~-------------------------------------------···--··--------~-- ---------------------~ I i 1 I ·j ) ! California State University, Northridge i ; ; BIOSYNTHESIS OF SERINE IN '' NEUROSPORA CRASSA A thesis submitted in partial satisfaction of the requirement for the degree of Master of Science in Biology by Fereshteh Khosravi Kline / May, 1973 .----,·----··----·-·----·------·--·---·--···--·-------··-··----··-·--·-----1 I I I I I I I The thesis of Fereshteh Khosravi Kline is approved: Committee Chairman California State University, Northridge May, 1973 ii r--~--- l ACKNOWLEDGE~lliNTS 1 ! I wish to extend my sincere appreciation and grat- I I j itude to Dr. Joyce Maxwell for her continuous encourage- :ment, assistance, and guidance on my behalf. Her dedi- 1 !cation and deep personal involvement in the preparation i of this "thesis made this effort possible. I also wish to extend my deep appreciation to Dr. · Bianchi and Dr. Spotts for their participation as members of my graduate committee. My sincere gratitude goes to Sam Muslin and Elaine Leboff for their technical assistance. iii --·-·--·----"--------------·-----·-----------·-------··-------, TABLE of CONTENTS page LIST o:f FIGURES ••••••••••••••••••••••••••••••••••••• vi LIST o :f TABLES • • • • • • • • • • .• • • • • • • • • • • • • • • • • • • • • • • . • • • • vii ABST.RACT • ••• • • • • • • • • • • • .. • • • • • • • • • • • • • • • • • • • • • • • • • • • • viii Chapters I. INTRODUCTION •••••••••••••••••••••• , • , •• 1 II. MATERIALS and METHODS •.•••••••••.•••••• 8 Strains . ............................. Chemicals . .. , ....•................•... Maintenance and Growth of Neurospora Cultures . ............................ Genetic Analysis •••••••••••••••••••••• Extraction of Soluble Enzymes ••••••••• En.z yme As says • • • • • •••••••••••••••••••• )-Phosphoglyceric Acid Dehydro­ genase and Glyceric Acid Dehy- drogenase ••.••.••••••••••••••••• Phosphoserine Transaminase and Serine Transaminase ••••••••••••• Phosphos~rine Phosphatase ••••••• III. RESULTS • ••••••.•••••••• I • • • • • • • • • • • • • • • 17 Genetic Analysis of The Mutant Ser-6 •• Mapping of Ser-6 •••••••.••••.••• Genetic Instability of Ser-6 Mutant • •.•..•.• , •.•.•.•••••••••• Mapping of Ser-6 on Linkage Group V • ••••••••.•••••.• , ••••••• I L__ ····~·· -iv Studies Concerning the Amino Acid Re- quirement of Ser-6 ••••••••••••••••.••• Determination of Serine Re- quirement •.•.••••••.•••••••••••• Growth of Ser-6 as a Function of Serine Concentration •••••••••••• Growth of Ser-6 as a Function of Time on Vogel's Minimal Medium and Minimal Medium Supplemented With Serine •••••••• The Effect of Inoculum Size on Utilization of Glycine by s er-6 . ........... a •••••••••••••• A Comparison of Serine Biosynthetic Enzymes in Ser-6 and in the Wild- type Culture . ...................... , .. )-Phosphoglyceric Acid Dehydro­ genase and Glyceric Acid Dehy- drogenase •.••••••.•.•••.•••••••• Phosphoserine Transaminase and Serine Transaminase ••••••.•.•.•• Phosphoserine Phosphatase ••••••• ~. DISCUSSION.. • • . • • • • • • • . • • . • . • • • • • . • • • • • • 48 Genetic Instability of Ser-6 ••••.••••• The Nature of the Serine Requirement • C' 6 1n uer- e 1 1 1 1 1 e e • 1 1 e 1 e e • e 1 1 1 1 • e 1 1 e 1 1 1 1 V. BIBLIOGRAPHY. • • • • • • • • • . • • • • • • . • • • • • • • • • • 59 v ---~---~-----l LIST of' FIGURES . I Figure page I 1. Pathways of serine biosynthesis f'rom i 4 ! products of glycolysis •••••••••.•••••••••••••• ' 2. Pathway of serine biosynthesis f'rom gly- oxylat e IJ •••••••••••••• e • • • • • • • • • • • • • • • • • • • • • • • 7 J. Calculated map of ser-6, his-1 and me-J on linkage group v ..•...••....•...•...•.•.•••• 22 4. Published map of his-1 and me-3 on linkage group V • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. • • • • • • • • 22 5. Growth of' ~er-6 and wild-type Neurospora as a function of serine concentration ••••••••• 27 6. Time course of' growth of ser-6 on serine •••••• 30 7. Glyceric acid dehydrogenase and phospho­ glyceric aci_d dehydrogenase activities in the wild-type grown on Vogel's minimal medium. • . • . • . • . • . • • .35 8. Glyceric acid dehydrogenase and phosphogly­ ceric acid dehydrogenase activities in the wild-type grown on Vogel's medium supple- mented with serine.. • . • • . • • • • • • . • • . • • • . • . • . • • 37 9. Glyceric acid dehydrogenase and phosphogly- ceric acid dehydrogenase activities in ser-6 •• 39 10. Serine transaminase and phosphoserine trans­ aminase activities in the wild-type........... 1+1 11. Serine transaminase and phosphoserine trans­ aminase activities in ser-6 ••••••••••••••••••• 43 _12. Phosphoserine phosphatase activity in the wild-type.. .. • . • • . • . • 45 13. Phosphoserine phosphatase activity in ser-6 ••• 47 vi -------------·- -----------·l LIST of TABLES I Table page I 1. Linkage data on random spores isolated from the cross: ser-6 X his--1, me-'3.... .. 