherichia coli. Here we report on the successful extension of these pathways and the C(30) carotenoid diaponeurosporene pathway with additional carotenoid genes. Extension of the known acyclic C(30) pathway with C(40) carotenoid enzymes-spheroidene monooxygenase and lycopene cyclase-yielded new oxygenated acylic products and the unnatural cyclic C(30) diapotorulene, respectively.

Crystallization of an alpha-amylase, AmyA, from the thermophilic halophile Halothermothrix orenii.

This report is the first crystallographic study of an amylase from an organism that is both thermophilic and halophilic. alpha-Amylase from the thermophilic halophile Halothermothrix orenii (AmyA) is a 515-residue protein. It is stable and significantly active at 338 K in starch solution containing NaCl [up to 25%(w/v)].

Cloning, sequencing and expression of an alpha-amylase gene, amyA, from the thermophilic halophile Halothermothrix orenii and purification and biochemical characterization of the recombinant enzyme.

A recombinant clone expressing an amylase was identified from an Escherichia coli generated genomic library of the thermophilic, moderately halophilic, anaerobic bacterium Halothermothrix orenii by activity screening, and the gene encoding the enzyme was designated AmyA. The amyA gene was 1545 bp long, and encoded a 515 residue protein composed of a 25 amino acid putative signal peptide and a 490 amino acid mature protein. It possessed the five consensus regions characteristic of the alpha-amylase family and showed the greatest homology to the Bacillus megaterium group of alpha-amylases.