The multifunctional 6‐methylsalicylic acid synthase gene of Penicillium patulum

Abstract

6-Methylsalicylic acid synthase (MSAS) from Penicillium patulum is a homomultimer of a single, multifunctional protein subunit. The enzyme is induced, at the transcriptional level, during the end of the logarithmic growth phase. After approximately 150-fold purification, a homogeneous enzyme preparation was obtained exhibiting, upon SDS gel electrophoresis, a subunit molecular mass of 188 kDa. By immunological screening of a genomic P . patulum DNA expression library, the MSAS gene together with its flanking sequences was isolated; 7131 base pairs of the cloned genomic DNA were sequenced. Within this sequence the MSAS gene was identified as a 5322-bp-long open reading frame coding for a protein of 1774 amino acids and 190731 Da molecular mass. Transcriptional initiation and termination sites were determined both by primer extension studies and from cDNA sequences specially prepared for the 5' and 3' portions of the gene. The same cDNA sequences revealed the presence of a 69-bp intron within the N-terminal part of the MSAS gene. The intron contains the canonical GT and AG dinucleotides at its 5'-and 3'-splice junctions. An internal TACTGAC sequence, resembling the TACTAAC consensus element of Succharomyces cerevisiae introns is suggested to represent the branch point of the lariat splicing intermediate. When compared to other known polyketide synthases, distinct amino acid sequence similarities of limited lengths were observed with some, though not all, of them. A comparatively low degree of similarity was detected to the yeast and Penicillium FAS or to the plant chalcone and resveratrol synthases. In contrast, a significantly higher sequence similarity was found between MSAS and the rat fatty acid synthase, especially at their transacylase, 2-oxoacyl reductase, 2-oxoacyl synthase and acyl carrier protein domains. Besides several dissimilar, interspersed regions probably coding for MSAS-and FAS-specific functions, the sequential order of the similar domains was colinear in both enzymes. The low similarity between the two P. putulum polyketide synthases, MSAS and FAS, possibly supports a convergent rather than a divergent evolution of both multienzyme proteins.

6‐Deoxyerythronolide‐B synthase 2 from Saccharopolyspora erythraea

“…Each of the N-terminal and C-terminal halves of the Orf B protein shows striking sequence similarity to their counterparts in Orf A. The alignment of these four 'half-proteins' with each other, with the sequence of rat fatty-acid synthase [27] and with the sequence of 6-methylsalicylate synthase from Penicillium patulum [21], is shown in Fig. 3.…”

“…The polyketide chain is then cyclised to form the 14-membered lactone 6-deoxyerythronolide B (Fig. 1, II), the first isolable intermediate in the pathway to erythromycin A [l, 21.…”

“…I , 1) is a clinically useful macrolide antibiotic produced by the Gram-positive bacterium Sacchurupnlyspora erythraeu [I, 21. The biosynthesis of erythromycin begins with the construction of a polyketide chain from one molecule of propionyl-CoA and six molecules of methylinalonyl-CoA, by a mechanism that resembles the biosynthesis of saturated fatty acids [I, 31.…”

Divergent sequence motifs correlated with the substrate specificity of (methyl)malonyl‐CoA:acyl carrier protein transacylase domains in modular polyketide synthases

“…Ery 1 6, Ave 2, Ole 5 and 6, Nem 6 and 9, and Sor 5, indicate AT domains present in the corresponding modules of the modular PKSs for erythromycin [3,4], avermectin, oleandomycin [13], nemadectin [14] and soraphen A [15] biosynthesis respectively. MSAS represents the 6-methylsalicylic acid synthase AT [16], and MAS that of mycocerosic acid synthase [17]. The sequence of the Escherichia coli malonyl-CoA:acyl carrier protein transacylase (MCAT) [6] is also shown.…”

“…When the acetate-specific AT domain of 6-methylsalicylic acid synthase from Penicillium patulum, [16] a typical type I fungal polyketide synthase, was aligned with the sequences in Fig. 1, it was seen to have a sequence SSDRV-7-QIGLSALL, a better match to the acetate-specific motif.…”