20 2. Linkage data on random spores isolated from the cross: ser-6 X his-1, me-3 •••••••••• 21 3. The effect of individual amino acids on the growth of ser-6 and nutritionally ;o·J wild-type al-2; cot-1 •.••.•••.•. I • I • • • • • • • • • • • 22 l ····~- ~--~---~-----·--~· ..... ·Vii ,------ ---· -------~------~---·---------l ABSTRACT I l I I BIOSYNTHESIS OF SERINE ! I IN NEUROSPORA CRASSA i by Fereshteh Khosravi Kline Master of Science in Biology May 197.3 Two pathways for serine biosynthesis in Neurospora crassa have been reported by Sojka and Garner. The phos­ phorylated pathway involves )-phosphoglyceric acid, phos­ phohydroxypyruvic acid, and phosphoserine as intermedi­ ates, whereas the non-phosphorylated pathway involves glyceric acid and hydroxypyruvic acid as intermediates (1).' The phosphorylated pathway was suggested to be the major pathway of serine biosynthesis in Neurosnora by these authors. In this investigation a serine-requiring mutant of Neuros:eor~ crassa was mapped on linkage group V and was I studied in order to determine the nature of its biochem- i ical lesion. Each enzyme in the two postulated pathways was extracted from the mutant strain and compared with the extract of the corresponding enzyme from the nutritionally·. wild-type strain of Neurospora crassa from which the mu­ tant was originally derived. All enzymes involv:ed in the two path~ays were comparable in activity in the mutant and in the wild-type, indicating that the serine requirement v.iii ,------·--------·---------------------.--·~--·--·--~---·-----, in the mutant does not arise from the loss or alteration I of any of the enzymes in these two pathways. f i I I i ix r--------------------------------~---- ----- --- -- ... ···--·---- .. _________ .. __ ··-··· -··-- ---------··--·-_ -------------~---------------1 I INTRODUCTION . !serine Biosynthesis I Synthesis of serine in Neurospora crassa has been studied by Sojka and Garner (1). The postulated pathways ' i are diagranuned in figure 1. The enzymes active in the ,Phosphorylated pathway are phosphoglyceric acid dehydro­ genase, involved in the conversion of 3-phosphoglyceric acid into 3-phosphohydroxypyruvic acid; phosphoserine : transaminase, active in transamination of 3-hydroxypyruvic :acid into 3-phosphoserine; and finally phosphoserine phos­ ! phatase which is involved in dephosphorylation of phospho- ! I ; serin6 (figure 1). · The enzymes implicated in the non-phos-• 1 · phorylated pathway are glyceric acid dehydrogenase, in­ : volved in conversion of glyceric acid to hydroxypyruvic 1 acid; and serine transaminase, active in transamination of ; hydroxypyruvic acid into serine (figure 1). Because of ·the higher activity of the enzymes involved in the phos­ phorylated pathway compared to the non-phosphorylated pathway under the conditions investigated by these authors, 1 Sojka and Garner suggested that the phosphorylated path­ way was the major pathway for serine biosynthesis in ' Neurospora crassa. The same two pathways for serine bio­ . synthesis were demonstrated originally in animal tissues (2, 3). In this case the relative contribution by each I pathway for the synthesis of serine varies from tissue to 1 2 ~~~:::~~~Y T~~l~:~~S ~~·~~~~~~e ~!h~:~~-::~:~~:s-~ j in higher plants (5). Studies using isotopic competition ! I land mutants blocked in serine biosynthesis indicated that i !the phosphorylated pathway is the only major source of i serine in E. coli and s. typhimurium (6). More recent studies confirming the phosphorylated pathway as the only significant source of serine were carried out in B. sub­ tilis (7), M. lysodeikticus (8), D. desulfuricans (9), and H. influenzae (10). A separate pathway leading to serine from glyoxy- late has been suggested in a variety of orgru1isms, The postulated pathway is shown in figure 2. The conversion of glyoxylate to serine has been shown to be the major pathway of serine biosynthesis in some animal and pla..nt tissues (1.1. 12, 13). Early studies supported the view that serine precede glycine in the pathway present in bakerts yeast (14), but later studies using short-term isotopic labeling indicated that the pathway from gly­ oxylic acid to serine predominates in this organism (15). In Neurosnora, Wright reported that a serine-glycine mu• tant studied by her grew better on glyoxylic acid or gly­ cine than on serine. She suggested that the biqsynthesis of serine in Neurospora involves the conversion of gly­ oxylic acid to glycine and subsequent conversion of gly­ cine to serine (16). The pathway suggested by Sojka and Garner clearly contradicts Wright's postulated pathway as Figure