No abstract

We introduce a method of in vitro recombination or "DNA shuffling" to generate libraries of evolved enzymes. The approach relies on the ordering, trimming, and joining of randomly cleaved parental DNA fragments annealed to a transient polynucleotide scaffold. We generated chimeric libraries averaging 14.0 crossovers per gene, a several-fold higher level of recombination than observed for other methods. We also observed an unprecedented four crossovers per gene in regions of 10 or fewer bases of sequence identity. These properties allow generation of chimeras unavailable by other methods. We detected no unshuffled parental clones or duplicated "sibling" chimeras, and relatively few inactive clones. We demonstrated the method by molecular breeding of a monooxygenase for increased rate and extent of biodesulfurization on complex substrates, as well as for 20-fold faster conversion of a nonnatural substrate. This method represents a conceptually distinct and improved alternative to sexual PCR for gene family shuffling.

Oxidation of C, -C, primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43 000-M, subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Znz+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mgz+ ions, MDH interacts with a previously identified 50 00044, activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.

؉ -dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD ؉ -dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed. Methanol dehydrogenase (MDH)1 of Bacillus methanolicus belongs to family III of NAD(P)-dependent alcohol dehydrogenases (ADHs) (2, 3), distinct from the zinc-containing medium chain dehydrogenases/reductases (family I) and the zinc-lacking short chain 32 ADHs (family II) (4, 5). The initial members of family III all were iron-dependent ADHs. In time, with an increasing number of member proteins characterized, it became clear that not all members were iron-dependent. Where investigated, other metals like zinc and magnesium also were found instead of iron (5). B. methanolicus MDH contains one Zn 2ϩ and one or two Mg 2ϩ ions/subunit (3). Identification of members of family III ADHs increasingly became based on overall sequence similarity. Three unique, conserved amino acid sequence motifs have been defined for this family, aiding in ADH classification (2, 6) ( Table I). Over 100 fully sequenced members of family III ADHs are now found in data bases. Many of these are putative proteins, with no biochemical data available.The genes encoding MDH of B. methanolicus, methanol:pnitroso-N,NЈ-dimethylaniline oxidoreductase (MNO) of Amycolatopsis methanolica, MNO of Mycobacterium gastri MB19, ADH of Desulfovibrio gigas, and ADH of Desulfovibrio HDv enzymes of B. methanolicus, A. methanolica, D. gigas, and Desulfovibrio HDv have been cloned and characterized by us (2).2 Classification of the M. gastri enzyme was based on Nterminal amino acid sequence analysis (Fig. 1A). Characterization of the five purified enzymes revealed that each of the proteins possesses a decameric quaternary structure (7-10). The first three are nicotinoproteins, containing a tightly but noncovalently bound NAD(P)(H)/subunit (8, 11). It is unknown whether other members of family III are nicotinoproteins as well. The bound NAD(P)(H) species of MDH and A. methanolica MNO act as cofactors; they become reduced when the enzymes oxidize primary alcohols to the respective aldehydes (8,11). B. methanolicus MDH requires a second, exogenous NAD ϩ for methanol oxidation, serving as a coenzyme and resulting in reoxidation of the NADH cofactor (11). These two NAD(H) molecules are not exchanged during the reaction (11). In vitro, the relatively low coenzyme NAD ϩ -dependent MDH activity is strongly stimulated by a M r 50,000 activator protein from the same organism, resulting in a 40-fold increase in the MDH turnover rate (11,12).Activator protein-mediated activation of MDH is characterized by hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) and converts the Ping-Pong type of reaction mechanism of MDH to a ternary complex mechanism, implyin...

Hydrophobins are a class of small proteins that fulfill a wide spectrum of functions in fungal growth and development. They do so by self-assembling into an amphipathic membrane at hydrophilic-hydrophobic interfaces. The SC3 hydrophobin of Schizophyllum commune is the best-studied hydrophobin. It assembles at the air-water interface into a membrane consisting of functional amyloid fibrils that are called rodlets. Here we examine the dynamics of SC3 assembly at an oil-water and air-water interface and the permeability characteristics of the assembled layer. Hydrophobin assembled at an oil-water interface is a dynamic system capable of emulsifying oil. It accepts soluble-state SC3 oligomers from water in a unidirectional process and sloughs off SC3 vesicles back into the water phase enclosing a portion of the oil phase in their hydrophobic interior. The assembled layer is impermeable to solutes >200 Da from either the water phase or the oil phase; however, due to the emulsification process, oil and the hydrophobic marker molecules in the oil phase can be transferred into the water phase, thus giving the impression that the assembled layer is permeable to the marker molecules. By contrast, the layer assembled at an air-water interface is permeable to water vapor from either the hydrophobic or hydrophilic side.