Cloning and charactesization of theDASgene encoding the major methanol assimilatory enzyme from the methylotrophic yeastHansenula polymorpha

Abstract

A gene library from the methanol utilizing yeast Hansenula polymorpha, constructed in a lambda Charon4A vector, was used to clone the gene encoding a key methanol assimilating enzyme, dihydroxyacetone synthase (DHAS) by differential plaque hybridization. The nucleotide sequence of the 2106 bp structural gene and the 5' and 3' non-coding regions was determined. The deduced amino acid sequence of the protein is in agreement with the apparent molecular weight and amino acid composition of the purified protein. The codon bias is not so pronounced as in some Saccharomyces cerevisiae genes.

Properties and functions of the thiamin diphosphate dependent enzyme transketolase

“…Less data are available for DHAS; the enzyme from H. polymorpha, C. boidinii and Acetinobacter sp. displays an even wider range of substrate speci®city, including all of the substrates mentioned for yeast TK, as well as formaldehyde and acetaldehyde as acceptors [70,74,128]. It appears [74] that the Michaelis constants for the donor (Dxylulose 5-phosphate, 1 mM) and acceptor (formaldehyde, 0.43 mM) substrates are similar to those measured for the substrates of TK (Table 1).…”

“…Genes coding for DHAS from the methanolutilising yeast Hansenula polymorpha [70] [132,155] and E. coli [68,152]; three di erent forms of TK have been cloned from the desiccation-tolerant plant Craterostigma plantagineum [4].…”

“…DHAS from C. boidinii is apparently a homotetramer with a subunit molecular weight of 55 kDa [74]. This is signi®cantly smaller than that of DHAS from H. polymorpha [70]. Recently, DHAS has been puri®ed from Acetinobacter sp.…”

A common structural motif in thiamin pyrophosphate‐binding enzymes

“…However, no sequence homology could be detected between this pyruvate decarboxylase-like group of enzymes and the Elp polypeptide chain of the pyruvate dehydrogenase complex from E. coli. This was taken to indicate that the pyruvate decarboxylase group may share a common ancestor dif- Hansenula polymorpha Ysmdas [11 ] ferent from that of E. coli Elp [10] and, by inference, that of E. coli Elo. What has been strikingly absent from the sequence comparisons thus far has been any indication of a sequence motif that might be related to be need of all these proteins to bind the essential cofactor, TPP.…”

“…What has been strikingly absent from the sequence comparisons thus far has been any indication of a sequence motif that might be related to be need of all these proteins to bind the essential cofactor, TPP. This has prompted us to reexamine the sequences and led us to uncover a structural motif found in the sequences of all these enzymes, and indeed in that of a hitherto unconsidered, but mechanistically related enzyme, dihydroxyacetone synthase [11]. This latter enzyme is exemplary of the transketolases.…”